2-Arylaminothiazoles as high-affinity corticotropin-releasing factor 1 receptor (CRF1R) antagonists: Synthesis, binding studies and behavioral efficacy

Article abstract of DOI:10.1016/j.bmcl.2003.08.055

2-Arylamino-4-trifluoromethyl-5-aminomethylthiazoles represent a novel series of high-affinity corticotropin releasing factor-1 receptor (CRF ₁R) antagonists that are prepared in three steps in good overall yields. Herein, we report binding SAR as well as anxiolytic activity of an exemplary compound (7a, K_i=8.6 nM) in a mouse canopy model.

Full text of DOI:10.1016/j.bmcl.2003.08.055

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3997–4000
2-Arylaminothiazoles as High-Affinity Corticotropin-Releasing
Factor 1 Receptor (CRF1R) Antagonists: Synthesis, Binding
Studies and Behavioral Efficacy
Gene M. Dubowchik,a,* Jodi A. Michne,a Dmitry Zuev,a Wendy Schwartz,a
Paul M. Scola,a Clint A. James,b Edward H. Ruediger,b Sokhom S. Pin,a
Kevin D. Burris,a,y Lynn A. Balanda,a Qi Gao,a Dedong Wu,a Lawrence Fung,a,{
Tracey Fiedler,a Kaitlin E. Browman,a,x Matthew T. Tabera and Jie Zhanga,{
aBristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5100, Wallingford, CT 06492-7660, USA
bBristol-Myers Squibb Pharmaceutical Research Institute, Candiac, Quebec, Canada J5R 1J1
Received 5 June 2003; revised 13 August 2003; accepted 27 August 2003
Abstract—2-Arylamino-4-trifluoromethyl-5-aminomethylthiazoles represent a novel series of high-affinity corticotropin releasing
factor-1 receptor (CRF1R) antagonists that are prepared in three steps in good overall yields. Herein, we report binding SAR as
well as anxiolytic activity of an exemplary compound (7a, Ki=8.6 nM) in a mouse canopy model.
# 2003 Elsevier Ltd. All rights reserved.
Corticotropin-releasing factor (CRF),
a
41-residue
brospinal fluid in a large portion of individuals diag-
nosed with major depression and anxiety disorders.
Furthermore, the CRF levels were shown to correlate
with severity of illness.5 Following antidepressant treat-
ment, the increased CRF levels observed in depressed
patients were reduced.6 Commensurate with its role as
the principal regulator of mammalian physiological and
behavioral responses to stress, CRF has also been
shown to mediate several immune system functions
through its effect on glucocorticoid plasma levels.7 The
neuropeptide carries out its diverse set of roles through
binding to CRF1 and CRF2 receptors,8 which belong to
the family of transmembrane G-protein-coupled receptors.
neuropeptide, first isolated from ovine hypothalamus,
coordinates the neuroendocrine, autonomic and beha-
vioral responses to stress by stimulating the release of
adrenocorticotropic hormone (ACTH) from the ante-
rior pituitary gland.1,2 Stress-induced secretion of
ACTH by CRF initiates the synthesis and release of
adrenal glucocorticoids, which subsequently suppress
the synthesis of CRF and ACTH, thereby restoring
homeostasis of the hypothalamic–pituitary–adrenal
(HPA) axis.3 Hypersecretion of CRF in the central ner-
vous system may lead to a variety of psychiatric and
stress-related illnesses, such as anxiety, depression,
obsessive-compulsive and post-traumatic stress dis-
orders.4 Support for this hypothesis is given by the
detection of marked elevations of CRF in the cere-
Various animal studies have suggested that antagonism
of the effects of CRF binding to CRF1 receptors
(CRF1R) present in the CNS represents a novel target
for the treatment of depression and anxiety. As a result,
numerous classes of non-peptide small molecules have
been reported as selective CRF1R antagonists.9,10
Extensive analysis of structure–activity relationships
(SARs) has led to the identification of structural fea-
tures common to most CRF1R antagonists.9 These are
exemplified by 111 and 2:12 a heteroaromatic core with
an sp2-hybridized nitrogen, a small alkyl group on the
atom next to that nitrogen, and an aryl ring attached to
*Corresponding author. Fax: +1-203-677-7702; e-mail: gene.
dubowchik@bms.com
yPresent address: Palatin Technologies Inc., 175 May St., Edison,
NJ 08837, USA.
{Present address: Neurogen Corp., 35 N.E. Industrial Rd., Branford,
CT 06405, USA.
xPresent address: Abbott Laboratories, Dept. R4N5, Bldg. AP9A, 100
Abbott Park Rd., Abbott Park, IL 60064-6125, USA.
{Present address: Aventis Pharmaceuticals, PO Box 6800, Bridge-
water, NJ 08807-0800, USA.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.055

