Design, synthesis, and structure-activity relationships of a series of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.07.047

We designed and synthesized a series of 2-Ar-8-methyl-5- alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF ₁) receptor antagonists. The structure-activity relationships of substituents at each position (R³, R⁵, R ^5′, and R⁸) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF ₁ receptor antagonists.

Full text of DOI:10.1016/j.bmcl.2012.07.047

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5372–5378
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and structure–activity relationships of a series
of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF1 receptor antagonists
Kunitoshi Takeda a, , Taro Terauchi a, Minako Hashizume a, Kogyoku Shin a, Mitsuhiro Ino b,
⇑
Hisashi Shibata b, Masahiro Yonaga a
a Medicinal Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
b Biopharmacology, Tsukuba Research Laboratories, Eisai Co., Ltd, 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-
Received 30 May 2012
Revised 10 July 2012
Accepted 13 July 2012
Available online 20 July 2012
releasing factor 1 (CRF1) receptor antagonists. The structure–activity relationships of substituents at each
0
position (R3, R5, R5 , and R8) was investigated. By derivatization, three compounds (6, 14b, and 14c) were
identified as orally active CRF1 receptor antagonists.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Corticotropin-releasing factor
Depression
Pharmacophore
Structure–activity relationships
Stress
Corticotropin-releasing factor (CRF), isolated by Vale et al. in
1981 as a 41-amino acid-long peptide, is the primary regulator of
the hypothalamic-pituitary-adrenal (HPA) stress response.1 Ele-
vated CRF concentration in patients with depression2 and a
blunted corticotropin response to CRF in patients with depression
and anxiety3 have been observed in clinical studies, which suggests
that CRF receptor antagonists may be useful in the treatment of
depression and anxiety. CRF exerts its biological functions by bind-
ing to 2 GPCR-subfamily receptors, the CRF1 and CRF2 receptors.4–7
The CRF1 receptor is abundantly found in the pituitary and is in-
volved in the regulation of adrenocorticotropic hormone (ACTH),
a key mediator of the stress response.8–10 Therefore, several phar-
maceutical research groups have focused on the discovery of CRF1
receptor antagonists for the treatment of depression or other
stress-related disorders, while the benefits of blocking the CRF2
receptor remain uncertain. To date, several CRF1 receptor antago-
nists have been reported; prototypical antagonists are illustrated
N
N
N
N
N
N
N
N
N
HCl
N
1 (R121919)
2 (CP-154526)
O
O
N
O
N
HN
in Figure 1. CRF1 receptor antagonists,
1 2
(R121919),11
N
N
N
(CP-154526),12,13 3 (DMP696),14 and 4 (CP-316311),15,16 exhibited
high in vitro affinity to the CRF1 receptor and demonstrated signif-
icant activity in animal models. However, the results of using these
antagonists in clinical studies remain ambiguous. Antagonist 1
exhibited efficacy in a small open-label clinical study on major
depression,17 but antagonist 4 failed in a double-blind study on
O
N
O
N
Cl
O
O
O
Cl
3 (DMP696)
4 (CP-316311)
5
⇑
Corresponding author. Tel.: +81 (0) 29 847 5842; fax: +81 (0) 29 847 4952.
E-mail address: k5-takeda@hhc.eisai.co.jp (K. Takeda).
Figure 1. Known CRF1 receptor antagonists.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.047

K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5372–5378
5373
Figure 2. Overlay of known CRF1 receptor antagonists and derived pharmacophores.
R
N
Top region
R
R
R
X
R
N R
5
Design
Ar
Central ring system
8
N
Small pocket
R
R
R
N
Y
Bottom region
Directly-linked series
Linker-series
Known antagonists
Figure 3. Design of a novel CRF1 receptor antagonist.
rectly linked series; various ways of docking to the pharmaco-
phores were obtained by changing only the substitution patterns,
while quinoline was retained as the core template (Fig. 3). Com-
pared to the compound 5 series (linker-series), the quinoline tem-
plate was inverted, and the Y linker atom has been removed. In
addition, the substituent at the C8 position may occupy the small
pocket of a pharmacophore. Consequently, the structure for com-
pound 6 was designed, and in silico simulation used to assess
whether this structure could dock to the pharmacophores, which
it appeared to be capable of doing (Fig. 4). We then set out to syn-
thesize compound 6 and its derivatives.
