Design, synthesis and structure-activity relationships of 5-alkylaminolquinolines as a novel series of CRF1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2012.05.066

A series of 5-alkylaminolquinolines was designed and synthesized as potential novel CRF₁ receptor antagonists. The structure-activity relationships (SARs) of the substituents on each position (R², R ³, R⁵ and R^5′) were investigated.

Full text of DOI:10.1016/j.bmcl.2012.05.066

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4756–4761
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and structure–activity relationships of
5-alkylaminolquinolines as a novel series of CRF1 receptor antagonists
Kunitoshi Takeda a, , Taro Terauchi 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:
A series of 5-alkylaminolquinolines was designed and synthesized as potential novel CRF1 receptor antag-
0
onists. The structure–activity relationships (SARs) of the substituents on each position (R2, R3, R5 and R5 )
Received 5 April 2012
Revised 16 May 2012
Accepted 17 May 2012
Available online 24 May 2012
were investigated.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
CRF
SAR
Pharmacophore
Stress
Depression
Corticotropin-releasing factor (CRF) is a 41-amino acid peptide
that acts as the prime regulator of the hypothalamic-pituitary-
adrenal (HPA) axis.1 CRF is a major modulator of the body’s overall
response to stressors2–6 and there is evidence supporting the
hypothesis that over production of CRF may underlie the pathology
of stress-related disorders, such as depression and anxiety.7–10 CRF
acts through a class-B G protein-coupled receptor (GPCR), which is
divided to two subtypes, CRF1 receptor and CRF2 receptor.11–14
CRF1 receptor is the most abundant subtype found in the pituitary
and is involved in the regulation of adrenocorticotropic hormone
(ACTH), a key mediator of the body’s response to stress.15–17 There-
fore a CRF1 receptor antagonist is hypothesized to be a valuable
target for the treatment of stress-related disorders. Prototypical
CRF1 receptor antagonists are illustrated in Figure 1. Preclinical
these trials. The accumulation of clinical data using a number of
structurally diverse CRF1 receptor antagonists is an efficient way
to investigate the role of CRF in humans and to discover new drugs
for treating depression and anxiety. Here we report the generation
of novel CRF1 receptor antagonists that have distinctive structural
features compared to the currently known antagonists.
First, we aligned low-energy conformers of the known antago-
nists shown in Figure 1 to extract pharmacophores using MOE
(Molecular Operating Environment) from Ryoka System Inc. The
analysis indicated the following structural features (Fig. 2): (1)
the top region is occupied by a lipophilic group such as a di-al-
kyl-amine, (2) the bottom region consists of substituted phenyl
rings, (3) there is a central ring system that fixes a certain confor-
mation between the top and bottom regions, (4) there is a small
pocket in which a methyl group fits. Potential variations in the
structures of the top and bottom regions are thought to be limited.
However, the central core appears to be relatively adaptable, with
the presence of a number of different hetreoaromatic systems ob-
served. Therefore, we examined the possibility of modifying the
central core structure, while leaving the positions of the top and
bottom regions intact. Molecular design was performed to find a
motif that fitted each pharmacophore and this suggested that a
6-6-membered ring was appropriate as the central ring system,
with side chains placed at the appropriate positions (Fig. 3). Quin-
oxaline derivative 10a was selected as a preliminary structure,
then in silico simulation was performed to clarify whether this
molecule fitted the pharmacophore model prior to synthesizing
the other derivatives. The results indicated that the compound
adequately occupied all of the pharmacophores (Fig. 4).
studies on CRF1 antagonists 1 (R121919),18 2 (CP-154526),19,20
3
(DMP696),21 and 4 (CP-316311)22,23 support the hypothesis that
CRF1 receptor antagonists have the potential to be used for the
treatment of stress-related disorders. However, clinical studies
looking at the role of CRF in the pathophysiology of depression
have been equivocal. CRF1 receptor antagonist
1 (R121919)
showed some efficacy in a small open label clinical study for major
depression,24 but 4 (CP-316311) was found to be ineffective in a
double-blind, placebo-controlled study for depression using sertra-
line as a positive control.25 Further clinical studies would be neces-
sary to elucidate the reasons for the differences in the success of
⇑
Corresponding author. Tel.: +81 29 847 5842; fax: +81 29 847 4952.
