6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives as novel corticotropin-releasing factor 1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2011.07.055

To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated. An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us to the discovery of an orally active CRF1 receptor antagonist. The results of a structure-activity relationship study are presented.

Full text of DOI:10.1016/j.bmc.2011.07.055

Bioorganic & Medicinal Chemistry 19 (2011) 5432–5445
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their
derivatives as novel corticotropin-releasing factor 1 receptor antagonists
⇑
Tetsuji Saito , Tetsuo Obitsu, Takashi Kondo, Toshiaki Matsui, Yuuki Nagao, Kensuke Kusumi,
Naoya Matsumura, Sonoko Ueno, Akihiro Kishi, Seishi Katsumata, Yoshifumi Kagamiishi, Hisao Nakai,
Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd, 3-1-1 Sakurai, Shimamoto, Mishima, Osaka 618-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-
cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated.
An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us
to the discovery of an orally active CRF1 receptor antagonist. The results of a structure–activity relationship
study are presented.
Received 3 July 2011
Revised 23 July 2011
Accepted 25 July 2011
Available online 31 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Corticotrophin-releasing factor 1 receptor
Antagonist
1. Introduction
aryl ring in an orthogonal orientation to the core ring, which is
minimally substituted in the ortho- and para-positions; a halide
The majority of research on the therapeutic utility of corticotro-
pin-releasing factor (CRF) receptor antagonists has focused on
treatment of stress-related disorders such as anxiety and depres-
sion.1–4 Thus, CRF, a 41-amino acid peptide first isolated in
1981,5 is considered to be one of the principal regulators of the
hypothalamic–pituitary–adrenal (HPA) axis, which coordinates
the endocrine, behavioral, and autonomic responses to stress.
CRF and its receptors play a key role in mediating the body’s re-
sponse to stress, and have provided compelling targets for novel
pharmacological approaches to treating depression, anxiety and
stress disorders.6–8 It is well known that CRF exerts its biological
functions through binding to type-1(CRF1) and type-2 (CRF2)
receptors.9 Major industry efforts have identified potent and selec-
tive antagonists for the CRF1 receptor, as summarized in recent re-
views.10–12 Since the report of antidepressant activity of one of
these, R121919, in an open-label, phase 2 trial conducted in de-
pressed patients,13,14 very few clinical results8 with CRF1 antago-
nists have been published. However, recent advances in CRF
neurobiology have further validated this target.15–17
or small alkyl group ortho to the sp2 nitrogen of the aromatic het-
erocyclic core; and a branched, lipophilic group, with limited toler-
ance for polar functional groups, para to the sp2 nitrogen of the
heterocyclic core. Many potent, small molecule CRF1 antagonists
from a variety of chemical classes have been reported since the dis-
closure of the CRF1 antagonist CP-154,526 1 in 1996 and 1997.18,19
Among the variety of chemotypes, representative bicyclic core
antagonists 1, 2a–b,20 3,21 and 422 are shown in Figure 1. This re-
port will focus on the discovery of novel CRF1 antagonists, specifi-
cally the aromatic heterotricyclic core antagonists 7a and 7b
(Scheme 1), which were designed based on the bicyclic core antag-
onists 2a and 2b, respectively.
As shown in Scheme 1, representative chemotypes containing
tricyclic cores have been designed based on the bicyclic core struc-
tures 2a and 2b. Cyclization ‘a’ resulted in the pyrrole- and pyra-
zole-based compounds 5a–b and others.23–31 Cyclization ‘b’
resulted in structure 6, which was reported to show potent CRF1
receptor binding.32 Cyclization ‘c’ resulted in novel structures as
illustrated by 7a–b, the biological profiles of which have not been
reported yet. In this paper, the discovery and biological evaluation
of a series of aromatic heterotricyclic core CRF1 antagonists is
reported.
The design and synthesis of non-peptidic small molecule CRF1
antagonists continues to be an active area of research. The key fea-
tures found in several known classes of CRF1 antagonists are an
aromatic heterocyclic core (monocyclic, bicyclic, or tricyclic) that
includes an sp2 nitrogen acting as a hydrogen bond acceptor; an
2. Chemistry
Synthesis of the test compounds listed in Tables 1–5 is outlined
in Schemes 2–6. Key intermediates 35a–g were prepared as
⇑
Corresponding author. Tel.: +81 75 961 1151; fax: +81 75 962 9314.
E-mail address: te.saitou@ono.co.jp (T. Saito).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.055

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5433
O
NH2
Me
Me
N
N
Me
Me
Me
HN
7
1
N
1a
N
N
N
6
N
2
Me
X
N
Me
Me
3
5
5a
Me
N
4
Me
Me
Cl
Me
OMe
1
3
2a X = Me
2b X = Cl
Cl
Me
N
Me
Me
N
N
N
Cl
Cl
Figure 1. CRF1 receptor antagonists containing bicyclic cores.
4
Scheme 1. Molecular design of CRF1 receptor antagonists containing tricyclic cores.
described in Scheme 2. Condensation reaction of benzyl nitriles 32
and 33 with an optional ethyl ester in the presence of base afforded
enolate anions of b-keto nitriles (34a–g). Cyclization reaction of
34a–g with hydrazine hydrate provided the 5-aminopyrazoles
35a–g.33 Synthesis of 7a–b, 8a–b, 9–10, 11a–b, 12, 18–28 and 30
is outlined in Scheme 3a–e. Coupling reaction of 5-amino-3-
methyl-4-phenylpyrazole (35b) and cyclic b-keto esters (36a–d)
in acetic acid under reflux temperature followed by chlorination
with phosphorous oxychloride afforded 37a–d. Aminolysis of
37a–d with 1-ethylpropylamine resulted in 7a, 8a, and 9–10,
respectively (Scheme 3a). Cyclization reaction of 35b 5-amino-3-
methyl-4-arylpyrazoles possessing an optionally substituted aryl
group and b-keto esters (38a–b) followed by treatment with phos-
phorous oxychloride afforded 39a and 39b, aminolysis of which
with 2-ethylpropylamine provided 11a and 12, respectively
(Scheme 3b). Compound 30 was synthesized from 35g (Scheme
3c) using a b-keto ester (40) instead of 36a–d and 38a–b, according
to the procedure (Scheme 3a–b) described above. Compounds 7b,

5434
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
Table 1
3. Results and discussion
Effect of the fused cyclic structure on the activity profiles
The compounds listed in Tables 1–5 were first tested for their
binding affinity to human CRF1 receptor and antagonist activity
in a CRF-stimulated adenylate cyclase assay.36 One of the leading
compounds with higher receptor binding and antagonist activity
was then evaluated for an in vitro activity and subjected to an
in vivo pharmacokinetic study in rats. Anxiolytic efficacy was also
assessed in the rat elevated plus-maze model.37–39
Me
Me
HN
X
N
N
Me
Me
N
As shown in Table 1, compounds 7a, 8a, 9, and 10, which pos-
sess 6,7-dihydrocyclopentapyrazolopyrimidine, 2,3-dihydrofuro-
pyrazolopyrimidine, 2,3-dihydropyrazolothienopyrimidine and
2,3-dihydropyrazolopyrrolopyrimidine cores, respectively, showed
moderate binding affinity. As reported previously,40,41 the newly
attached cyclopenta moiety seemed to be very sensitive to bulki-
ness while compound 7a still exhibited equipotent activity with
the chemical lead 2a, which showed potent binding affinity
(Ki = 1.0 02 nM)42 and antagonist activity (IC50 = 10 0.01 nM).42
Fine tuning of its bulkiness and polarity by introducing a hetero-
atom into the cyclopenta moiety was predicted to result in an
interesting investigation. Thus, replacement of one of the carbon
atoms of the fused carbocyclic five-membered ring with a more
hydrophilic hetero atom such as oxygen, sulfur and nitrogen affor-
ded 8a, 9 and 10, respectively, with a tendency for decreased bind-
ing affinity and/or antagonist activity. Compound 8a exhibited
unexpectedly reduced antagonist activity given its potent binding
affinity. Table 2 shows the activity profiles of compounds 11a,
and 12, which possess 5H,7H-furo[3,4-d]pyrazolopyrimidine, and
5H,7H-pyrazolo[1,5a]thienopyrimidine. Compounds 11a and 12,
which contain more Lewis basic heteroatoms O and S than the cor-
responding isomers 8a and 9, respectively, tended to show equipo-
tent to slightly less potent binding affinity. Accordingly, the more
Lewis basic property of the ether and the sulfide moieties of 11a
and 12 were found to be more beneficial relative to the corre-
sponding moieties of 8a and 9, respectively, especially in effort to
improve antagonist activity of 11b. Transformation of the bicyclic
core of the known antagonists (2a–b, Fig. 1) into the tricyclic core
led us to readjustment of the size and/or polarity of the methyl res-
idue attached to the pyrazole moiety of the tricyclic core.
OMe
Compd
X
Binding affinity IC50 (nM)
Human
Antagonist activity EC50 (nM)
Human
7a
8a
9
CH2
O
S
37
31
80
170
640
NTa
NTa
10
NH
170
a
Not tested.
Table 2
Effect of the fused cyclic structure on the activity profiles
Me
Me
HN
N
N
X
Me
Me
N
OMe
The effect of the substituent attached to the pyrazole moiety on
activity profiles was investigated and the results are summarized
in Table 3. Removal of the methyl group from the pyrazole moiety
of 7a afforded the unsubstituted pyrazole analog 13 with a 6.5-fold
reduction in binding affinity, while replacement of the methyl
group with an ethyl group and cyclobutyl group afforded 14 and
15, respectively, with reduced binding affinity. Replacement of
the methyl group of 7a with a trifluoromethyl group afforded 16
with nearly equipotent binding affinity. Replacement of the methyl
group of 7a with a methylthiomethyl group provided 17 with re-
duced binding affinity. As a result, the methyl group was found
to be the most optimized substituent among the substituents
tested, in terms of both bulkiness and hydrophobicity.
The tricyclic core was predicted to require another optimal aryl
structure different from the one required by the parent bicyclic
core. Based on the above-mentioned prediction, the effect of the
substituents of the aryl ring on activity profiles was investigated.
Among 2-methylphenyl analogs (7a, 18–21), the 2,4-disubstituted
phenyl analogs 7a and 18–20 tended to show relatively stronger
binding affinities than the 2,5-disubstituted phenyl analog 21.
The 4-methoxyphenyl analogs 22–25 were tested to determine
binding affinity and antagonist activity. Among the tested analogs,
the 2,6-dimethyl-4-methoxyphenyl analog 23, the 2-chloro-4-
methoxyphenyl analog 7b and the 2,5-dimethyl-4-methoxyphenyl
analog 22 showed potent binding affinity and antagonist activity.
The 2-methylthio-4-methoxyphenyl analog 24 showed moderate
Compd
X
Binding affinity IC50 (nM)
Human
Antagonist activity EC50 (nM)
Human
7a
11a
12
CH2
O
S
37
35
37
70
75
150
8b, and 11b were synthesized from 35g (Scheme 3d) using 36a–b
and 38b as b-keto esters according to the procedures described
above. Compounds 18–28 were prepared from an optional 5-amin-
opyazole20 (Scheme 3e) using 36a as a b-keto ester according to
the procedure described above.
As shown in Scheme 4, compounds 13–17 were synthesized from
5-aminopyrazoles 35a and 35c–f (Scheme 2), respectively, using
36a as a cyclic b-keto ester according to the procedure described
above. Another tricyclic core analog 31 was prepared as outlined
in Scheme 5. Coupling reaction of cyclic b-enaminoketonitrile 4334
with 5-amino-4-arylpyrazole 35g afforded an aminoheterotricyclic
core structure, 44, N-alkylation of which with 3-bromopentane in
N,N-dimethylformamide in the presence of tetra-n-butyl ammo-
nium iodide afforded 31. Synthesis of 29 is described in Scheme 6.
Demethylation of 7b with boron tribromide in dichloromethane
provided 45. Difluoromethylation of phenol 48 with chlorodifluo-
romethane under basic condition afforded 29.35