The synthesis of compound 11 is described in Scheme 1. Nitra-
tion of commercially available 7 was conducted in the presence of
HNO3/H2SO4 to obtain intermediate 8. The position of the nitro-
group was determined by NMR analysis.20 In the next reaction, this
intermediate was coupled to a phenol moiety in the presence of a
palladium catalyst, to yield intermediate 9. Reduction of the nitro-
group of intermediate 9, using Fe, followed by reductive amination
in the presence of NaBH(OAc)3 as a reducing reagent, yielded target
compound 11. A Suzuki-Miyaura cross-coupling reaction of inter-
mediate 8 with boronic acid produced 12.21 Reduction of the
nitro-group of 12, using Fe, followed by reductive amination with
aldehydes or ketones, or a substitution reaction with alkyl halides,
generated target compounds 6 and 14a–i. Compound 20 was syn-
thesized as described in Scheme 2. Compound 18 was synthesized
in a manner similar to that described for compounds 6 and 14a–i,
starting with compound 15, which was prepared using a method
analogous to that previously reported.19 Iodation of 18 afforded 8-
iodoquinoline 19. Target compound 20 was prepared by conversion
of the iodine group to an ethyl group. Intermediates 22, 25, and 30
were prepared in a manner similar to that described for compound
12, starting with 21, 23,22 and 28, respectively (Scheme 3). Interme-
diate 28 was prepared using a method analogous to that reported by
Figure 4. Molecular simulation of compounds 5 and 6.
depression.18 Thus, it is essential to discover structurally diverse
CRF1 receptor antagonists, and to amass the clinical data conveyed
by the results of their usage, in order to clarify the role of CRF in
humans.
We have previously reported a 5-alkylaminoquinoline deriva-
tive 5 as a novel CRF1 receptor antagonist, which is structurally
distinct from other antagonists because this compound possesses
6–6-membered ring with a characteristic substitution pattern,19
and have been exploring compound designs to obtain further struc-
turally diverse CRF1 receptor antagonists. This report describes a
novel series of such compounds. Using the same method previously
reported by us, we extracted four pharmacophores from known
antagonists (Fig. 2) and performed molecular design to find struc-
tures that could dock to these pharmacophores. The analyses were
performed using molecular operating environment (MOE) from
Ryoka System Inc. As a result, we designed a novel structure in a di-

5374
K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5372–5378
NO2
NH2
N
NO2
a
b
c
d
N
N
O
O
N
N
N
O
O
O
O
O
O
O
Cl
Cl
O
O
O
7
8
9
10
11
R5
N
NH2
NO2
R5'
O
N
N
e
f
g
N
8
O
O
O
O
O
O
O
O
12
13
6, 14a-i
Scheme 1. Reagents and conditions: (a) HNO3, H2SO4, ꢀ20 °C, (27%); (b) 2,6-dimethoxy-4-(methoxymethyl)phenol, Pd(OAc)2, 2-di-tert-butylphosphino-20,40,60-
triisopropylbiphenyl, K3PO4, PhMe, reflux, (34%); (c) Fe, saturated aqueous NH4Cl, EtOH reflux, (83%); (d) propionaldehyde, NaBH(OAc)3, AcOH, THF, RT, (69%); (e) 2,6-
dimethoxy-4-(methoxymethyl)phenyl boronic acid, Pd(OAc)2, PPh3, K2CO3, H2O, DME, reflux, (95%); (f) Fe, saturated aqueous NH4Cl, EtOH reflux, (99%); (g) aldehyde or
ketone, NaBH(OAc)3, AcOH, THF, RT, (for 6, 19%, for 14a, 26%, for 14b, 35%, for 14c, 72%, for 14f, 25%, for 14g, 76%, for 14h, 21%), or 2-bromoethyl methyl ether, K2CO3, DMF,
100 °C, (14i, 65%), then aldehyde, NaBH(OAc)3, AcOH, THF, RT, (for 14d, 83%, for 14e, 75%).