E-mail address: k5-takeda@hhc.eisai.co.jp (K. Takeda).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.066

K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4756–4761
4757
N
N
N
N
N
N
N
N
N
HCl
N
1 (R121919)
2 (CP-154526)
O
O
O
HN
N
N
N
N
O
N
Cl
Cl
3 (DMP696)
4 (CP-316311)
Figure 1. Known CRF1 receptor antagonists.
Figure 4. Molecular simulation of compound 10a.
The synthesis of compounds 10a–b is described in Scheme 1.
Intermediate 6 was prepared by condensation of commercially
available 3-nitro-o-phenylenediamine (5) with pyruvic acid.
Chlorination using POCl3 followed by a substitution reaction with
ArNH2 afforded intermediate 8. Hydrogenation of the nitro group
in 8 gave the amine 9 and the following reductive amination in
the presence of
a-picoline-borane as a reducing agent, gave the
target derivatives 10a–b. Compounds 16–18 were prepared as
depicted in Scheme 2. Chlorination using POCl3 of commercially
available 5-nitro-1,4-dihydro-quinoxaline-2,3-dione (11) afforded
di-chloro 12, which was transformed into intermediate 13 using
sodium methoxide. A substitution reaction of 13 with ArNH2
Top region
Small pocket Central ring system
Bottom region
Overlay of known antagonists 1, 2, 3 and 4
Derived pharmacophores
Figure 2. Overlay of known CRF1 receptor antagonists and derived pharmacophores.
R
N
Top region
R
R
X
R
6
Design
Ar
Central ring system
6
Small pocket
R
R
Y
Bottom region
Known antagonists
Figure 3. Design of a novel CRF1 receptor antagonist.

4758
K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4756–4761
O2N
N
O2N
N
O2N
O2N
H2N
a
c
b
d
N
N
N
N
HN
NH2
OH
Cl
5
6
7
8
R'
N
H2N
N
R
e
N
N
N
HN
HN
9
10a-b
Scheme 1. Reagents and conditions: (a) Pyruvic acid, oxalyl chloride, DMF, DCM, rt, (46%); (b) POCl3, N,N-diethylaniline, reflux, (59%); (c) 2,4,6-trimethylaniline, NMP, 150 °C;
(d) H2, Pd/C, EtOH, THF, rt, (22%, two steps); (e) aldehyde or ketone,
a-picoline-borane, AcOH, MeOH, rt.
O2N
O2N
N
O2N
N
O2N
b
c
d
a
HN
O
N
N
N
N
N
HO
Cl
MeO
HN
OH
Cl
Cl
11
12
13
14
H2N
N
N
N
N
N
e
f
HN
O
HN
N
N
N
N
O
O
O
HN
HN
HN
N
15
16
17
18
Scheme 2. Reagents and conditions: (a) POCl3, N,N-diethylaniline, reflux; (b) NaOMe, MeOH, DMF, rt, (18%, two steps); (c) 2,4,6-trimethylaniline, NMP, 150 °C; (d) H2, Pd/C,
EtOH, THF, rt, (61%, two steps); (e) propionaldehyde, -picoline-borane, AcOH, MeOH, rt, (75%); (f) NaH, MeI, DMF, rt, (17:26%, 18:36%).
a
produced intermediate 14 with the accompanying demethylation
of the methoxy-quinoxaline moiety. Hydrogenation of 14 followed
reducing agent, afforded compounds 23–38. The synthesis of 2,6-
dimethoxy-4-methoxymethylaniline (40) and 2,6-dimethoxy-4-
methoxymethylphenol (42) is described in Scheme 4. Compound
40 was synthesized from compound 39 via azidation. Hydrolysis
of 41 using H2O2 afforded compound 42.