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5435
Table 3
Effect of the substituent on the pyrazole moiety on activity profiles
Me
N
Me
HN
N
N
R
Me
OMe
Compd
R
Binding affinity IC50 (nM)
Human
Antagonist activity EC50 (nM)
Human
13
7a
14
H
Me
Et
240
37
97
NTa
170
NTa
15
490
NTa
16
17
CF3
CH2SMe
59
620
NTa
NTa
a
Not tested.
Table 4
Effect of the substituent on the aryl ring on the activity profiles
Me
Me
HN
N
N
Me
N
Ar
Compd
Ar
X and/or Y
Binding affinity IC50 (nM)
Human
Antagonist activity EC50 (nM)
Human
7a
18
19
20
21
4-OMe
4-Me
4-F
4-NMe2
5-Me
37
72
93
38
820
170
920
Me
NTa
NTa
NTa
X
22
23
2,5-Dimethyl
2,6-Dimethyl
22
9
47
80
X
7b
24
25
2-Cl
2-SMe
3-OMe
4
49
600
4 (7)b
71
15
37
3
40
Y
NTa
NTa
OMe
Cl
27
29
7b
26
28
4-OMe
4-OEt
4-OCF3
4-Cl
40 (40)c
NTa
NTa
200
200
X
4-OCHF2
a
b
c
Not tested.
Binding assay for rat CRF.
cAMP assay for rat.
binding affinity. The 3,4-dimethoxyphenyl analog 25 showed rela-
tively weaker binding affinity (IC50 = 600 nM) because the aryl ring
was unsuitably oriented to the core ring. Based on the results de-
scribed above, further optimization of the 2-chloro-4-substituted
phenyl moiety, which was found to be the most optimal among
those tested, was carried out. This screening led us to the discovery
of the 2-chloro-4-difluoromethoxy analog 29, which exhibited very
potent binding affinity (IC50 = 3 nM) but only weak antagonist
activity (IC50 = 200 nM). Analogs 26–28, which exhibited moderate
to weak binding affinity, were also discovered. Compounds 28 and

5436
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
Table 5
SAR of 2-chloro-4-methoxyphenyl analogs
Me
Me
HN
1
R1
R2
7
1a
N
N
2
6
5
Me
Cl
3
N 5a
4
OMe
Compd
R1, R2
Binding affinity IC50 (nM)
Human
Antagonist activity EC50 (nM)
Human
2b
30
R1 = H, R2 = Me
R1 = R2 = Me
6a
31a
49a
100a
7b
4
40
4
O
O
8b
7
11b
22
84
66
8
31
O
a
In-house data.
O
R
HN N
H2N
NC
NC
R
X
X
X
a or b
c
OMe
32 X = Me
33
OMe
OMe
35a-g
34a-g
X = Cl
X = Cl
X = Me
g
R = Me
a
R = cyclobutyl
R = H
b R = Me
d
e
R = CF3
R = CH2SMe
c
R = Et
f
Scheme 2. Synthesis of intermediates 35a–g. Reagents: (a) NaOEt, RCO2Et, reflux; (b) Na, ethyl acetate, reflux; (c) NH2NH2ꢀH2O, acetic acid/toluene, reflux.
29 showed equipotent antagonist activity, although their binding
affinities differed by more than 10 times. Among the tested com-
pounds, the 2-chloro-4-methoxyphenyl moiety of 7b was found
to be the most optimized aryl moiety in terms of both binding
affinity and antagonist activity.
While it is difficult to clarify the SAR between the bicyclic core
30 and the tricyclic core 7b, the intramolecular steric hindrance
of the 6-methyl of 30 may be slightly alleviated by forming the tri-
cyclic core system 7b. Second, fine readjustment of the physico-
chemical properties of 7b was also carried out as described in
Table 5. Replacement of the aliphatic carbon atom of the newly
fused ring with ether oxygen is considered to lower the c-log P va-
lue of the highly lipophilic 7b. Thus, introduction of ether oxygen
into the newly fused five-membered ring of 7b afforded the three
isomers 8b, 11b and 31, all of which possess a 2-chloro-4-
methoxyphenyl group as an optimized aryl moiety. The isomer
8b exhibited increased binding affinity and antagonist activity,
while 11b and 31 showed decreased binding affinity relative to
7b. The isomer 11b exhibited nearly equipotent antagonist activity
relative to 7b, while 31 showed unexpectedly strong antagonist
activity for its decreased binding affinity. As a result, the structural
fine tuning described above resulted in discovery of 8b as the most
optimized structure in the in vitro tests. Compounds 2b and 7b
A study of the structure–activity relationships of the 5-methyl
bicyclic core analogs 2a–b, the 5,6-dimethyl bicyclic core analog
30 and the tricyclic core analog 7b was conducted as shown in Ta-
ble 5. First, replacement of the 2-methyl-4-methoxyphenyl moiety
of 2a with a 2-chloro-4-methoxyphenyl moiety afforded 2b with
very strong binding affinity and antagonist activity, while intro-
duction of another methyl residue into the 6-position of the bicy-
clic core provided 30 with significant loss of these activities.
Accordingly, introduction of an alkyl group into the 6-position is
not beneficial while the loss of activity was recovered in the tricy-
clic core antagonist 7b. Based on this result, the 6-position of the
bicyclic core system was found to be one of the very sensitive posi-
tions for these small molecules to interact with the CRF receptor.

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5437
a) Synthesis of 7a, 8a and 9-10
Cl
CO2R
O
X
N
X
a, b
c
c
N
7a, 8a and 9-10
35b
Me
+
N
Ar
36a-d
c
X = CH2, R = Et
a
X = S, R = Me
X = NZ, R = Et
37a-d
d
b X = O, R = Me
b) Synthesis of 11a and 12
Cl
CO2Me
O
N
a, b
N
11a and 12
+
35b
X
Me
X
N
Ar
38a-b
39a-b
a
b
X = O
X = S
c) Synthesis of 30
35g
Cl
Me
Me
N
N
Me
Cl
Me CO2Et
c
a, b
N
30
+
O
Me
40
OMe
41
d) Synthesis of 7b, 8b and 11b
35g
a, b, c
a, b, c
36a, 36b and 38b
7b, 8b and 11b
18-28
+
e) Synthesis of 18-28
HN N
H2N
Me
36a
+
Ar
Scheme 3. Synthesis of 7a–b, 8a–b, 9–10, 11a–b, 12, 18–28 and 30. Reagents: (a) acetic acid, reflux; (b) POCl3, N,N-diethylaniline, toluene, reflux; (c) 1-ethylpropylamine,
isopropyl alcohol, 90 °C.
Cl
N
N
R
a, b
c
N
Me
13-17
36a
+
35a and 35c-f
OMe
42a-e
R = H
R = Et
R = cyclobutyl
a
R = CF3
R = CH2SMe
d
e
b
c
Scheme 4. Synthesis of 13–17. Reagents: (a) acetic acid, reflux; (b) POCl3, N,N-diethylaniline, toluene, reflux; (c) 1-ethylpropylamine, isopropyl alcohol, 90 °C.
NH2
N
N
Me
Cl
CN
a
b
O
N
31
+
35g
O
NH2
OMe
44
43
Scheme 5. Synthesis of 31. Reagents: (a) pyridine, reflux; (b) 3-bromopentane, NaH, tetra-n-butylammonium iodide, DMF, 50–70 °C.

5438
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
Table 6
Me
Me
Pharmacokinetic parameters of compound 7b
HN
N
Parameter
iv
po
N
N
Dose (mg/kg)
AUCinfinity (ng h/ml)
Tmax (h)
0.3
278
3
Me
Cl
a
b
608
1.0
87.1
29
7b
Cmax (ng/mL)
CLtotal (ml/min/kg)
T1/2 (h)
Vss (ml/kg)
F (%)
18
4.7
1780
OH
7.8
22
45
Scheme 6. Synthesis of 29. Reagents: (a) BBr3, CH2Cl2, ꢁ50–0 °C; (b) ClCHF2, NaOH,
THF, H2O, 70 °C.
n = 4.
showed equipotency in both the binding affinity and antagonist
activity while 7a showed less potency relative to 2a (in-house
data). Based on the data, replacement of the 2-methyl-4-methoxy-
phenyl moiety of 7a with 2-chloro-4-methoxyphenyl moiety was
estimated to contribute for the increased potency although the ef-
fect was found to reach the ceiling as illustrated by the results ob-
tained for 2a, 2b, and 7b. Thus, 2-chloro-4-methoxyphenyl moiety
was considered to have more contribution to the increased binding
affinity than 2-methyl-4-methoxy moiety as illustrated by the po-
tency of their receptor binding: 7a < 7b and 8a < 8b. Hydrophilic
moiety such as the ether moieties also considered to be one of
the important factors influencing in vitro activities. Hydrophilicity
and Lewis basicity of the ether function, both of which are ex-
pected to be higher in 11a and 11b relative to 8a and 8b because
of the more delocalization of the lone pairs of the ether oxygen
of 8a and 8b. As a result, the more hydrophilic and more Lewis ba-
sic ether oxygen is presumed to have more chances to result in an
irrelevant interaction with off-targets. Such an unfavorable effect
may result in the unexpected SAR of 11a and 11b, which showed
equipotency in the in vitro assay. In evaluation of antagonist activ-
ity, it may be relatively more difficult to get exact activity in such a
cell-based assay, that is, influenced during evaluation process
starting from the addition of the test compound to the assay sys-
tem till measurement of the released cAMP. For such a reason,
antagonist activity sometimes does not show EC50 values expected
from the corresponding binding assay. Thus, weaker antagonist
activity for the corresponding binding affinity is usually explained
by protein-binding while stronger antagonist activity for the corre-
sponding binding affinity as illustrated by 31 may suggest the
plausible existence of other mechanism of inhibiting cAMP release.
Compound 31 showed weaker binding affinity relative to 7b and
8b. Based on the result, the ether oxygen in 31, which corresponds
to the methyl substituent in the reported bicyclic core system such
as 2b, was considered to give a negative effect to the binding affin-
ity while the ether oxygen in 8b was regarded as a linker, which
could be replaced with the methylene moiety without reduction
of the binding affinity as illustrated by 7b.
3.1. In vivo study of one of the representative compounds
The anxiolytic efficacy of compound 7b, which showed good
binding affinity to rat brain membranes (IC50 = 7 nM) and good
antagonist activity in a cyclic AMP assay (EC50 = 40 nM), was as-
sessed using the rat elevated plus-maze test (Fig. 2). Vehicle-trea-
ted animals spent significantly less time in the open arms (p < 0.05)
relative to control animals. Pretreatment with compound 7b at
doses of 3 and 10 mg/kg significantly increased the time spent in
open arms (p = 0.023 and 0.032, respectively) relative to vehicle-
treated animals.
3.2. Rat pharmacokinetic study
Pharmacokinetic data for compound 7b were investigated after
administration of single doses to rats (Table 6). Intravenous admin-
istration of compound 7b to rats (0.3 mg/kg, n = 4) resulted in
detectable plasma levels (T1/2 = 4.7 h), while oral administration
of 7b to rats (3 mg/kg, n = 4) resulted in a T1/2 of 7.8 h. The AUC va-
lue of 7b was 278 ng h/ml after intravenous administration versus
608 ng h/ml after oral administration. The steady state volume of
distribution (Vss) was calculated to be 1780 ml/kg indicating that
this compound showed good distribution to tissues. Systemic
clearance was 18 ml/min/kg. The Cmax value after oral dosing was
87.1 ng/ml while the Tmax value was 1.0 h. Bioavailability of 7b
was 22%. Kp value (brain content of 7b/plasma concentration of
7b) was 0.4 (1.0 h after oral dosing).
4. Conclusion
#
#
40
30
20
10
0
For the purpose of finding an orally active CRF1 receptor antag-
onist, a series of heterotricyclic derivatives were designed, synthe-
sized and evaluated. After substantial chemical modification and
evaluation, the unsubstituted carbocyclic analogs 7a–b, and the
oxa-analogs 8a–b, 11a–b and 31 were identified as CRF1 antago-
nists possessing potent to moderate in vitro activity. In particular,
the 2-chloro-4-methoxyphenyl analogs 7b, 8b and 31 exhibited
potent in vitro activity. Among them, 7b was found to show orally
effective CRF1 antagonist activity.
*
Control
Vehicle
1
3
10
5. Experimental
5.1. Chemistry
Compound 7b (mg/kg, p.o.)
Non-stress
Swim stress
5.1.1. General procedures
Analytical samples were homogeneous as confirmed by TLC,
and afforded spectroscopic results consistent with the assigned
Figure 2. Effect of compound 7b in the rat elevated plus-maze test in swim stress-
loaded rats. Each column represents mean standard errors of 16 rats. #p <0.05
versus control group (t-test). ⁄p <0.05 versus vehicle group (Dunnett test).