3998
G. M. Dubowchik et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3997–4000
Table 1. hCRF1R binding affinities of thiazole amides 6
it containing one or two ortho-substituents to enforce
what has been proposed to be the active, mutually
orthogonal conformation. In addition, most potent
CRF1R antagonists possess a para-substituent on the
pendant aryl ring as well as a branched side chain
attached to the core heterocycle.
Compd
R1
R2
Ki (nM)
6a
6b
6c
6d
6e
6f
cPrCH2
nPr
nBu
nPr
nPr
Et
Et
Et
360
820
2800
5000
16,000
51,000
Et
MeOCH2CH2
MeOCH2CH2
MeOCH2CH2
Table 2. hCRF1R binding affinities of thiazoles 6–8
In search of novel antidepressive/anxiolytic agents, we
have synthesized 2-arylamino-4-trifluoromethyl-5-ami-
nomethylthiazoles that are high-affinity CRF1R
antagonists. These compounds were easily prepared in
three steps from commercially available starting mate-
rials. As shown in Scheme 1, the aminothiazole was
assembled by condensation of ethyl 2-chloro-3-oxobu-
tyrates 3 with aryl thioureas 4.13 Hydrolysis of the esters
5 was attempted (LiOH, H2O, THF, rt, 12 h), but the
resulting carboxylates were easily decarboxylated.
Instead, direct amidation under Weinreb conditions14
provided amides 6 cleanly and in good yields.
Compd
X
R1
R2
R3
R4
R5
Ki (nM)
6g
6h
6i
O
O
O
Me
Et
Me
Cl
Cl
Cl
Cl
Cl
Br
Cl
Cl
C
C
Br
Me
Me
Br
Br
Br
Cl
Me
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
Me
Cl
Cl
Cl
H
H
H
H
H
H
H
H
Me
Me
2600
11,000
31,000
8.6
C
F
3
7a
7b
7c
7d
7e
8a
8b
7f
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
H2
CF3
Me
a
Cl
—
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Me
Me
Me
Me
Me
C Cl F H
Br
Me
Me
Br
H
16
21
23
100
CRF1R binding affinities were determined by displace-
ment of [125I]Tyr-o-CRF from hCRF1R endogenously
expressed on IMR-32 human neuroblastoma cells.15
Initial work (Table 1) showed that the (N-cyclopro-
pylmethyl-N-n-propyl)amide 6a was superior to some
other small branched structural motifs commonly used
by others.9 In addition, the very low affinity demon-
strated by MeO-containing compounds 6e and 6f sug-
gested that polar atoms in this region might not be well
tolerated by the receptor.
l
l
Et
67
28
28
60
73
7.8
2900
56
3
7g
7h
7i
Me
Me
Cl
H
H
H
H
H
Et
H
H
H
7j
Br
7k
8c
7l
7m
7n
iPr
iPr
Cl
MeO
H
H
H
H
Et
4600
5100
MeO
Et
>40,000
aCompound was unstable to purification.
We decided to explore structural changes in other parts
of the chemotype while retaining the N-cyclopro-
pylmethyl-N-n-propyl side chain (Table 2). The moder-
ate activity of 6a was reduced by replacement of all the
chlorines with CH3 (6g). The Dupont group had repor-
ted that an ethyl group often confers best affinity for a
CRF1R antagonist containing a five-membered A-ring,
while methyl substitution is optimal when the A-ring is
a six-membered aromatic heterocycle.18 Surprisingly,
the 4-ethylthiazole 6h showed a 30-fold loss of activity
in comparison with 6a. Replacement with CF3 (6i) fur-
ther reduced affinity.
Concern that the amide group in 6 might be imposing
unfavorable conformational rigidity, or that the polarity
of the carbonyl oxygen might be disfavored in the
binding pocket, led us to reduce it to give the corre-
sponding 5-aminomethylthiazoles 7. For the 4-tri-
fluoromethylthiazoles, 1 M BH3–THF followed by
methanolysis (Scheme 2) gave high yields of stable pro-
ducts (7a and 7c–p).19 However, in the case of 6a, these
Scheme 1. Reagents and conditions: (a) EtOH, reflux, 16 h, 85–97%;
(b) Me3Al, R5-NH-R6, PhCH3, reflux, 14 h, 68–92%.