The compounds in this study were screened for their ability to
inhibit [125I] CRF binding to cell membranes expressing the human
CRF1 receptor.28 Table 1 summarizes the results obtained for the
synthesized quinoxaline derivatives. Compound 10a exhibited a
high binding affinity. This result not only showed the validity of
our pharmacophore model but also encouraged further derivatiza-
tion of the quinoxaline template to advance current understanding
of the structure–activity relationship (SAR) involved. Both second-
ary and tertiary amines were found to be well tolerated in the top
by reductive amination in the presence of
a-picoline-borane as a
reducing agent, afforded target compound 16. The results of NOESY
analysis of compound 15 confirmed the position of 2,4,6-trimeth-
ylaniline moiety.26 Methylation of 16 in the presence of MeI/NaH
produced a mixture of 17 and 18, which was separable by silica
gel column chromatography. The synthesis of the quinoline deriv-
atives is illustrated in Scheme 3. Nitration of commercially avail-
able starting material 19 was performed with HNO3/H2SO4 to
afford intermediate 20. The position of the nitro group was deter-
mined by NMR analysis.27 A coupling reaction with ArNH2 or ArOH
afforded intermediate 21a–e. Hydrogenation of 21a–e followed by
reductive amination in the presence of
a-picoline-borane as a

K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4756–4761
4759
R'
N
O2N
H2N
O2N
R
a
b
c
d
N
N
N
N
N
Cl
Cl
19
20
21a-e
22a-e
23-38
O
O
HN
HN
O
HN
O
a
d
b
e
c
O
O
O
O
O
O
O
Scheme 3. Reagents and conditions: (a) HNO3, H2SO4, ꢀ20 °C, (46%); (b) For 21a, 2,4,6-trimethylaniline, NMP, 220 °C (Microwave irradiation), (39%), for 21b, 2,6-dimethoxy-
4-methoxymethylaniline, Pd2(dba)3, 2-(di-tbutylphosphino)-biphenyl, tBuONa, PhMe, reflux, (33%), for 21c, 2,4,6-trimethoxyaniline, Pd2(dba)3, 2-(di-tbutylphosphino)-
biphenyl, tBuONa, PhMe, reflux, (34%), for 21d, 2,4,6-trimethylphenol, K3PO4, Pd(OAc)2, 2-di- tbutylphosphino-20,40,60-triisopropylbiphenyl, PhMe, reflux, (19%), for 21e, 2,6-
dimethoxy-4-methoxymethylphenol, K3PO4, Pd(OAc)2, 2-di- tbutylphosphino-20,40,60-triisopropylbiphenyl, PhMe, reflux, (27%); (c) H2, Pd/C, EtOAc, rt; (d) aldehyde or ketone,
a-picoline-borane, AcOH, MeOH, rt.
Table 1
Effects of R2, R3, R4, R5 and R5 on the quinoxaline and the quinoline
Br
NH2
0
O
a
O
O
O
R5'
N
R5'
N
R5
R4
R5
R3
O
O
N
X
39
40
N
N
O
N
N
R2
R2
HO
OH
OH
B
b
O
O
0
O
O
No.
X
R5
R5
R3
R4
R2
Binding IC50 (nM)
10a
10b
16
17
18
N
N
—
—
—
C
n-Propyl
4-Heptyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
H
n-Propyl
n-Propyl
n-Propyl
n-Propyl
Me
Me
—
—
—
—
—
H
Me
Me
—
H
H
H
H
Me
H
155
197
100
672
>1000
88
O
O
41
23
Me
Scheme 4. Reagents and conditions: (a) n-BuLi, diphenylphosphoryl azide, THF,
ꢀ78 °C to ꢀ15 °C then Sodium bis(2-methoxyethoxy)aluminum hydride, ꢀ78 °C to
rt, (60%); (b) 30% H2O2, H2O, rt, (96%).