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5439
structures. Proton nuclear magnetic resonance spectra (1H NMR)
were taken on a Varian Mercury 300 spectrometer using deuter-
ated chloroform (CDCl3) or deuterated dimethylsulfoxide (DMSO-
d6) as the solvent. Fast atom bombardment (FAB-MS, HRMS) and
electron ionization (EI) mass spectra were obtained on a JEOL
JMS-DX303HF spectrometer. Atmospheric pressure chemical ioni-
reaction mixture was allowed to be heated at reflux and stirred
for 3 h. The reaction mixture was cooled and evaporated. The
resulting residue was dissolved in 2 N HCl aq and washed with
hexane/EtOAc (3:1). By adding ammonium hydroxide solution
(28%), pH value of the solution was adjusted to 11. The solution
was extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4 and concentrated in vacuo to give 35b
(5.15 g, 90% yield) as a yellow oil, which was used for the next
reaction without further purification. TLC Rf = 0.4 (AcOH/MeOH/
CHCl3, 1:4:15); 1H NMR (300 MHz, CDCl3) d 7.08 (d, J = 8.4 Hz,
1H), 6.84 (d, J = 3 Hz, 1H), 6.77 (dd, J = 8.4, Hz, 1H), 3.82 (s, 3H),
2.19 (s, 3H), 2.08 (s, 3H).
zation (APCI) mass spectra were determined on
a HITACHI
MI200H spectrometer. Infrared spectra (IR) were measured in a
Perkin-Elmer FT-IR 1760X spectrometer. Melting points and results
of elemental analyzes were uncorrected. Column chromatography
was carried out on silica gel [Merck Silica gel 60 (0.063–0.200 mm),
Wako gel C-200, or Fuji Silysia FL60D]. Thin layer chromatography
was performed on silica gel (Merck TLC or HPTLC plates, Silica gel
60 F254). The following abbreviations for solvents and reagents are
used diethyl ether (Et2O), N,N-dimethylformamide (DMF), ethyl
acetate (EtOAc), acetic acid (AcOH), methanol (MeOH), dichloro-
methane (CH2Cl2), chloroform (CHCl3), tert-butylmethyl ether (t-
BuOMe), sodium hydride (NaH), tetrahydrofuran (THF).
5.1.9. 4-(4-Methoxy-2-methylphenyl)-2H-pyrazol-3-ylamine
(35a)
A reddish yellow oil:TLC Rf = 0.45 (MeOH/CHCl3, 1:10); 1H NMR
(300 MHz, CDCl3) d 7.30 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 6.83 (d,
J = 3.0 Hz, 1H), 6.77 (dd, J = 3.0, 8.4 Hz, 1H), 3.82 (s, 3H), 2.27 (s,
3H).
5.1.2. 2-(2-Methyl-4-methoxyphenyl)-3-oxo-butyronitrile (34b)
To a stirred solution of 32 (5.0 g, 31 mmol) in EtOAc (70 mL)
was portionwisely added sodium (929 mg) at room temperature
and the reaction mixture was allowed to be heated at reflux. After
being stirred over night, the reaction mixture was cooled to room
temperature. The precipitates were collected by filtration and then
dissolved in water. The solution was acidified with 2 N HCl (pH 5–
6) and extracted with EtOAc. The combined organic layers were
washed with water, brine, dried over MgSO4 and evaporated to
give 34b (5.38 g, 85% yield) as a pale yellow oil, which was used
for the next reaction without further purification. TLC Rf = 0.65
5.1.10. 5-Ethyl-4-(4-methoxy-2-methylphenyl)-2H-pyrazol-3-
ylamine (35c)
A brown oil; MS (MALDI, Pos) m/z 232 (M+H)+; 1H NMR
(300 MHz, CDCl3) d 7.08 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 2.7 Hz, 1H),
6.76 (dd, J = 8.7, 2.7 Hz, 1H), 3.82 (s, 3H), 2.45 (dq, J = 3.3, 7.5 Hz,
2H), 2.18 (s, 3H), 1.11 (t, J = 7.5 Hz, 3H).
5.1.11. 5-Cyclobutyl-4-(4-methoxy-2-methylphenyl)-2H-pyraz
ol-3-ylamine (35d)
A yellow oil; MS (MALDI, Pos) m/z 258 (M+H)+; 1H NMR
(300 MHz, CDCl3) d 7.05 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 2.7 Hz, 1H),
6.75 (dd, J = 8.4, 2.7 Hz, 1H), 3.82 (s, 3H), 3.32 (m, 1H), 2.22–1.72
(m, 9H).
(EtOAc/hexane, 1:1), 1H NMR (300 MHz, CDCl3)
d 7.30 (d,
J = 8.4 Hz, 1H), 6.85–6.75 (m, 2H), 4.75 (s, 1H), 3.81 (s, 3H), 2.33
(s, 3H), 2.22 (s, 3H).
5.1.3. 2-(4-Methoxy-2-methylphenyl)-3-oxo-propionitrile (34a)
A reddish yellow oil; TLC Rf = 0.45 (MeOH/CHCl3, 1:10); 1H NMR
(300 MHz, CDCl3) d 7.30 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 6.83 (d,
J = 3.0 Hz, 1H), 6.77 (dd, J = 3.0, 8.4 Hz, 1H), 3.82 (s, 3H), 2.27 (s, 3H).
5.1.12. 4-(4-Methoxy-2-methylphenyl)-5-trifluoromethyl-2H-
pyrazol-3-ylamine (35e)
A pale yellow oil; TLC Rf = 0.61 (MeOH/CHCl3, 1/20); 1H NMR
(300 MHz, CDCl3) d 7.12 (d, J = 8.4 Hz, 1H),6.84 (d, J = 3.0 Hz, 1H),
6.78 (dd, J = 3.0, 8.4 Hz, 1H), 3.83 (s, 3H), 2.16 (s, 3H).
5.1.4. 2-(4-Methoxy-2-methylphenyl)-3-oxo-pentanenitrile (34c)
A brown oil; 1H NMR (300 MHz, CDCl3) d 7.29 (m, 1H), 6.79 (m,
2H), 4.76 (s, 1H), 3.81 (s, 3H), 2.54 (m, 2H), 2.32 (s, 3H), 1.05 (t,
J = 6.9 Hz, 3H).
5.1.13. 4-(4-Methoxy-2-methylphenyl)-5-methylsulfanylmeth
yl-2H-pyrazol-3-ylamine (35f)
TLC Rf = 0.40 (MeOH/CH2Cl2, 1:9); 1H NMR (300 MHz, CDCl3) d
7.07 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 2.7 Hz, 1H), 6.77 (dd, J = 8.4,
2.7 Hz, 1H), 3.82 (s, 3H), 3.51 (d, J = 13.5 Hz, 1H), 3.46 (d,
J = 13.5 Hz, 2H), 2.19 (s, 3H), 1.98 (s, 3H).
5.1.5. 3-Cyclobutyl-2-(4-methoxy-2-methylphenyl)-3-oxo-
propionitrile (34d)
A yellow oil; TLC Rf = 0.75 (EtOAc/benzene, 1:5) ; 1H NMR
(300 MHz, CDCl3) d 7.24 (d, J = 8.1 Hz, 1H), 6.78 (m, 2H), 4.73 (s,
1H), 3.80 (s, 3H), 3.40 (m, 1H), 2.38–1.77 (m, 9H).
5.1.14. 4-(2-Chloro-4-methoxyphenyl)-5-methyl-2H-pyrazol-3-
ylamine (35g)
A pale yellow powder; TLC Rf = 0.26 (MeOH /CH2Cl2, 1:9); 1H
NMR(300 MHz, CDCl3) d 7.19 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 2.4 Hz,
1H), 6.86 (dd, J = 8.7, 2.4 Hz, 1H), 3.83 (s, 3H), 2.13 (s, 3H).
5.1.6. 4,4,4-Trifluoro-2-(4-methoxy-2-methylphenyl)-3-oxo-but
yronitrile (34e)
A pale yellow oil; TLC Rf = 0.61 (MeOH/CHCl3, 1:20); 1H NMR
(300 MHz, CDCl3) d 7.12 (d, J = 8.4 Hz, 1H),6.84 (d, J = 3.0 Hz, 1H),
6.78 (dd, J = 3.0, 8.4 Hz, 1H), 3.83 (s, 3H), 2.16 (s, 3H).
5.1.15. 3-(4-Methoxy-2-methylphenyl)-2-methyl-6,7-dihydro-
5H-1,4,8a-triaza-s-indacen-8-ol
To a stirred solution of 35b (3.0 g, 13.8 mmol) in AcOH (20 mL)
was added ethyl 2-oxocyclopentanecarboxylate (2.37 g, 15.2 mmol)
at room temperature and the reaction mixture was allowed to be
heated at reflux and stirred for 3 h. The reaction mixture was cooled
to room temperature and diluted with Et2O/hexane (1:3). The pre-
cipitates were collected by filtration to give 3-(2-methyl-4-
methoxyphenyl)-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrazol-
o[1,5-a]pyrimidin-8-ol (3.88 g, 91% yield) as an off-white powder,
which was used for the next reaction without further purification.
TLC Rf = 0.47 (MeOH/CHCl3, 1:10); 1H NMR (300 MHz, DMSO-d6) d
11.90 (br s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 3.0 Hz, 1H), 6.83
5.1.7. 2-(2-Chloro-4-methoxyphenyl)-3-oxo-butyronitrile (34g)
TLC Rf = 0.13 (EtOAc/hexane, 1:3); 1H NMR(300 MHz, CDCl3) d
7.38 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.4,
2.4 Hz, 1H), 5.11 (s, 1H), 3.83 (s, 3H), 2.29 (s, 3H).
5.1.8. 4-(2-Methyl-4-methoxyphenyl)-3-methyl-1H-pyrazol-5-
amine (35b)
To a stirred solution of 34b (5.38 g, 26.5 mmol) in toluene
(95 mL) were added AcOH (5.16 mL, 90.1 mmol) and hydrazine
monohydrate (2.92 mL, 59.6 mmol) at room temperature and the