NO2
NH2
N
NO2
N
N
a
b
c
d
N
O
O
O
O
N
O
O
Cl
O
O
O
15
16
17
18
N
N
e
I
N
N
O
O
O
O
O
O
19
20
Scheme 2. Reagents and conditions: (a) 2,6-dimethoxy-4-(methoxymethyl)phenyl boronic acid, Pd(OAc)2, PPh3, K2CO3, H2O, DME, reflux, (87%); (b) H2, Pd/C, EtOAc, RT; (c)
propionaldehyde, -picoline-borane, AcOH, MeOH, RT, (75%, 2 steps); (d) NIS, DMF, RT, (79%); (e) Et2Zn, bis(tri-tert-butylphosphine)palladium, 1,4-dioxane, 70 °C, (21%).
a

K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5372–5378
NO2
5375
NO2
a
N
O
O
N
Cl
O
21
22
NO2
NO2
F
N
b
c
F
O
O
N
N
F
I
I
O
23
24
25
NO2
NO2
N
d
e
f
g
Cl
O
I
O
N
N
N
N
Cl
Cl
Cl
Cl
I
I
O
30
26
27
28
29
Scheme 3. Reagents and conditions: (a) 2,6-dimethoxy-4-(methoxymethyl)phenyl boronic acid, Pd(PPh3)4, 1 M Na2CO3 aq, EtOH, PhMe, reflux, (quant.); (b) fuming HNO3,
H2SO4, 0 °C; (c) 2,6-dimethoxy-4-(methoxymethyl)phenyl boronic acid, Pd(PPh3)4, 1 M Na2CO3 aq, EtOH, PhMe, reflux, (30%, 2 steps); (d) I2, LDA, THF, ꢀ78 °C, (49%); (e) LDA,
THF, ꢀ78 °C, (56%); (f) fuming HNO3, H2SO4, 0 °C; (g) 2,6-dimethoxy-4-(methoxymethyl)phenyl boronic acid, Pd(PPh3)4, 1 M Na2CO3 aq, EtOH, PhMe, reflux, (37%, 2 steps).
NO2
NH2
N
N
N
I
N
a
N
b
c
d
R3
O
R3
O
N
N
N
R3
O
R3
O
R3
O
O
O
O
O
O
O
O
O
O
O
3
22
31a,b,c
32a,b,c
33a,b,c
34a,b,c
(R = H)
25 (R3 = F)
a : R3 = H
b : R3 = F
c : R3 = Cl
3
30
(R = Cl)
Scheme 4. Reagents and conditions: (a) Fe, saturated aqueous NH4Cl, EtOH reflux, (for 31a, 99%, for 31b, quant., for 31c, 97%); (b) acetaldehyde, NaBH(OAc)3, AcOH, THF, RT,
(for 32a, 72%, for 32b, 72%, for 32c, 69%); (c) NIS, DMF, RT, (for 33a, 79%, for 33b, 75%, for 33c, 77%); (d) Me2Zn, bis(tri-tert-butylphosphine)palladium, 1,4-dioxane, 70 °C, (for
34a, 95%, for 34b, 85%, for 34c, 98%).
Patrick Rocca et al.,22 starting with 3-chloro-4-iodoquinoline 27.
The position of the nitro-groups of compounds 24 and 29 were
determined by NMR analysis.23 The synthesis of compounds 34a,
34b, and 34c is described in Scheme 4. By using a procedure similar
to that described for 20, compounds 22, 25, and 30 were trans-
formed to yield target compounds 34a, 34b, and 34c, respectively.
The compounds in this study were first screened for their ability
to inhibit [125I] CRF binding to the membranes of cells expressing
the human CRF1 receptor.24 Compounds with high-binding affini-
ties were then evaluated in further functional studies. The func-
tional assay evaluated the CRF antagonistic function of the
compounds, that is, inhibition of CRF-induced cyclic adenosine

5376
K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5372–5378
Table 1
Table 2
0
Effects of R8
Effects of R5 and R5
R5
N
R5'
O
N
N
8
8
R8
R8
N
N
N
O
O
2
2
O
O
O
O
O
O
O
0
No.
R5
R5
Binding
IC50 (nM)
Functional
IC50 (nM)
A
B
6
n-Propyl
n-Propyl
Me
Ethyl
88
212
79
88
89
109
286
246
>1000
>1000
70
>1000
24
46
174
23
NT
NT
NT
NT
No.