G. M. Dubowchik et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3997–4000
3999
Scheme 2. Reagents and conditions: (a) (i) BH3–THF (3 equiv), reflux,
16 h; (ii) MeOH, reflux, 1 h (56–88%); (b) NaH, MeI or EtI, THF, rt,
14 h, 95%-quant.
conditions led to decomposition, presumably because
the resulting 4-methyl-2-aminothiazole 7b is so electron-
rich that loss of the side-chain amine is facile. Using a
non-Lewis acidic reducing agent (Red-Al, PhMe, rt, 8
h), 7b could be generated and even purified by pre-
parative LC. However, decomposition occurred upon
concentration of the product-containing fractions.
Clearly, the 5-aminomethyl-2-aminothiazole core
requires at least one electron-withdrawing group to be
viable under ambient conditions.
Figure 1. X-ray structure of 7a (HCl salt).
As shown in Table 2, amide reduction of 6i resulted in a
3500-fold gain in CRF1R affinity for the product, 7a.
An X-ray crystal structure of the HCl salt of 7a (Fig. 1)
shows an orthogonal relationship between the two rings
in the crystal form. The oxazole analogue (9) of 7a was
prepared using a different synthetic sequence.20 An
almost 400-fold loss of binding affinity for 9 (Ki=3.5
mM) suggests that the large sulfur atom is lipophilic
enough to be tolerated by the CRF1R and may serve to
maintain mutual orthogonality between the rings.
Table 3. Rat PK Parameters for 7a (10 mg/kg, po; 5 mg/kg, iv)
T1/2
Cl
Fpo
Vd
7.6 h
16 mL/min/kg
9% (ꢀ3%)
1.1 L/kg
2.0
1730 ng/mL*h
B/P (2 h)
AUC(plasma), 0-4 h
propylphenyl compound 7k was increased 50-fold by
N-ethyl substitution (8c).
Compound 7a was chosen for further study. Table 3
shows the results of a pharmacokinetic study in rats.
The HCl salt of 7a showed moderate clearance and
volume of distribution with good brain uptake and an
acceptable plasma half-life. However, oral bioavail-
ability was low.
Sequential replacement of aryl chlorines with CH3 led to
a systematic diminution of activity for 7c, 7e, 7i, and 7h.
No advantage was seen with the larger o-Br substituents
in 7d or 7j. Removal of one o-Cl from 7a resulted in a
500-fold reduction in activity for 7l, while two other 2,4-
disubstitution patterns (7k and 7m) gave similarly low
activity. A single example of a 2,6-disubstitution pattern
(7n) was inactive.
In order to determine its potential as an anxiolytic
agent, 7a (HCl salt) was tested in a mouse canopy
stretched attend posture (SAP) model.21 In this para-
digm, mice are placed on a well-lit black platform, a
portion of which is covered by a clear red canopy
attached by a central pillar. SAPs are characterized by
forward elongations of the body, exhibited when the
mouse is standing still or moving slowly forward. This
behavior is investigative in nature, and is considered an
important behavioral indicator of anxiety in mice.
Active compounds reduce the number of SAPs, indicat-
ing anxiolytic potential. When given ip at 32 and 64 mg/
kg, 7a significantly reduced SAPs in a dose-dependent
manner in comparison with vehicle-treated mice (Fig.
2). CP-154,526 1 showed comparable activity at 32 mg/
kg, as did buspirone at 2 mg/kg.
Alkylation of the linking nitrogen atom between the
core heterocycle and aryl ring with methyl or ethyl
groups has been found to be advantageous for some
CRF1R antagonist chemotypes. This was easily effected
by treatment of 7 with excess NaH in THF followed by
methyl- or ethyl iodide. The remarkably low nucleophil-
icity of the aminomethyl nitrogen was attested to by the
fact that 6 equiv of alkyl iodide could be used without
formation of detectable quaternized product. Using 7f
as a test case for trisubstituted phenyl compounds, we
found that N-alkylation with methyl (8a) or ethyl (8b)
had a somewhat deleterious effect on CRF1R binding.
In contrast, the affinity of the 2-bromo-4-iso-
In summary, we have discovered a new class of high-affi-
nity CRF1R antagonists, 2-arylamino-4-trifluoromethyl-
5-aminomethylthiazoles, that can be prepared in three