5440
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
(dd, J = 8.0, 3.0 Hz, 1H), 3.78 (s, 3H), 2.81 (t, J = 7.5 Hz, 2H), 2.66 (t,
J = 7.5 Hz, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 2.03 (m, 2H).
J = 2.7 Hz, 1H), 6.83 (dd, J = 2.7, 8.1 Hz, 1H), 3.83 (s, 3H), 3.09 (t,
J = 7.8 Hz, 2H), 3.09 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.26 (m, 2H).
5.1.16. 8-Chloro-3-(4-methoxy-2-methylphenyl)-2-methyl-6,7-
dihydro-5H-1,4,8a-triaza-s-indacene (37a)
5.1.24. 8-Chloro-2-ethyl-3-(4-methoxy-2-methylphenyl)-6,7-
dihydro-5H-1,4,8a-triaza-s-indacene (42b)
To a stirred suspension of 3-(2-methyl-4-methoxyphenyl)-2-
methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-
ol (3.88 g, 12.6 mmol) in toluene (40 mL) were added diethylaniline
(2.4 mL, 15.1 mmol) and phosphorus oxychloride (6.1 mL,
65.3 mmol) at room temperature and the reaction mixture was
allowed to be heated at reflux and stirred for 5 h. The reaction
mixture was cooled to room temperature, poured into ice-water,
stirred for 20 min and extracted with EtOAc. The combined organic
layers were washed with water, NaHCO3 aq, brine, dried over
MgSO4 and evaporated to give 37a (3.73 g, 91% yield) as a yellow
powder. TLC Rf = 0.42 (EtOAc/hexane, 1:2); MS (APCI, Pos) m/z
328 (M+H)+; 1H NMR (300 MHz, CDCl3) d 7.15 (d, J = 8.4 Hz, 1H),
6.87 (d, J = 2.4 Hz, 1H), 6.81 (dd, J = 2.4, 8.4 Hz, 1H), 3.83 (s, 3H),
3.05 (m, 4H), 2.40 (s, 3H), 2.23 (m, 2H), 2.15 (s, 3H).
TLC Rf = 0.89 (EtOAc/hexane, 1:1); MS (MALDI, Pos) m/z 342
(M+H)+; 1H NMR (300 MHz, CDCl3) d 7.15 (d, J = 8.4 Hz, 1H), 6.87
(d, J = 2.4 Hz, 1H), 6.80 (dd, J = 8.4, 2.4 Hz, 1H), 3.83 (s, 3H), 3.04
(m, 4H), 2.77 (m, 2H), 2.22 (m, 2H), 2.13 (s, 3H), 1.18 (t,
J = 7.8 Hz, 3H).
5.1.25. 8-Chloro-2-cyclobutyl-3-(4-methoxy-2-methylphenyl)-
6,7-dihydro-5H-1,4,8a-triaza-s-indacene (42c)
A solid; MS (MALDI, Pos) m/z 368 (M+H)+
;
1H NMR (300 MHz,
CDCl3) d 7.09 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 2.7 Hz, 1H), 6.79 (dd,
J = 8.4, 2.7 Hz, 1H), 3.83 (s, 3H), 3.60 (m, 1H), 3.05 (t, J = 5.7 Hz,
2H), 3.03 (t, J = 6.3 Hz, 2H), 2.44 (m, 2H), 2.22 (m, 4H), 2.11 (s,
3H), 1.92 (m, 2H).
5.1.26. 8-Chloro-3-(4-methoxy-2-methylphenyl)-2-trifluorome
thyl-6,7-dihydro-5H-1,4,8a-triaza-s-indacene (42d)
5.1.17. 8-Chloro-5-(4-methoxy-2-methylphenyl)-6-methyl-2,3-
dihydro-1-oxa-4,7,7a-triaza-s-indacene (37b)
An ivory powder; TLC Rf = 0.37 (EtOAc/hexane, 1:3); 1H NMR
(300 MHz, CDCl3) d 7.17 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 2.7 Hz, 1H),
6.80 (dd, J = 2.7, 8.7 Hz, 1H), 3.83 (s, 3H), 3.11 (t, J = 6.9 Hz, 2H),
3.09 (t, J = 7.8 Hz, 2H), 2.28 (m, 2H), 2.11 (s, 3H).
TLC Rf = 0.55 (EtOAc/hexane, 1:1); 1H NMR (300 MHz, CDCl3) d
7.14 (d, J = 8.1 Hz, 1H), 6.88 (d, J = 2.7 Hz, 1H), 6.81 (dd, J = 8.1,
2.7 Hz, 1H), 4.82 (t, J = 8.4 Hz, 2H), 3.83 (s, 3H), 3.40 (t, J = 8.4 Hz,
1H), 2.38 (s, 3H), 2.15 (s, 3H).
5.1.27. 8-Chloro-3-(4-methoxy-2-methylphenyl)-2-methylsulfa
nylmethyl-6,7-dihydro-5H-1,4,8a-triaza-s-indacene (42e)
TLC Rf = 0.40 (EtOAc/hexane, 1:3); 1H NMR (300 MHz, CDCl3) d
7.23 (d, J = 8.1 Hz, 1H), 6.87 (d, J = 2.7 Hz, 1H), 6.81 (dd, J = 8.1,
2.7 Hz, 1H), 3.84 (d, J = 13.5 Hz, 1H), 3.83 (s, 3H), 3.71 (d,
J = 13.5 Hz, 2H), 3.06 (t, J = 7.5 Hz, 2H), 3.04 (t, J = 7.5 Hz, 2H),
2.23 (m, 2H), 2.15 (s, 3H), 2.09 (s, 3H).
5.1.18. 8-Chloro-5-(4-methoxy-2-methylphenyl)-6-methyl-2,3-
dihydro-1-thia-4,7,7a-triaza-s-indacene (37c)
TLC Rf = 0.38 (EtOAc/hexane, 1:2); 1H NMR (300 MHz, CDCl3) d
7.14 (d, J = 8.5 Hz, 1H), 6.88 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.5,
2.5 Hz, 1H), 3.84 (s, 3H), 3.50–3.38 (m, 4H), 2.38 (s, 3H), 2.15 (s, 3H).
5.1.19. 8-Chloro-5-(4-methoxy-2-methylphenyl)-6-methyl-2,3-
dihydro-1H-1,4,7,7a-tetraaza-s-indacene (37d)
5.1.28. (1-Ethyl-propyl)-[3-(4-methoxy-2-methylphenyl)-2-met
hyl-6,7-dihydro-5H-1,4,8a-triaza-s-indacen-8-yl]-amine (7a)
To a stirred solution of 37a (1.0 g, 3.0 mmol) in 2-propanol
(10 mL) was added 1-ethylpropylamine (1.78 mL, 15.3 mmol) at
room temperature and the reaction mixture was allowed to be
heated at 90 °C and stirred for 4 h. The reaction mixture was
evaporated. The residue was purified by column chromatography
on silica gel (EtOAc/hexane, 1:3) to yield 7a (1.16 g, 100% yield)
as a white powder. TLC Rf = 0.57 (EtOAc/hexane, 1/1); MS (APCI,
Pos) m/z 379 (M+H)+; FABHRMS calcd for C23H30N4O: 379.2498.
Found: 379.2495;1H NMR (300 MHz, CDCl3) d 7.16 (d, J = 8.4 Hz,
1H), 6.85 (d, J = 2.7 Hz, 1H), 6.78 (dd, J = 8.4, 2.7 Hz, 1H), 6.22
(d, J = 10.5 Hz, 1H), 3.82 (s, 3H), 3.79 (m, 1H), 3.08 (t, J = 7.2 Hz,
2H), 2.89 (t, J = 7.8 Hz, 2H), 2.30 (s, 3H), 2.19 (s, 3H), 2.13 (m,
2H), 1.72 (m, 4H), 1.02 (m, 6H); IR (KBr) 3361, 2960, 2933,
1625, 1557, 1509, 1467, 1305, 1235, 1165, 1011, 845 cmꢁ1; mp
156–157 °C.
TLC Rf = 0.31 (EtOAc/hexane, 1:3); 1H NMR (300 MHz, CDCl3) d
7.14 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 3.0 Hz, 1H), 6.83 (dd, J = 3.0,
8.4 Hz, 1H), 4.45 (m, 2H), 3.84 (s, 3H), 3.27 (m, 2H), 2.41 (s, 3H),
2.14 (s, 3H).
5.1.20. 8-Chloro-3-(4-methoxy-2-methylphenyl)-2-methyl-5H,
7H-6-oxa-1,4,8a-triaza-s-indacene (39a)
TLC Rf = 0.31 (EtOAc/hexane, 1:2); 1H NMR (300 MHz, CDCl3) d
7.15 (d, J = 8.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.83 (dd, J = 8.5,
2.5 Hz, 1H), 5.21 (s, 2H), 5.01 (s, 2H), 3.84 (s, 3H), 2.43 (s, 3H),
2.15 (s, 3H).
5.1.21. 8-Chloro-3-(4-methoxy-2-methylphenyl)-2-methyl-5H,
7H-6-thia-1,4,8a-triaza-s-indacene (39b)
A yellow solid; TLC Rf = 0.42 (EtOAc/hexane, 1:2); 1H NMR
(300 MHz, CDCl3) d 7.15 (d, J = 8.5 Hz, 1H), 6.89 (d, J = 3.0 Hz, 1H),
6.82 (dd, J = 8.5, 3.0 Hz, 1H), 4.28–4.22 (m, 4H), 3.84 (s, 3H), 2.42
(s, 3H), 2.14 (s, 3H).
5.1.29. (1-Ethyl-propyl)-[5-(4-methoxy-2-methylphenyl)-6-met
hyl-2,3-dihydro-1-oxa-4,7,7a-triaza-s-indacen-8-yl]-amine (8a)
The title compound was synthesized from 35b as a white pow-
der according to the same manner as described for the prepara-
tion of 7a from 35b using 36b instead of 36a. TLC Rf = 0.43
(EtOAc/hexane, 1:2); MS (APCI, Pos) m/z 381 (M+H)+; FABHRMS
5.1.22. 7-Chloro-3-(4-methoxy-2-methylphenyl)-2,5,6-trimeth
yl-pyrazolo[1,5-a]pyrimidine (41)
A yellow oil; Rf = 0.67 (EtOAc/hexane = 1/2); MS (APCI, Pos) 335
(M+H)+; 1H NMR (300 MHz, CDCl3) d 7.29 (d, J = 8.4 Hz, 1H), 7.08
(d, J = 2.7 Hz, 1H), 6.91 (dd, J = 8.4, 2.7 Hz, 1H), 3.85 (s, 3H), 2.55
(s, 3H), 2.44 (s, 3H), 2.42 (s, 3H).
calcd for
C
22H29N4O2: 381.2291. Found: 381.2286; 1H NMR
(300 MHz, CDCl3) d 7.31 (br s, 1H), 7.12 (d, J = 8.4 Hz, 1H), 6.89
(d, J = 2.7 Hz, 1H), 6.82 (dd, J = 8.4, 2.7 Hz, 1H), 4.76 (t,
J = 9.0 Hz, 2H), 4.30 (m, 1H), 3.83 (s, 3H), 3.74 (t, J = 9.0 Hz, 2H),
2.34 (s, 3H), 2.19 (s, 3H), 1.90–1.70 (m, 4H), 1.04 (m, 6H); IR
(KBr) 3434, 2925, 1657, 1604, 1493, 1453, 1320, 1240, 1029,
5.1.23. 8-Chloro-3-(4-methoxy-2-methylphenyl)-6,7-dihydro-
5H-1,4,8a-triaza-s-indacene (42a)
A yellow powder; TLC Rf = 0.42 (EtOAc/hexane, 1:3); 1H NMR
(300 MHz, CDCl3) d 8.12 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 6.87 (d,
756, 698 cmꢁ1
.