Type
R8
Binding IC50 (nM)
Functional IC50 (nM)
14a Me
14b Ethyl
14c Cyclopropylmethyl Cyclopropylmethyl
14d Me
14e Ethyl
14f
14g Cyclopropylmethyl 4-THP
14h
14i
5
11
18
6
A
A
B
B
B
H
Me
H
Me
Et
138
>1000
276
88
115
163
NT
97
70
375
Methoxyethyl
Methoxyethyl
20
Cyclopropylmethyl CH2-4-THP
NT = not tested.
H
H
3-Pentyl
Methoxyethyl
NT = not tested.
monophosphate (cAMP) production in human CRF1 receptor-
expressing HEK293 cells.25
Our initial investigation aimed to assess the effects of substitu-
ents at the C2 and C8 positions. To this end, several derivatives,
shown in Table 1, were prepared. As seen for compounds 5 and
18, binding affinity was reduced by removing the linker atom. It
was noteworthy that introduction of a methyl group to the C8 po-
sition resulted in quite opposite effects between the compounds in
the linker-series (Type A) and those in the directly linked series
(Type B). When compared with compound 5, compound 11 exhib-
ited a markedly reduced binding affinity. However, compound 6
exhibited approximately 3-fold higher binding affinity than did
compound 18. This opposite effect of the methyl group supports
the results of the pharmacophore analysis shown in Figure 4. The
methyl group at the C8 position in type B compounds could occupy
the small pocket of a pharmacophore, leading to improved binding
affinity. On the other hand, the methyl group in type A compounds
was not related to the structure of any pharmacophores and may
have a negative impact on the conformation of Ar–O group in the
bottom region, leading to a reduction in binding affinity. In brief,
we hypothesized that a series of compounds with different modes
of binding to the CRF1 receptor could be obtained by simply remov-
ing the linker atom of a previously reported series of compounds.
By removing the linker atom, the conformation of compound 6 be-
comes more rigid than that of compound 5, which might be a rea-
son for higher binding affinity and more potent antagonistic
activity observed in compound 6. Replacement of methyl at the
C8 position with an ethyl group (20) resulted in a retained binding
affinity, but reduction in antagonistic activity. Thus far, methyl was
the most suitable substituent at the C8 position.
dropyran moiety reduced the binding affinity (14f, 14g), which
indicated that large size of a cyclic group was intolerable at this po-
sition. Secondary amines were also examined, but resulted in
markedly lowered activity (14h and 14i). Among these derivatives,
compound 14b, a di-ethyl amine derivative, exhibited the highest
binding affinity and the most potent antagonistic activity.
The effects of R3 were also examined (Table 3). To allow com-
parison to compound 14b, methyl and di-ethyl amine were re-
tained at the C8 and C5 positions, respectively. Removing the
methyl or replacement of the methyl group with fluorine led to a
decreased binding affinity and antagonistic activity (34a, 34b). In
addition, a chloro derivative, 34c, retained the binding affinity
and antagonistic activity compared to 14b. These results indicated
that substituents at this position influenced activity, although this
position was not related to any pharmacophores according to our
pharmacophore analysis (Fig. 4). Furthermore, the dihedral angle
between the quinoline and Ar group in the bottom region may
Table 3
Effects of R3
5 N
8
N
R3
To further comprehend the SAR of the directly linked series, the
O
O
0
effects of R5 and R5 were examined with the methyl substituent re-
tained at the C8 position (Table 2). Replacement of di-n-propyl with
di-methyl resulted in a lowered binding affinity as well as antago-
nistic activity (14a). Di-ethyl (14b) and di-cyclopropylmethyl (14c)
derivatives again improved the binding affinity as well as the
antagonistic activity. From the results of compound 14d and 14e,
it appears that a methoxyethyl substituent is also tolerated. These
results indicated that substituents larger than a dimethyl amine
group are requisite at this position for high-binding affinity and
potent antagonistic activity. Moreover, introduction of a tetrahy-
O
No.
R3
Binding IC50 (nM)
Functional IC50 (nM)
14b
34a
34b
34c
Me
H
F
79
184
144
81
24
381
131
45
Cl

K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5372–5378
5377
Table 4
Effects of orally administered compounds on CRF-induced fecal pellet output in rats
No.