(n-Pr)2N- moiety in the quinoline template could give higher levels
of activity than that in the quinoxaline template. Thus, further
derivatization was conducted using the quinoline template.
0
region (10a and 10b, respectively), with little change in activity ob-
served. Changing the core template from quinoxaline to quinoxali-
none was also tolerated (16). However, the introduction of a
methyl group to R4 (17) resulted in a decrease in activity. More-
over, introduction of a methyl group to R2 caused complete loss
of activity (18). This loss of activity is possibly caused by the con-
formational change of the (n-Pr)2N- or Ar–N– moiety on the central
ring system. Further modification of the central core was carried
out in order to fine-tune the conformation for maximum activity.
Alteration of the conformation of the (n-Pr)2N- moiety was at-
tempted by replacing the nitrogen atom next to the C3 position
in the quinoxaline core with a carbon atom (quinoline 23). This
quinoline exhibited a higher binding affinity than the original
quinoxaline (10a vs 23), suggesting that the conformation of
The effects of the substituents R5, R5 and R2 were next explored
(Table 2). The comparison between compounds 23 and 24 demon-
strates that a small alkyl group was well tolerated, with very little
difference in activity apparent. The large disparity in binding affin-
ity between compounds 25 and 26 indicates that the length or size
0
of the substituents at R5 and R5 is highly restricted. It is hypothe-
sized that the lipophilic nature of the compounds may be one of
the issues hampering the clinical development of nonpeptide
CRF1 receptor antagonists.29,30 The compounds developed here also
exhibit high lipophilicity (ClogP 9.17 for 23 calculated by Daylight
version 4.94). Thus, we attempted to convert the 2,4,6-trimethyl-
phenyl, a highly lipophilic substituent, to a more hydrophilic Ar
group such as 2,6-dimethoxy-4-methoxymethylphenyl (27) or
2,4,6-trimethoxyphenyl (28). From our previous experience, we

4760
K. Takeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4756–4761
Table 2
0
Effects of R2, R5 and R5 on the nitrogen-linked quinoline and oxygen-linked quinoline
R5'
N
R5
3
N
X
R2
0
No.
X
R5
R5
R2
Binding IC50 (nM)
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
NH
NH
NH
NH
NH
NH
O
O
O
O
O
n-Propyl
n-Propyl
2,4,6-Trimethylphenyl
2,4,6-Trimethylphenyl
2,4,6-Trimethylphenyl
2,4,6-Trimethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,4,6-Trimethoxyphenyl
2,4,6-Trimethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
2,6-Dimethoxy-4-methoxymethylphenyl
88
75
Cyclopropylmethyl
Cyclopropylmethyl
Cyclopropylmethyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
Cyclopropylmethyl
Ethyl
Cyclopropylmethyl
Ethyl
3-Pentyl
Cyclopropylmethyl
CH2-4-THP
4-THP
n-Propyl
n-Propyl
n-Propyl
n-Propyl
CH2-4-THP
CH2-4-THP
4-THP
4-THP
H
H
Pyridin-4-ylmethyl
Ethyl
742
90
472
>1000
79
138
762
646
157
323
>1000
>1000
>1000
70
O
O
O
O
Cyclohexyl
Ethyl
Ethyl
O
found that these substituents could successfully reduce lipophilic-
ity without loss of activity. In the results obtained here, although
the ClogP values of 27 and 28 were lower (7.45 and 7.60, respec-
tively) than that of 23 (9.17), both compounds exhibited less activ-
ity. We speculated that this loss of activity might be caused by the
planarity of the Ar-NH- moiety, as the methoxy substituent on the
ortho-position of the phenyl ring forms an intramolecular hydro-
gen bond with the NH group. This interpretation is supported by
our pharmacophore model (Fig. 4). To confirm the effect of a linker
atom, oxygen-linked derivatives were prepared (29 and 30). Com-
pound 29 retained a level of binding affinity comparable to 23. On
the other hand, compound 30, which had 2,6-dimethoxy-4-meth-
oxymethylphenyl on R2, exhibited higher binding affinity than
27. This result supports the hypothesis that the flat angle caused
by the intramolecular hydrogen bond between NH and the ortho-
substituent of the phenyl ring, is an unfavorable conformation.