4000
G. M. Dubowchik et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3997–4000
min at 25 ꢁC(assay buffer: 50 mM Tris (pH 7.2), 10 mM
MgCl2, 0.5% BSA, 0.005% Triton X-100, 10 mg/mL aprotinin
and 10 mg/mL leupeptin). Assays were stopped by addition of
ice-cold buffer. Non-specific binding was defined with 10 mM
o-CRF. These compounds are full antagonists of the CRF1R,
as determined by their ability to inhibit CRF stimulated
cAMP production in IMR-32 cells.16 For 7a, functional
EC50=42 nM (90% inhibition). Compound 7a was also tested
against the CRF2R and found to have IC50>10 mM.17
16. Dieterich, K. D.; DeSouza, E. B. Brain Res. 1996, 733,
113.
Figure 2. Canopy test results in which a reduction in stretched attend
postures corresponds to putative anxiolytic activity.20 Data represents
the meanꢀSEM of 10 mice (BALBc) per group. Asterices indicate
significant difference from vehicle, p<0.05 (Dunnett’s test).
17. Suman-Chauhan, N.; Carnell, P.; Franks, R.; Webdale,
L.; Gee, N. S.; McNulty, S.; Rossant, C. J.; Van Leeuwen, D.;
MacKenzie, R.; Hall, M. D. Eur. J. Pharmacol. 1999, 379, 219.
18. Wilde, R. G.; Klaczkiewicz, J. D.; Carter, K. L.; Gilligan,
P. J.; Olson, R. E.; Frietze, W. E.; Buckner, W. H.; Curry, M.
A.; Robertson, D. W.; Sun, J. H.; Arneric, S. P.; Hartig, P.;
Fitzgerald, L. W.; Marshal, W. J. 219th ACS National Meet-
ing, San Francisco, CA, 2000; MEDI 317.
19. After overnight reflux in 1:1 concd HCl/MeOH, 7a was
found to be unchanged. All new compounds gave satisfactory
analytical data. For 7a: 1H NMR (CDCl3) d 0.05 (2H, q), 0.45
(2H, m), 0.84 (4H, t and m), 1.42 (2H, m), 2.34 (2H, d), 2.48
(2H, ABq), 3.75 (2H, d), 7.45 (2H, s), 8.42 (1H, brs). Mass
spec.: 474.12 (MH)+. Anal. calcd for C18H19N3SF3Cl3: C
42.45, H 3.96, N 8.25. Found: C42.56, H 3.94, N 8.07.
20. Synthesis of 9 (yields are unoptimized):
steps in good overall yields. An exemplary compound,
7a, demonstrated anxiolytic activity in a mouse beha-
vioral model, suggesting potential use of these com-
pounds as anxiolytic agents.
References andNotes
1. Owens, M. J.; Nemeroff, C. B. Pharmacol. Rev. 1991, 43, 425.
2. Grigoriadis, D. E.; Haddach, M.; Ling, N.; Saunders, J.
Curr. Med. Chem. CNS Agents 2001, 1, 63.
3. Plotsky, P. M. J. Neuroendocrinol. 1991, 3, 1.
4. Kasckow, J. W.; Baker, D.; Geracioti, T. D. Peptides 2001,
22, 845.
5. Holsboer, F. J. Psychiatric Res. 1999, 33, 181.
6. Banki, C. M.; Karmasci, L.; Bissette, G.; Nemeroff, C. B.
Eur. Neuropsychopharmacol. 1992, 2, 107.
7. Webster, E. L.; Torpy, D. J.; Elenkov, I. J.; Chrousos, G. P.
Ann. N.Y. Acad. Sci. 1998, 840, 21.
8. Takahashi, L. K. Neurosci. Behav. Rev. 2001, 25, 627.
9. Gilligan, P. J.; Robertson, D. W.; Zaczek, R. J. Med.
Chem. 2000, 43, 1641.
10. McCarthy, J. R.; Heinrichs, C.; Grigoriadis, D. E. In
Annual Reports in Medicinal Chemistry; Bristol, J. A., Ed.;
Academic: San Diego, CA, 1999; Vol. 34, p 11.
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., 3rd;
Winston, E. N.; Chen, Y. L.; Heym, J. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 10477.
12. Chen, C.; Dagnino, R.; De Souza, E. B.; Grigoriadis,
D. E.; Huang, C. Q.; Kim, K. I.; Liu, Z.; Moran, T.; Webb,
T. R.; Whitten, J. P.; Xie, Y. F.; McCarthy, J. R. J. Med.
Chem. 1996, 39, 4358.
13. Dodson, R. M.; King, L. C. J. Am. Chem. Soc. 1945, 67,
2242.
14. Levin, J. I.; Turos, E.; Weinreb, S. M. Synth. Commun.
1982, 12, 989.
15. Membranes were prepared from IMR-32 cells as pre-
viously described16 and incubated with [125I]Tyr-o-CRF (100
pM) and increasing concentrations of test compound for 100
Reagents and conditions: (a) 3,4,5-trichloronitrobenzene,
K2CO3, DMF, 80 ꢁC, 48 h (80%); (b) (i) SnCl2, EtOH, reflux,
1 h (79–96%); (ii) CuCl2, tBuONO, MeCN, 65 ꢁC, 1 h (35%);
(c) LiAlH4, THF, 0 ꢁCto rt (18%); (d) Dess–Martin, CH 2Cl2,
rt (59%); (e) (i) c-PrCH2NHnPr, (MeO)3CH, DMF, AcOH;
(ii) NaBH(OAc)3, (10%).
21. Grewal, S. S.; Shepherd, J. K.; Bill, D. J.; Fletcher, A.;
Dourish, C. T. Psychopharmacology 1997, 133, 29.