Fecal weight (g)
Vehicle + CRF + compound
% Inhibition
Statistical significance
Vehicle + CRF
⁄
⁄
⁄
6
14b
14c
1.323 0.283
1.323 0.283
1.216 0.152
0.481 0.137
0.492 0.146
0.567 0.218
64
63
53
Each value represents the mean S.E.M., 6–7 rats/group (⁄P <0.05, unpaired t test).
Compounds (10 mg/kg) were orally administered 1 h before intravenous injection of CRF (10 lg/kg). Fecal weight was measured 4 h after CRF injection.
9. Millan, M. A.; Jacobowitz, D. M.; Hauger, R. L.; Catt, K. J.; Aguilera, G. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 1921.
10. Grigoriadis, D. E.; Dent, G. W.; Turner, J. G.; Uno, H.; Shelton, S. E.; DeSouza, E.
B.; Kalin, N. H. Dev. Neurosci. 1995, 17, 357.
11. Chen, C.; Wilcoxen, K. M.; Huang, C. Q.; Xie, Y. F.; McCarthy, J. R.; Webb, T. R.;
Zhu, Y.-F.; Saunders, J.; Liu, X. J.; Chen, T. K.; Bozigian, H.; Grigoriadis, D. E. J.
Med. Chem. 2004, 47, 4787.
12. 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.; Winston, E. N.; Chen, Y. L.; Heym, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
10477.
13. Mansbach, R. S.; Brooks, E. N.; Chen, Y. L. Eur. J. Pharmacol. 1997, 323, 21.
14. He, L.; Gilligan, P. J.; Zaczek, R.; Fitzgerald, L. W.; McElroy, J. F.; Shen, H.-S. L.;
Saye, J. A.; Kalin, N. H.; Shelton, S.; Christ, D.; Trainor, G.; Hartig, P. J. Med. Chem.
2000, 430, 449.
15. Chen, Y. L.; Braselton, J.; Forman, J.; Gallaschun, R. J.; Mansbach, R.; Schmidt, A.
W.; Seeger, T. F.; Sprouse, J. S.; Tingley, F. D.; Winston, E.; Schulz, D. W. J. Med.
Chem. 2008, 51, 1377.
16. Chen, Y. L.; Obach, R. S.; Braselton, J.; Corman, M. L.; Forman, J.; Freeman, J.;
Gallaschun, R. J.; Mansbach, R.; Schmidt, A. W.; Sprouse, J. S.; Tingley, F. D.;
Winston, E.; Schulz, D. W. J. Med. Chem. 2008, 51, 1385.
17. Zobel, A. W.; Nickel, T.; Kunzel, H. E.; Ackl, N.; Sonntag, A.; Ising, M.; Holsboer,
F. J. Psychiatr. Res. 2000, 34, 171.
be important for antagonistic activity, because the substituents at
this position would have a considerable impact on the angle. More-
over, a twisted conformation for the Ar moiety in the bottom re-
gion may be favored, judging from the finding that substituted
derivatives (14b, 34b, and 34c) exhibited better binding affinities
and antagonistic activities than an unsubstituted derivative 34a.
The in vivo functional antagonism of compounds with potent
in vitro affinity was determined using a CRF-induced defecation
model. It is well known that exogenously injected CRF increases fe-
cal weight in rats and that CRF1 receptor antagonists can block this
CRF-induced defecation.26 Three compounds (6, 14b, and 14c)
were evaluated in this model. Compounds (10 mg/kg) were orally
administered 1 h before intravenous injection of CRF (10 lg/kg)
and the effect on CRF-induced fecal pellet output was evaluated;
the results are shown in Table 4. All compounds caused a statisti-
cally significant decrease in fecal weight compared to a group who
received only vehicle + CRF (6–7 rats/group; P <0.05, unpaired t-
test). These results confirmed the antagonistic activity of these
compounds against the CRF1 receptor in vivo.
18. Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger, T. Am. J. Psychiatry
2008, 165, 617.
19. Takeda, K.; Terauchi, T.; Shin, K.; Ino, M.; Shibata, H.; Yonaga, M. Bioorg. Med.
Chem. 2012, 22, 4756.
20.