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5441
5.1.30. (1-Ethyl-propyl)-[5-(4-methoxy-2-methylphenyl)-6-met
hyl-2,3-dihydro-1-thia-4,7,7a-triaza-s-indacen-8-yl]-amine (9)
The title compound was synthesized from 35b as a white pow-
der according to the same manner as described for the preparation
of 7a from 35b using 36c instead of 36a. TLC Rf = 0.40 (EtOAc/hex-
ane, 1:2); MS (APCI, Pos) m/z 397 (M+H)+; FABHRMS calcd for
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b.
TLC Rf = 0.50 (EtOAc/benzene, 1:10); MS (MALDI, Pos) m/z 363
(M+H)+; FABHRMS calcd for C23H31N4: 363.2549. Found:
363.2571; 1H NMR (300 MHz, CDCl3) d 7.13 (d, J = 7.5 Hz, 1H),
7.11 (br s, 1H), 7.03 (m, 1H), 6.21 (d, J = 10.8 Hz, 1H), 3.80 (m,
1H), 3.08 (t, J = 6.9 Hz, 2H), 2.89 (t, J = 7.5 Hz, 2H), 2.34 (s, 3H),
2.31 (s, 3H), 2.18 (s, 3H), 2.13 (m, 2H), 1.56–1.82 (m, 4H), 1.02
(m, 6H); IR (KBr) 3447, 2961, 1624, 1560, 1459, 1317, 1281,
C
22H29N4OS: 397.2062. Found: 397.2071; 1H NMR (300 MHz,
CDCl3) d 7.15 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 3.0 Hz, 1H), 6.79 (dd,
J = 8.5, 3.0 Hz, 1H), 6.17 (d, J = 10.0 Hz, 1H), 3.99 (m, 1H), 3.82 (s,
3H), 3.36–3.20 (m, 4H), 2.30 (s, 3H), 2.18 (s, 3H), 1.82–1.56 (m
4H), 1.03 (t, J = 7.5 Hz, 6H); IR (KBr) 3339, 2962, 2930, 1619,
1013 cmꢁ1
.
1558, 1504, 1469, 1314, 1290, 1237, 1006 cmꢁ1
.
5.1.35. N-(1-Ethylpropyl)-3-(4-fluoro-2-methylphenyl)-2-meth
yl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-
amine hydrochloride (19)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b.
TLC Rf = 0.44 (EtOAc/hexane, 1:3); MS (MALDI, Pos) m/z 367
(M+H)+; FABHRMS calcd for C22H28FN4: 367.2298. Found:
367.2284; 1H NMR (300 MHz, DMSO-d6) d 7.34–7.24 (m, 2H),
7.20–7.10 (m, 1H), 4.03–3.85 (m, 1H), 3.14 (br t, J = 8.1 Hz, 2H),
2.95 (br t, J = 8.1 Hz, 2H), 2.25 (s, 3H), 2.25–2.10 (m) and 2.12 (s)
total 5H, 1.85–1.60 (m, 4H), 0.95–0.85 (m, 6H); IR (KBr) 2967,
1645, 1593, 1543, 1460, 1357, 1325, 1255, 1154, 1129 cmꢁ1; mp
126.5–131.5 °C.
5.1.31. (1-Ethyl-propyl)-[5-(4-methoxy-2-methylphenyl)-6-met
hyl-2,3-dihydro-1H-1,4,7,7a-tetraaza-s-indacen-8-yl]-amine (10)
The title compound was synthesized from 35b as a viscous oil
according to the same manner as described for the preparation of
7a from 35b using 36d instead of 36a. TLC Rf = 0.37 (EtOAc/hexane,
1:1); MS (APCI, Pos) m/z 380 (M+H)+; FABHRMS calcd for
C
22H30N5O: 380.2450. Found: 380.2440; 1H NMR (300 MHz, CDCl3)
d 7.16 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 2.7 Hz, 1H), 6.78 (dd, J = 2.7,
8.4 Hz, 1H), 5.86 (d, J = 10.5 Hz, 1H), 4.07 (m, 1H), 3.82 (s, 3H),
3.58 (t, J = 8.1 Hz, 2H), 3.06 (t, J = 8.1 Hz, 2H), 2.30 (s, 3H), 2.19 (s,
3H), 1.52–1.82 (m, 4H), 1.01 (m, 6H) ; IR (neat) 3348, 2963,
1638, 1557, 1512, 1464, 1314, 1236, 1161, 1054, 732 cmꢁ1
.
5.1.32. (1-Ethyl-propyl)-[3-(4-methoxy-2-chlorophenyl)-2-met
hyl-6,7-dihydro-5H-1,4,8a-triaza-s-indacen-8-yl]-amine (7b)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation
of 7a from 36a using 35g instead of 35b. TLC Rf = 0.45 (EtOAc /hex-
ane, 1:2); MS (APCI, Pos) m/z 399 (M+H)+; 1H NMR(300 MHz,
CDCl3) d 7.31 (d, J = 8.4 Hz, 1H), 7.05 (d, J = 2.7 Hz, 1H), 6.88 (dd,
J = 2.7, 8.4 Hz, 1H), 6.22 (br d, J = 10.5 Hz, 1H), 3.82 (s, 3H), 3.80
(m, 1H), 3.08 (t, J = 7.2 Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.34 (s,
3H), 2.14 (m, 2H), 1.52–1.82 (m, 4H), 1.01 (t, J = 7.5 Hz, 3H); 13C
NMR (75 MHz, CDCl3) d 10.36, 13.74, 23.65, 28.76, 29.33, 34.70,
55.59, 55.86, 97.42, 104.91, 113.04, 114.91, 124.05, 133.62,
135.44, 142.69, 146.92, 151.23, 159.14, 168.62; Anal. Calcd for
5.1.36. 3-[4-(Dimethylamino)-2-methylphenyl]-N-(1-ethylpro
pyl)-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyr
imidin-8-amine (20)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b.
TLC Rf = 0.17 (EtOAc/hexane, 1:3); MS (APCI, Pos) m/z 392
(M+H)+; FABHRMS calcd for C24H34N5: 392.2814. Found:
392.2825; 1H NMR (300 MHz, CDCl3) d 7.11 (d, J = 8.1 Hz, 1H),
6.70 (d, J = 2.7 Hz, 1H), 6.64 (dd, J = 8.1, 2.7 Hz, 1H), 6.19 (d,
J = 10.2 Hz, 1H), 3.80 (m, 1H), 3.08 (t, J = 7.5 Hz, 2H), 2.95 (s, 6H),
2.89 (t, J = 7.5 Hz, 2H), 2.32 (s, 3H), 2.18 (s, 3H), 2.18–2.08 (m,
2H), 1.80–1.56 (m, 4H), 1.01 (br s, 6H); IR (KBr) 3340, 2965,
1624, 1562, 1515, 1478, 1348, 1320, 1273, 1134, 1011, 841,
C22H27ClN4O; C, 66.24; H, 6.82; N, 14.04. Found: C, 66.11; H,
6.76; N, 14.15; IR (KBr) 33351, 2962, 1622, 1557, 1508, 1473,
1368, 1321, 1271, 1231, 1135, 1061, 1040, 1008, 875, 819,
611 cmꢁ1; mp 148–149 °C.
806 cmꢁ1
.
5.1.33. 5-(2-Chloro-4-methoxyphenyl)-N-(1-ethylpropyl)-6-meth
yl-2,3-dihydrofuro[3,2-d]pyrazolo[1,5-a]pyrimidin-9-amine (8b)
The title compound was synthesized from 35g as a white pow-
der according to the same manner as described for the preparation
of 7a from 35b using 36b instead of 36a. TLC Rf = 0.46 (EtOAc/hex-
ane, 1:1); MS (APCI, Pos) m/z 401 (M+H)+; FABHRMS calcd for
5.1.37. 3-(2,5-Dimethylphenyl)-N-(1-ethylpropyl)-2-methyl-6,7-
dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-amine
hydrochloride (21)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 36b.
C
21H26ClN4O2 :401.1744. Found: 401.1746; 1H NMR (300 MHz,
DMSO-d6) d 7.30 (d, J = 8.42 Hz, 1H), 7.06 (d, J = 2.56 Hz, 1H), 6.88
(dd, J = 8.42, 2.56 Hz, 1H), 5.76 (d, J = 10.06 Hz, 1H), 4.61 (t,
J = 8.60 Hz, 2H), 4.02–4.18 (m, 1H), 3.83 (s, 3H), 3.26 (t,
J = 8.60 Hz, 2H), 2.35 (s, 3H), 1.50–1.80 (m, 4H), 1.00 (t,
J = 7.41 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 10.35, 13.73, 28.87,
32.40, 55.60, 56.47, 70.71, 104.97, 113.10, 114.96, 123.89, 124.72,
133.11, 133.54, 135.46, 145.00, 151.17, 154.19, 159.26; IR (KBr)
1029, 1060, 1080, 1136, 1227, 1261, 1290, 1317, 1409, 1516,
1567, 1653, 2876, 2926, 2965, 3360 cmꢁ1; mp 149–150 °C.
TLC Rf = 0.54 (acetone/benzene, 1:10); MS (MALDI, Pos) m/z 363
(M+H)+; FABHRMS calcd for C23H31N4: 363.2549. Found: 363.2557;
1H NMR(300 MHz, CDCl3) d 7.31 (br d, J = 10.2 Hz, 1H), 7.24 (d,
J = 7.5 Hz, 1H), 7.15 (br dd, J = 1.2, 7.5 Hz, 1H), 7.01 (br s, 1H),
3.99 (m, 1H), 3.49 (t, J = 7.5 Hz, 2H), 3.14 (t, J = 6.9 Hz, 2H), 2.35
(s, 3H), 2.32 (s, 3H), 2.29 (m, 2H), 2.18 (s, 3H), 1.64–1.94 (m, 4H),
1.07 (t, J = 7.5 Hz, 6H), 1.06 (t, J = 7.2 Hz, 6H); IR (KBr) 3444,
2964, 1644, 1593, 1460, 1354 cmꢁ1
.
5.1.38. N-(1-Ethylpropyl)-3-(4-methoxy-2,5-dimethylphenyl)-2-
methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimid
in-8-amine (22)
The title compound was synthesized from 36a as an ivory
powder according to the same manner as described for the prepa-
ration of 7a from 36a using an optional 4-phenyl-3-methyl-
5.1.34. 3-(2,4-Dimethylphenyl)-N-(1-ethylpropyl)-2-methyl-6,7-
dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-amine
(18)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation

5442
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
1H-pyrazol-5-amine instead of 35b. TLC Rf = 0.43 (EtOAc/benzene,
1:10); MS (MALDI, Pos) m/z 393 (M+H)+; FABHRMS calcd for
C
J = 7.5 Hz, 3H), 1.05 (t, J = 7.2 Hz, 3H); IR (KBr) 3428, 2968, 1650,
1596, 1548, 1462, 1325, 1287, 1227, 1060 cmꢁ1
.
24H33N4O: 393.2654. Found: 393.2668; 1H NMR(300 MHz, CDCl3)
d 6.99 (s, 1H), 6.76 (s, 1H), 6.20 (d, J = 10.5 Hz, 1H), 3.84 (s, 3H), 3.82
(m, 1H), 3.08 (t, J = 6.9 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.31 (s, 3H),
2.19 (6.9 Hz, 3H), 2.17 (s, 3H), 2.14 (m, 2H), 1.01 (m, 6H); IR (KBr)
5.1.43. 3-[2-Chloro-4-(trifluoromethoxy)phenyl]-N-(1-ethylpro
pyl)-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyr
imidin-8-amine hydrochloride (27)
3345, 2965, 1625, 1559, 1511, 1469, 1318, 1233, 1109 cmꢁ1
.
The title compound was synthesized from 36a as a colorless
powder according to the same manner as described for the prepa-
ration of 7a from 36a using an optional 4-phenyl-3-methyl-1H-
pyrazol-5-amine instead of 35b. TLC Rf = 0.52 (EtOAc/hexane,
1:3); MS (MALDI, Pos) 453 (M+H)+; FABHRMS calcd for
5.1.39. N-(1-Ethylpropyl)-3-(4-methoxy-2,6-dimethylphenyl)-2-
methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimi
din-8-amine (23)
The title compound was synthesized from 36a as an ivory pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b. TLC Rf = 0.33 (EtOAc/benzene, 1:10); MS
(APCI, Pos) m/z 393 (M+H)+; FABHRMS calcd for C24H33N4O:
393.2654. Found: 393.2650; 1H NMR(300 MHz, CDCl3) d 6.68 (s,
2H), 6.21 (d, J = 10.5 Hz, 1H), 3.81 (m, 1H), 3.80 (s, 3H), 3.09 (t,
J = 7.2 Hz, 2H), 2.88 (t, J = 7.8 Hz, 2H), 2.19 (s, 3H), 2.13 (m, 2H),
2.04 (s, 6H), 1.55–1.83 (m, 4H), 1.03 (t, J = 7.5 Hz, 6H); IR (KBr)
3337, 2966, 1620, 1557, 1498, 1466, 1321, 1274, 1146, 1068,
C
22H25ClF3N4O: 453.1669. Found: 453.1663; 1H NMR (300 MHz,
Pyridine-d5 0.5 mL CDCl3 0.1 mL) d 7.71 (d, J = 8.4 Hz, 1H), 7.57
(m, 1H), 7.28 (m, 1H), 6.77 (d, J = 10.5 Hz, 1H), 3.74 (m, 1H), 2.95
(t, J = 7.5 Hz, 2H), 2.85 (t, J = 7.8 Hz, 2H), 2.46 (s, 3H), 1.98 (m,
2H), 1.64–1.48 (m, 4H), 0.92 (t, J = 7.5 Hz, 6H); IR (KBr) 3425,
3320, 2969, 1804, 1637, 1599, 1544, 1460, 1360, 1281, 1224,
1153 cmꢁ1
.
5.1.44. 3-(2,4-Dichlorophenyl)-N-(1-ethylpropyl)-2-methyl-6,7-
dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-amine
(28)
1011 cmꢁ1
.
The title compound was synthesized from 36a as an ivory pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b. TLC Rf = 0.50 (EtOAc/hexane, 1:2); MS
(MALDI, Pos) m/z 403 (M+H)+; FABHRMS calcd for C21H25Cl2N4:
403.1456. Found: 403.1443 ; 1H NMR (300 MHz, CDCl3) d 7.50 (d,
J = 2.0 Hz, 1H), 7.35 (d, J = 8.5 Hz, 1H), 7.29 (dd, J = 8.5, 2.0 Hz,
1H), 6.23 (d, J = 10.5 Hz, 1H), 3.81 (m, 1H), 3.09 (t, J = 7.5 Hz, 2H),
2.91 (t, J = 7.5 Hz, 2H), 2.34 (s, 3H), 2.15 (m, 2H), 1.82–1.55 (m
4H), 1.01 (t, J = 7.5 Hz, 6H); IR (KBr) 3315, 2957, 1626, 1562,
5.1.40. N-(1-Ethylpropyl)-3-[4-methoxy-2-(methylsulfanyl)phe
nyl]-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyr
imidin-8-amine hydrochloride (24)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the prepara-
tion of 7a from 36a using an optional 4-phenyl-3-methyl-1H-
pyrazol-5-amine instead of 35b. TLC Rf = 0.10 (EtOAc/hexane,
1:3); MS (APCI, Pos) 411 (M+H)+; FABHRMS calcd for C23H31N4OS
:411.2219. Found: 411.2212; 1H NMR(300 MHz, CDCl3) d 7.26–
7.16 (m, 1H), 6.83 (m, 1H), 6.84–6.76 (m, 1H), 3.97 (m, 1H),
3.86 (s, 3H), 3.48 (m, 2H), 3.12 (m, 2H), 2.44 (s, 3H), 2.33 (s,
3H), 2.28 (m, 2H), 1.95–1.44 (m, 4H), 1.11–0.99 (m, 6H); IR
(KBr) 3412, 3276, 2967, 1645, 1596, 1462, 1360, 1325, 1288,
1500, 1463, 1377, 1320, 1280, 1094, 1069 cmꢁ1
.
5.1.45. N-(1-Ethylpropyl)-3-(4-methoxy-2-methylphenyl)-2-met
hyl-5H,7H-furo[3,4-d]pyrazolo[1,5-a]pyrimidin-8-amine (11a)
The title compound was synthesized from 35b as an ivory pow-
der according to the same manner as described for the preparation
of 7a from 35b using 38a instead of 36a. TLC Rf = 0.33 (EtOAc/hex-
ane, 1:2); MS (MALDI, Pos) m/z 381 (M+H)+; FABHRMS calcd for
C22H29N4O2: 381.2291. Found: 381.2288; 1H NMR (300 MHz,
CDCl3) d 7.15 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 6.79 (dd,
J = 8.5, 2.5 Hz, 1H), 6.32 (d, J = 10.0 Hz, 1H), 5.29 (s, 2H), 4.90 (br
s, 2H), 3.82 (s, 3H), 3.24 (m, 1H), 2.33 (s, 3H), 2.18 (s, 3H), 1.84–
1.56 (m 4H), 1.02 (t, J = 7.5 Hz, 6H) ; IR (KBr) 3344, 2960, 2873,
1239, 1037 cmꢁ1
.
5.1.41. 3-(3,4-Dimethoxyphenyl)-N-(1-ethylpropyl)-2-methyl-6,
7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-amine
(25)
The title compound was synthesized from 36a as an off-white
powder according to the same manner as described for the prepa-
ration of 7a from 36a using an optional 4-phenyl-3-methyl-1H-
pyrazol-5-amine instead of 35b. TLC Rf = 0.56 (EtOAc/hexane,
1:1); MS (APCI, Pos) 395 (M+H)+; FABHRMS calcd for
1632, 1581, 1514, 1467, 1326, 1304, 1234, 1056 cmꢁ1
.
C
23H31N4O2: 395.2447. Found: 395.243; 1H NMR(300 MHz, CDCl3)
d 7.29 (d, J = 2.1 Hz, 1H), 7.19 (dd, J = 2.1, 8.1 Hz, 1H), 6.96 (d,
J = 8.1 Hz, 1H), 6.20 (br d, J = 10.5 Hz, 1H), 3.93 (s, 3H), 3.91 (s,
3H), 3.80 (m, 1H), 3.09 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.5 Hz, 2H),
2.55 (s, 3H), 2.16 (m, 2H), 1.53–1.81 (m, 4H), 1.00 (t, J = 7.2 Hz,
6H); IR (KBr) 3457, 2955, 1621, 1571, 1552, 1463, 1254, 1220,
5.1.46. N-(1-Ethylpropyl)-3-(4-methoxy-2-methylphenyl)-2-met
hyl-5H,7H-pyrazolo[1,5-a]thieno[3,4-d]pyrimidin-8-amine (12)
The title compound was synthesized from 35b as a yellow
amorphous solid according to the same manner as described for
the preparation of 7a from 35b using 38b instead of 36a. TLC
Rf = 0.51 (EtOAc/hexane, 1:2); MS (MALDI, Pos) m/z 397 (M+H)+;
FABHRMS calcd for C22H29N4OS: 397.2062. Found: 397.2061; 1H
1142, 1029 cmꢁ1
.
5.1.42. 3-(2-Chloro-4-ethoxyphenyl)-N-(1-ethylpropyl)-2-meth
yl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-
amine hydrochloride (26)
NMR (300 MHz, CDCl3) d 7.15 (d, J = 8.5 Hz, 1H), 6.86 (d,
J = 2.5 Hz, 1H), 6.79 (dd, J = 8.5, 2.5 Hz, 1H), 6.44 (m, 1H), 4.32 (br
s, 2H), 4.14 (br s, 2H), 3.82 (s, 3H), 3.76 (m, 1H), 2.32 (s, 3H),
2.18 (s, 3H), 1.84–1.57 (m 4H), 1.03 (t, J = 7.0 Hz, 6H); IR (KBr)
The title compound was synthesized from 36a as an ivory pow-
der according to the same manner as described for the preparation
of 7a from 36a using an optional 4-phenyl-3-methyl-1H-pyrazol-
5-amine instead of 35b. TLC Rf = 0.49 (EtOAc/hexane, 1:2); MS
(APCI, Pos) 413(M+H)+; FABHRMS calcd for C23H30ClN4O:
413.2108. Found: 413.2114; 1H NMR (300 MHz, CDCl3) d 7.34 (d,
J = 8.7 Hz, 1H), 7.06 (d, J = 2.4 Hz, 1H), 6.95 (dd, J = 2.4, 8.7 Hz,1H),
4.07 (m, 2H), 3.99 (m, 1H), 3.34–3.65 (m, 2H), 3.13 (t, J = 7.8 Hz,
2H), 2.35 (s, 3H), 2.29 (m, 2H), 1.42 (t, J = 6.9 Hz, 3H), 1.06 (t,
3331, 2963, 1616, 1557, 1506, 1466, 1319, 1237 cmꢁ1
.
5.1.47. 3-(2-Chloro-4-methoxyphenyl)-N-(1-ethylpropyl)-2-met
hyl-5H,7H-furo[3,4-d]pyrazolo[1,5-a]pyrimidin-8-amine (11b)
The title compound was synthesized from 35g as a white solid
according to the same manner as described for the preparation of
7a from 38b using 36b instead of 36a. TLC Rf = 0.41 (EtOAc/ben-
zene, 1:4); MS (APCI, Pos) m/z 401 (M+H)+; 1H NMR (300 MHz,