Changing the linker atom to oxygen would keep the twisted con-
formation of Ar-O- moiety, even in compounds with a methoxy
substituent at the ortho-position, by avoiding the formation of an
intramolecular hydrogen bond. To further understand the SAR of
this oxygen-linked series, derivatization of the dialkylamine moi-
ety of compound 30 was carried out. From the results of 31, 32,
33 and 34, a 4-tetrahydropyranyl (THP) substituent was more fa-
vored than a CH2-4-tetrahydropyranyl substituent, which was sim-
ilar to the nitrogen-linked series (25 and 26). Introduction of
secondary amines or replacement of a di-alkyl-amine with a pyri-
dine moiety resulted in a remarkable reduction in activity (35, 36
and 37). Among these compounds, 38, which has diethyl amine
moiety, exhibited both a better binding affinity and a lower ClogP
value (5.37), indicating that the oxygen-linked series has the
potential to overcome the trade-off relationship between lipophil-
icity and activity.
Among those derivatives with high binding affinities, com-
pounds 10a, 16, 23, 26, 30, and 38 were subjected to a functional
assay to evaluate their antagonism to the CRF1 receptor. The inhi-
bition of CRF-induced cAMP (cyclic adenosine monophosphate)
production was measured for human CRF1 receptor-expressing
HEK293 cells.31 The results are summarized in Table 3. Compounds
30 and 38 exhibited potent activity, while compounds 10a, 16, 23,
and 26 were slightly less active.
In summary, the pharmacophore was extracted by analysis of
the common structural features of the currently known CRF1
receptor antagonists. Molecular design based on the pharmaco-
phore subsequently identified 5-dialkyaminoquinolines and 5-
dialkylaminoquinoxalines as potential novel CRF antagonists.
Through preparation and evaluation of these identified derivatives,
0
the SAR of substituents on each position (R2, R3, R4, R5, R5 ) was
grasped. As a result, a number of compounds with high binding
affinities and potent antagonistic activities were found (10a, 16,
23, 26, 30, and 38). Future work will aim to optimize these com-
pounds further in order to achieve the development of a novel
CRF1 receptor antagonist for clinical application.
Acknowledgments
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 in silico
simulation.
References and notes
Table 3
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No.
Functional IC50 (nM)
10a
16
23
26
30
38
563
469
372
255
163
167
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NO2
HMBC
NOESY
COSY
N
Cl
.
28. For the binding assay and functional assay, HEK293 cells expressing the human
CRF1 receptor were cloned.11 Screening of CRF1 receptor binding was
performed using the scintillation proximity assay (SPA™
,
Amersham
g/well), wheat germ
agglutinin coated SPA beads (1 mg/well), [125I] human/rat CRF (0.1 nM), and
diluted test compound solution were suspended in 150 L of assay buffer
Pharmacia, UK) using 96-well plates. Cell membrane (5
l
l
(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 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.
29. Hsin, L. W.; Tian, X.; Webster, E. L.; Coop, A.; Caldwell, T. M.; Jacobson, A. E.;
Chrousos, G. P.; Gold, P. W.; Habib, K. E.; Ayala, A.; Eckelman, W. C.; Contoreggi,
C.; Rice, K. C. Bioorg. Med. Chem. 2002, 10, 175.
30. Zorrilla, E. P.; Koob, G. F. Drug Discovery Today 2010, 15, 371.
31. 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.11 cAMP was
measured using an enzyme immunoassay (EIA) kit (Amersham Pharmacia, UK).
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
25. Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger, T. Am. J. Psychiatry
2008, 165, 617.
26.
H2N
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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
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.
N
NOESY
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.