In summary, 2-Ar-8-methyl-5-alkylaminoquinoline derivatives
were designed as novel CRF1 receptor antagonists in this study.
When compared to a previously reported 2-aryloxy-5-alkylamino-
quinoline series, higher binding affinity and more potent antago-
nistic activity were obtained in in vitro assays by removing the
linker atom and by introducing substituents at the C8 position.
The core template quinoline remained unchanged, but we expect
that its binding mode to the receptor may have been changed,
according to the results shown in Figure 4 and Table 1. We ob-
tained compounds with a high in vitro potency (6, 14b, 14c, 14e,
NO2
HMBC
NOESY
COSY
N
Cl
8
and 34c) through the preparation and evaluation of the derivatives
21. Miyaura, N.; Yamada, K.; Suzuki, A. Tetrahedron Lett. 1979, 20, 3437.
0
at each position (R3, R8, R5, and R5 ). Among these, compounds 6,
22. Arzel, E.; Rocca, P.; Marsais, F.; Godard, A.; Queguiner, G. Tetrahedron Lett. 1998,
39, 6465.
14b, and 14c exhibited in vivo functional antagonism when orally
administered in a CRF-induced defecation model. Further optimi-
zation will be reported in due course.
23.
NO2
F
NO2
Acknowledgments
N
N
Cl
The authors would like to thank all of their colleagues who
helped to generate the data reported in this manuscript. In partic-
ular, special thanks are due to Kazuya Nagaoka for the help with in
silico simulation.
HMBC
COSY
I
24
I
29
24. For the binding assay, HEK293 cells expressing the human CRF1 receptor were
cloned.4 Screening of CRF1 receptor binding was performed using the
scintillation proximity assay (SPA™, Amersham Pharmacia, UK) using 96-
well plates. Cell membrane (5
beads (1 mg/well), [125I] human/rat CRF (0.1 nM), and diluted test compound
solution were suspended in 150 L of assay buffer (137 mM NaCl, 8.1 mM
Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 10 mM MgCl2, 2 mM EGTA, 1.5% bovine
serum albumin (BSA), Protease inhibitor cocktail (Roche, Diagnostics GmbH),
pH 7.0). Total binding and nonspecific binding were measured in the absence
lg/well), wheat germ agglutinin coated SPA
References and notes
l
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Kilts, C. D.; Loosen, P. T.; Vale, W. Science 1984, 226, 1342.
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Med. 1984, 311, 1127.
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and presence of 0.4 lM unlabeled Sauvagine, respectively. Plates were shaken
gently and incubated for over 2 h at room temperature. The plates were
centrifuged (260 ꢁ g, 5 min, room temperature), and the radioactivity was
detected using a TopCount (Perkin Elmer, MA, USA) 1 min counting time per
well. Each count was corrected by subtracting the non-specific binding, and
was represented as a percentage of total binding. The IC50 value of each
compound was calculated using a concentration-response curve.
25. To determine the activities of the antagonists, their effects on CRF-stimulated
intracellular cyclic AMP (cAMP) accumulation were examined on HEK293 cells
expressing the human CRF1 receptor, as described previously.4 cAMP was

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measured using an enzyme immunoassay (EIA) kit (Amersham Pharmacia, UK).
were lysed with the EIA kit lysis buffer. The amount of intracellular cAMP was
measured according to the manufacturer’s instructions. Basal levels of cAMP
(i.e., in the absence of CRF) were subtracted from the measured values and
these were then expressed as a percentage of total production. The IC50 value of
each compound was calculated using a concentration–response curve.
26. Saunders, P. R.; Maillot, C.; Million, M.; Tache, Y. Eur. J. Pharmacol. 2002, 435,
231.
HEK293 cells expressing the human CRF1 receptor were seeded into 96-well
plates (5 ꢁ 104 cells/well) in DMEM containing 0.1% fetal bovine serum and
1 mM 3-isobutyl-1-methylxanthine, which is a phosphodiesterase inhibitor.
After 30 min of pre-incubation, the diluted test compounds were added to the
wells and incubated for
a further 30 min at 37 °C. The cells were then
stimulated with 1 nM human/rat CRF for 30 min at 37 °C and collected by
centrifugation (630 ꢁ g, 5 min, 4 °C). After aspiration of the medium, the cells