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5443
CDCl3) d 7.30 (d, J = 8.52 Hz, 1H), 7.06 (d, J = 2.75 Hz, 1H), 6.89 (dd,
J = 8.52, 2.75 Hz, 1H), 6.34 (d, J = 10.71 Hz, 1H), 5.29 (s, 2H), 4.92 (s,
2H), 3.83 (s, 3H), 3.23 (m, 1H), 2.36 (s, 3H), 1.70 (m, 4H), 1.02 (t,
J = 7.42 Hz, 6H); 13C NMR (75 MHz, CDCl3) d 10.29, 13.62, 28.68,
55.56, 56.86, 70.80, 72.58, 93.43, 105.71, 113.18, 115.07, 123.50,
133.55, 135.51, 141.50, 152.45, 152.47, 159.50, 163.41; Anal. Calcd
for C21H25ClN4O2; C, 62.92; H, 6.29; N, 13.97. Found: C, 63.11; H,
6.23; N, 14.04; mp 138–139 °C.
1.70–2.22 (m, 4H), 1.08 (m, 6H); IR (KBr) 3428, 2968, 1650, 1597,
1507, 1461, 1363, 1242, 1180, 1134 cmꢁ1
.
5.1.52. N-(1-Ethylpropyl)-3-(4-methoxy-2-methylphenyl)-2-
[(methylsulfanyl)methyl]-6,7-dihydro-5H-cyclopenta[d]pyrazo
lo[1,5-a]pyrimidin-8-amine hydrochloride (17)
A colorless powder. TLC Rf = 0.31 (EtOAc/hexane, 1:3); MS
(MALDI, Pos) m/z 425 (M+H)+; FABHRMS calcd for C24H33N4OS:
425.2375. Found: 425.2383; 1H NMR (300 MHz, CDCl3) d 7.31 (br
d, J = 10.8 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 6.89 (d, J = 2.4 Hz, 1H),
6.80 (dd, J = 8.4, 2.4 Hz, 1H), 4.00 (br s, 1H), 3.83 (s, 3H), 3.70 (d,
J = 13.5 Hz, 1H), 3.60 (d, J = 13.5 Hz, 1H), 3.50 (m, 2H), 3.14 (t,
J = 7.2 Hz, 2H), 2.29 (m, 2H), 2.32 (s, 3H), 2.04 (s, 3H), 1.95–1.65
(m, 4H), 1.07 (t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.5 Hz, 3H); IR (KBr)
5.1.48. N-(1-Ethylpropyl)-3-(4-methoxy-2-methylphenyl)-6,7-
dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-amine
hydrochloride (13)
The title compound was synthesized from 36a as an off-white
powder according to the same manner as described for the prepa-
ration of 7a from 36a using 35a instead of 35b. TLC Rf = 0.46
(EtOAc/hexane, 1:3); MS (FAB, Pos) m/z 365 (M+H)+; FABHRMS
calcd for C22H29N4O: 365.2341. Found: 365.2318; 1H NMR
2964, 1644, 1591, 1543, 1459, 1359, 1307, 1237, 1043 cmꢁ1
.
5.1.53. 3-(2-Chloro-4-methoxyphenyl)-N-(1-ethylpropyl)-2,5,6-
trimethylpyrazolo[1,5-a]pyrimidin-7-amine (30)
(300 MHz, DMSO-d6)
d 9.25 (m, 1H), 8.31 (s, 1H), 7.23 (d,
J = 8.1 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 2.4, 8.1 Hz,
1H), 3.99 (m, 1H), 3.78 (s, 3H), 3.15 (m, 2H), 3.02 (t, J = 7.8 Hz,
2H), 2.20 (s, 3H), 2.18 (m, 2H), 1.60–1.88 (m, 4H), 0.89 (t,
J = 7.5 Hz, 6H); IR (KBr) 3421, 2966, 1646, 1596, 1459, 1361,
The title compound was synthesized from 35g as a white pow-
der according to the same manner as described for the preparation
of 7a from 35g using 41 instead of 36a.
TLC Rf = 0.43 (EtOAc/hexane, 1:3); MS (APCI, Pos) 387 (M+H)+;
1H NMR (300 MHz, CDCl3) d 7.32 (d, J = 8.52 Hz, 1H), 7.05 (d,
J = 2.47 Hz, 1H), 6.88 (dd, J = 8.52, 2.75 Hz, 1H), 5.98 (d,
J = 10.44 Hz, 1H), 3.89 (m, 1H), 3.83 (s, 3H), 2.46 (s, 3H), 2.35 (s,
3H), 2.28 (s, 3H), 1.67 (m, 4H), 0.99 (t, J = 7.42 Hz, 6H); Anal. Calcd
for C21H27ClN4O; C, 65.19; H, 7.03; N, 14.48. Found: C, 65.30; H,
7.06; N, 14.47; mp 117–119 °C.
1241, 1189, 1037 cmꢁ1
.
5.1.49. 2-Ethyl-N-(1-ethylpropyl)-3-(4-methoxy-2-methylphen
yl)-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidin-8-
amine (14)
The title compound was synthesized from 36a according to the
same manner as described for the preparation of 7a from 36a using
35c instead of 35b. TLC Rf = 0.59 (EtOAc/benzene, 1:5) ; MS (MAL-
5.1.54. 3-(2-Chloro-4-methoxyphenyl)-2-methyl-6,7-dihydrofu
ro[2,3-d]pyrazolo[1,5-a]pyrimidin-8-amine (44)
DI, Pos) m/z 393 (M+H)+
; FABHRMS calcd for C24H33N4O:
393.2654. Found: 393.2646; H NMR (300 MHz, CDCl3) d 7.15 (d,
J = 8.1 Hz, 1H), 6.85 (d, J = 2.7 Hz, 1H), 6.77 (dd, J = 8.1, 2.7 Hz,
1H), 6.27 (d, J = 10.5 Hz, 1H), 3.82 (s, 3H), 3.80 (m, 1H), 3.08 (t,
J = 7.5 Hz, 2H), 2.89 (t, J = 7.8 Hz, 2H), 2.67 (m, 2H), 2.17 (s, 3H),
2.13 (m, 2H), 1.81–1.52 (m, 4H), 1.16 (t, J = 7.2 Hz, 3H), 1.04 (t,
J = 7.5 Hz, 3H), 1.01 (t, J = 7.8 Hz, 3H); IR (neat) 2963, 1623, 1557,
A solution of 43 (700 mg, 6.3 mmol) and 38 g in pyridine
(0.9 mL) was heated up to reflux temperature and stirred for
14 h. The solvent was removed by evaporation. The resulting resi-
due was purified by column chromatography on silica gel (MeOH/
CH2Cl2, 1:10) to give 44 (756 mg, 36% yield) as an ivory powder.
TLC Rf = 0.40 (MeOH/CH2Cl2, 1:9); 1H NMR (300 MHz, CDCl3) d
7.26 (m, 1H), 7.01 (d, J = 2.6 Hz, 1H), 6.84 (dd, J = 8.5, 2.6 Hz, 1H),
5.47 (s, 2H), 4.68 (m, 2H), 3.80 (s, 3H), 3.11 (t, J = 8.2 Hz, 2H),
2.32 (s, 3H).
1508, 1459, 1275, 1235, 1160 cmꢁ1
.
5.1.50. 2-Cyclobutyl-N-(1-ethylpropyl)-3-(4-methoxy-2-methyl
phenyl)-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-
a]pyrimidin-8-amine (15)
5.1.55. 3-(2-Chloro-4-methoxyphenyl)-N-(1-ethylpropyl)-2-meth
yl-6,7-dihydrofuro[2,3-d]pyrazolo[1,5-a]pyrimidin-8-amine (31)
To a stirred solution of 44 (200 mg, 0.60 mmol) in DMF (3.0 mL)
was added NaH (26 mg, 0.67 mmol , 60% in mineral oil) and tetra-
butylammoniun iodide (catalytic amount) at ambient temperature
under argon atmosphere. After 1.5 h, the reaction mixture was al-
lowed to be heated at 50 °C for 1 h, then at 80 °C for 22 h. The reac-
tion mixture was cooled to ambient temperature and diluted with
hexane/EtOAc (1:1). The organic layer was washed with brine and
dried over MgSO4, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel using Et2O/hex-
ane (1:2–1:3) to give 31 (36 mg, 15% yield) as an ivory powder.
TLC Rf = 0.67 (EtOAc/hexane, 1:1) ; MS (APCI, Pos) m/z 401
(M+H)+ ; FABHRMS calcd for C21H26ClN4O2: 401.1744. Found:
401.1743; 1H NMR (300 MHz, CDCl3) d 7.26 (m, 1H), 7.01 (d,
J = 2.6 Hz, 1H), 6.84 (dd, J = 8.5, 2.6 Hz, 1H), 6.18 (d, J = 10.8 Hz,
1H), 4.62 (m, 2H), 3.81 (s, 3H), 3.60 (m, 1H), 3.34 (t, J = 8.2 Hz,
2H), 2.30 (s, 3H), 1.68 (m, 4H), 1.01 (m, 6H); mp 139–141 °C.
The title compound was synthesized from 36a as an off-white
powder according to the same manner as described for the prep-
aration of 7a from 36a using 35d instead of 35b. TLC Rf = 0.62
(EtOAc/benzene, 1:5); MS (MALDI, Pos) m/z 419 (M+H)+; FAB-
HRMS calcd for
NMR (300 MHz, CDCl3)
C
26H35N4O: 419.2811. Found: 419.2835; 1H
7.09 (d, J = 8.1 Hz, 1H), 6.83 (d,
d
J = 2.7 Hz, 1H), 6.75 (dd, J = 8.1, 2.7 Hz, 1H), 6.35 (d, J = 10.5 Hz,
1H), 3.82 (s, 3H), 3.81 (m, 1H), 3.53 (m, 1H), 3.08 (t, J = 7.5 Hz,
2H), 2.88 (t, J = 7.8 Hz, 2H), 2.41 (m, 2H), 2.28–2.06 (m, 4H),
2.15 (s, 3H), 2.01–1.58 (m, 6H), 1.05 (t, J = 7.5 Hz, 3H), 1.02 (t,
J = 7.8 Hz, 3H); IR (neat) 2963, 1623, 1556, 1508, 1465, 1276,
1238, 1160 cmꢁ1
.
5.1.51. N-(1-Ethylpropyl)-3-(4-methoxy-2-methylphenyl)-2-(tri
fluoromethyl)-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-
a]pyrimidin-8-amine hydrochloride (16)
The title compound was synthesized from 36a as a white pow-
der according to the same manner as described for the preparation
of 7a from 36a using 35e instead of 35b. TLC Rf = 0.42 (EtOAc/hex-
ane, 1:3); MS (APCI, Pos) m/z 433 (M+H)+; FABHRMS calcd for
5.1.56. 3-Chloro-4-[8-(1-ethylpropylamino)-2-methyl-6,7-dihyd
ro-5H-1,4,8a-triaza-s-indacen-3-yl]-phenol (45)
To a stirred solution of 7b (1.0 g, 2.5 mmol) in CH2Cl2 was added
BBr3 (7.5 mL, 7.5 mmol, 1.0 M CH2Cl2 solution) at ꢁ50 °C under ar-
gon atmosphere and allowed to be warmed at 0 °C. After being stir-
red for 5.5 h, the reaction mixture was quenched with NaHCO3 aq
C
23H28F3N4O: 433.2215. Found: 433.2223; 1H NMR (300 MHz,
CDCl3) d 7.33 (br d, J = 10.2 Hz, 1H), 7.13 (d, J = 8.7 Hz, 1H), 6.89
(d, J = 2.4 Hz, 1H), 6.81 (dd, J = 2.4, 8.7 Hz, 1H), 4.04 (m, 1H), 3.83
(s, 3H), 3.56 (m, 2H), 3.20 (m, 2H), 2.33 (m, 2H), 2.19 (s, 3H),

5444
T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
and extracted with CH2Cl2. The organic layer was washed with
brine, dried over MgSO4 and evaporated. The resulting residue
was purified by column chromatography on silica gel to yield 45
(730 mg, 76% yield) as an ivory solid. TLC Rf = 0.31 (EtOAc/hexane,
1:2); 1H NMR (300 MHz, CDCl3) d 11.43 (br s, 1H), 7.02 (d,
J = 8.4 Hz, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.50 (dd, J = 8.3, 2.5 Hz,
1H), 6.42 (d, J = 10.4 Hz, 1H), 3.85 (m 1H), 3.12 (t, J = 7.1 Hz, 2H),
3.00 (t, J = 7.9 Hz, 2H), 2.30 (s, 3H), 2.20 (tt, J = 8.9, 7.3 Hz, 2H),
1.76 (m, 2H), 1.66 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H), 1.03 (t,
J = 7.4 Hz, 3H).
rat CRF. Specific binding was determined by subtracting nonspe-
cific binding from total binding. Concentration of the test com-
pound that caused 50% inhibition of specific radiolabeled ligand
binding (IC50 values) was determined from each concentration–re-
sponse curve.
5.2.3. cAMP assay
CHO-K1 cells expressing CRF receptor type 1 dispersed at
1 ꢂ 104 cells/well in 96-well plates were incubated overnight.
The culture medium was removed, the cells were washed twice
with F-12 nutrient mixture, and then 178 ll of assay medium
5.1.57. 3-[2-Chloro-4-(difluoromethoxy)phenyl]-N-(1-ethylprop
yl)-2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyr
imidin-8-amine (29)
(F12 nutrient mixture containing 1 mmol/l 3-isobutyl-1-methyl-
xanthine, a phosphodiesterase inhibitor) was added. After incuba-
tion for 10 min at 37 °C, cells were treated with 2
ll of test
In a three-neck flask (50 mL) equipped with a condenser (dry-
ice), a gas-inlet tube, and a magnetic stirring bar, NaOH (310 mg,
7.8 mmol) was added to a solution of 45 (600 mg, 1.6 mmol) in a
mixture of THF (11.0 mL) and H2O (0.7 mL) at room temperature.
The reaction mixture was stirred under the continuous flow of
ClF2CH for 3 h at reflux temperature. The resulting mixture was
cooled and extracted with t-BuOMe. The organic layer was washed
with water, brine, dried over MgSO4 and evaporated. The resulting
residue was purified by column chromatography on silica gel to
yield 29 (380 mg, 56% yield) as a white solid. TLC Rf = 0.49
(EtOAc/hexane, 1:2); MS (APCI, Pos) 435 (M+H)+; FABHRMS calcd
compound (1–10,000 nmol/l) and 20 l of assay medium contain-
l
ing 10 nmol/l CRF. After the treated cells were incubated for
15 min at 37 °C, supernatants were aspirated, and cells were
immediately chilled to terminate further reactions. cAMP formed
in the cells was determined using a cAMP enzyme immunoassay
system.
The cAMP level under respective treatments was determined by
mean of corresponding two wells of the blank group from that of
treatment group. Concentration of the test compound that caused
50% inhibition of cAMP production (IC50 values) was determined
from each concentration–response curve.
for
C
22H26ClF2N4O: 435.1763. Found: 435.1744; 1H NMR
(300 MHz, CDCl3) d 7.41 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 2.4 Hz, 1H),
7.09 (dd, J = 8.4, 2.4 Hz, 1H), 6.52 (t, J = 73.5 Hz, 1H), 6.24 (d,
J = 10.5 Hz, 1H), 3.81 (m, 1H), 3.09 (m, 2H), 2.91 (m, 2H), 2.35 (s,
3H), 2.15 (m, 2H), 1.82–1.54 (m, 4H), 1.02 (t, J = 7.5 Hz, 6H); IR
(KBr) 3343, 2962, 2875, 1625, 1558, 1503, 1391, 1322, 1271,
5.2.4. Elevated plus-maze test in swim stress-loaded rats
5.2.4.1. Animals.
Male Sprague–Dawley rats weighing 230–
280 g (Charles River, Japan) were used. Rats were accommodated
for more than a week in a room at 24 2 °C, 55 15% relative
humidity with controlled 12 h dark–light cycles (alternating 12 h
cycles with illumination from fluorescent light: 08:00–20:00 h)
and were allowed free access to food and water. They were housed
in groups of five or six rats per cage until experiments. All experi-
mental procedures were approved by the Animal Care and Use
Committee of Ono Pharmaceutical Co. Ltd and conducted in accor-
dance with the ‘Guidelines on the Use of Experimental animals’.
1221, 1132, 1036 cmꢁ1
.
5.1.58. 3-Oxo-pyrrolidine-1,2-dicarboxylic acid 1-benzyl ester 2-
ethyl ester (36d)
The titled compound was prepared as detailed in the
literature.43
TLC Rf = 0.40 (EtOAc/hexane 1:2); 1H NMR(300 MHz, CDCl3) d
7.23–7.44 (m, 5H), 5.06–5.28 (m, 2H), 4.61 and 4.56 (s, total 1H),
3.80–4.33 (m, 4H), 2.70 (t, J = 7.5 Hz, 2H),1.30 and 1.16 (t,
J = 7.2 Hz, total 3H).
5.2.4.2. Elevated plus-maze test in rats, swim stress induced
anxiety-like behavior.
The elevated plus-maze apparatus
was made of Plexiglas and consisted of four arms (50 cm
long ꢂ 10 cm wide): two had 40 cm high walls (closed arms), and
two had no walls (open arms). The maze was elevated to a height
of 50 cm. In the experiments of swim stress paradigm, rats were
forced to swim for 90 s in a pool (40 ꢂ 30 ꢂ 38 cm) filled with
water (depth of 25 cm) maintained at 22 2 °C prior to the ele-
vated plus-maze test. In non-stress conditions, the rats in the con-
trol group were not subjected to forced swim stress. After forced
swim stress for 90 s, rats were removed from the pool and dried
with a paper towel. For testing, rats were placed individually onto
the center of the maze facing a closed arm and behavior was re-
corded for 5 min with a video camera. The images were analyzed
using a computerized behavior tracking and analysis system (Etho-
Vision Version 3.0, Noldus Information Technology). The primary
measures were the time spent in open arms and the number of en-
tries in open arms.
5.2. Biology
5.2.1. Membrane preparations
CHO-K1 cells expressing CRF receptor type 1 were washed with
phosphate buffered saline (PBS), scraped and pelleted by centrifu-
gation. Cell pellets were homogenized with binding assay buffer
(50 mmol/l Tris–HCl buffer containing 10 mmol/l MgCl , 2 mmol/l
EDTA, and 10 TIU/L aprotinin) and centrifuged at 10,000 g for
15 min at 4 °C. The pellet was suspended in the assay buffer, and
used as crude membrane preparations for binding studies. Protein
concentration was determined according to Bradford, 1976.
5.2.2. Binding assay
Binding assays for 125I human/rat CRF were done according to
reported method (De Souza, 1987; Grigoriadis et al., 1996) but
with slight modification. The reaction was initiated by incubating
Experiments were performed 1 h after oral administration of
vehicle (0.5% MC (w/v)), test compounds (1, 3, and 10 m/kg).
49
ll of membrane preparations with 50
l
l of 0.5 nmol/l 125I hu-
man/rat CRF and 1
ll of test compound (1–10,000 nmol/l). The
5.2.4.3. Data and statistical analysis.
The results of the ele-
reaction mixture was incubated for 2 h at room temperature, and
terminated by centrifugation at 15,000 g for 10 min, and pellet
was washed with PBS containing with 0.01% Triton X-100. The
vated plus-maze test were expressed as mean S.E. values. Com-
parisons between the vehicle and control groups were performed
using the t-test, while differences between the vehicle and test
compound groups were compared with the Dunnett test. Probabil-
ities of <5% (p <0.05) were considered statistically significant.
radioactivity was measured in a
c-counter. Nonspecific bindings
were determined in the presence of unlabeled 1
lmol/l human/

T. Saito et al. / Bioorg. Med. Chem. 19 (2011) 5432–5445
5445
D.; Winston, E. N.; Chen, Y. L.; Heym, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
10477.
5.3. Single dose rat pharmacokinetic study of 7b
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Zamani-Kord, S.; Grigoriadis, D. E.; Crowe, P. D.; Chen, T. K.; Williams, J. P. J.
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Single dose pharmacokinetics of 7b was studied in rats. Formu-
lation for intravenous injection was prepared using saline contain-
ing 30% HP-b-CD (w/v). Formulation for oral dosing was prepared
using saline containing 0.5% MC (w/v). Test compounds (0.3 mg/
kg) were dosed intravenously to the fasted male rats (n = 4). Test
compounds (3 mg/kg) were dosed orally to the fasted male rats
(n = 4). After dosing, blood samples (250 ll) were collected from
the jugular vein using a heparinized syringe at the selected time
points (iv: pre-dosing, 2, 5, 15, 30 min and 1, 2, 3, 4, 8, 24, 48,
72 h; po: 1, 2, 4, 6, 8, 24, 48, 72 h, respectively). The blood samples
were ice-chilled and then centrifuged at 12,000 rpm for 2 min at
room temperature to obtain plasma, which was preserved at
ꢁ70 °C in a freezer. The AUC, Cmax, Tmax, T1/2, Vss and CL were ob-
tained by measuring the time course of the plasma concentration
of the test compounds. Bioavailability (F) was calculated according
to the following equation:
25. Gross, R. S.; Guo, Z.; Dyck, B.; Coon, T.; Huang, C. Q.; Lowe, R. F.; Marinkovic, D.;
Moorjani, M.; Nelson, J.; Zamani-Kord, S.; Grigoriadis, D. E.; Hoare, S. R. J.;
Crowe, P. D.; Bu, J. H.; Haddach, M.; McCarthy, J.; Saunders, J.; Sullivan, R.;
Chen, T.; Williams, J. P. Med. Chem. 2005, 48, 5780.
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Fð%Þ ¼ ðAUCpo=DpoÞ=AUCiv=DivÞ ꢂ 100
AUCpo: AUC after oral dosing; AUCiv: AUC after intravenous dosing;
28. Gentile, G.; Di Fabio, R.; Pavone, F.; Sabbatini, F. M.; St-Denis, Y.; Zampori, M.
G.; Vitulli, G.; Worby, A. Bioorg. Med. Chem. Lett. 2007, 17, 5218.
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Gentile, G.; Ghirlanda, D.; Marchionni, C.; Messina, F.; Micheli, F.; Pavone, F.;
Pasqarello, A.; Sabbatini, F. M.; Zampori, M. G.; Arban, R.; Vitulli, G. Bioorg. Med.
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D
po: Dosage of oral administration; Div: Dosage of intravenous
administration.
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