Chemical modification of aryl-1,2,3,6-tetrahydropyridinopyrimidine derivative to discover corticotropin-releasing factor1 receptor antagonists: Aryl-1,2,3,6-tetrahydropyridino-purine, -3H-1,2,3-triazolo[4,5-d]pyrimidine, -purin-8-one, and -7H-pyrrolo[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1016/S0968-0896(01)00005-0

Structure-affinity relationships (SARs) of non-peptide CRF₁ antagonists suggest that such antagonists can be constructed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area). Recently, various non-peptide corticotropin-releasing factor₁ (CRF₁) receptor antagonists obtained by modification of the Central-Area have been reported. In contrast, we modified the Up-Area and presented 4- or 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine derivatives including potent CRF receptor ligands 1a-c, and proposed that the 4- or 5-aryl-1,2,3,6-tetrahydropyridino moiety might be useful as a substituent in the Up-Area. Our interest shifted to the chemical modification in which the pyrimidine ring of 1a-c was replaced by other heterocycles, purine ring of 2, 3H-1,2,3-triazolo[4,5-d]pyrimidine ring of 3, purin-8-one ring of 4 and 7H-pyrrolo[2,3-d]pyrimidine ring of 5. Among them, 5-aryl-1,2,3,6-tetrahydropyridinopurine compound 6j (CRA0186) had the highest affinity for CRF₁ receptors (IC₅₀=20 nM). We report here the synthesis and SARs of derivatives 6-9.

Full text of DOI:10.1016/S0968-0896(01)00005-0

Bioorganic & Medicinal Chemistry 9 (2001) 1357±1363
Chemical Modi®cation of
Aryl-1,2,3,6-tetrahydropyridinopyrimidine Derivative to Discover
Corticotropin-Releasing Factor1 Receptor Antagonists:
Aryl-1,2,3,6-tetrahydropyridino-purine,
-3H-1,2,3-triazolo[4,5-d]pyrimidine, -purin-8-one,
and -7H-pyrrolo[2,3-d]pyrimidine Derivatives
Toshihito Kumagai, Taketoshi Okubo, Hiromi Kataoka-Okubo, Shigeyuki Chaki,
Shigeru Okuyama and Atsuro Nakazato*
1st Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd.,1-403 Yoshino-cho, Ohmiya,
Saitama 330-8530, Japan
Received 8November 2000; accepted 6 January 2001
AbstractÐStructure±anity relationships (SARs) of non-peptide CRF1 antagonists suggest that such antagonists can be con-
structed of three units: a hydrophobic unit (Up-Area), a proton accepting unit (Central-Area), and an aromatic unit (Down-Area).
Recently, various non-peptide corticotropin-releasing factor1 (CRF1) receptor antagonists obtained by modi®cation of the Central-
Area have been reported. In contrast, we modi®ed the Up-Area and presented 4- or 5-aryl-1,2,3,6-tetrahydropyridinopyrimidine
derivatives including potent CRF receptor ligands 1a±c, and proposed that the 4- or 5-aryl-1,2,3,6-tetrahydropyridino moiety
might be useful as a substituent in the Up-Area. Our interest shifted to the chemical modi®cation in which the pyrimidine ring of
1a±c was replaced by other heterocycles, purine ring of 2, 3H-1,2,3-triazolo[4,5-d]pyrimidine ring of 3, purin-8-one ring of 4 and
7H-pyrrolo[2,3-d]pyrimidine ring of 5. Among them, 5-aryl-1,2,3,6-tetrahydropyridinopurine compound 6j (CRA0186) had the
highest anity for CRF1 receptors (IC50=20 nM). We report here the synthesis and SARs of derivatives 6±9. # 2001 Elsevier
Science Ltd. All rights reserved.
Introduction
for the treatment of depression, anxiety, or other dis-
eases related to stress.
Corticotropin-releasing factor (CRF), a 41-amino acid
peptide, is a principal modulator for the responses to
stress through the release of adrenocorticotropic (ACTH)
from the anterior pituitary.1,2 CRF moderates various
physiological responses to sustained stress3À7 by binding
to two subtypes of seven-transmembrane G-protein-
coupled CRF receptors, CRF1 and CRF2 receptors,8À11
and has higher anity for CRF1 receptors than CRF2
receptors.10 Clinical evidence, the elevation of CRF
concentration in patients with depression12 or chronic post-
traumatic stress disorder,13 and the blunted corticotropin
response to CRF in patients with depression,14 anxiety,
anorexia nervosa, or post-traumatic stress disorder15
suggest that CRF receptor antagonists may be useful
Structure±anity relationships (SARs) of non-peptide
CRF1 receptor antagonists suggest that such antagonists
can be constructed of three parts, a hydrophobic unit
(Up-Area), a proton accepting unit (Central-Area) and
an aromatic unit (Down-Area), and N,N-dialkylamino
groups containing an alkoxyalkyl group may be typical
Up-Area moieties (Fig. 1).16 Recently, various non-peptide
CRF1 receptor antagonists produced by modi®cation of
the Central-Area have been presented.16À28 Among
them, pyrimidine,19,20,24,26,28,29 purine,22 3H-triazolo[4,5-d]
pyrimidine,22 purin-8-one23 and 7H-pyrrolo[2,3-d]pyr-
imidine17 may be typical Central-Area residues.
We found that pyrimidine derivatives 1b (CRA1001),28
1c (CRA1000)28 and 1d (CRA0165)29 containing a 4- or
5-aryl-1,2,3,6-tetrahydropyridino group in the Up-Area
*Corresponding author. Tel.: +81-48-663-1111; fax: +81-48-652-
7254; e-mail: s10968@ccm.taisho.co.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00005-0

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T. Kumagai et al. / Bioorg. Med. Chem. 9 (2001) 1357±1363
exhibited high anities for CRF1 receptors (IC50=10,
22 and 11 nM, respectively). As part of our ongoing
e€ort to discover novel CRF1 receptor antagonists, we
have continued to change the pyrimidine ring of derivative
1 with other Central-Area moieties, purine, 3H-triazolo
[4,5-d]pyrimidine, purin-8-one and 7H-pyrrolo[2,3-d]
pyrimidine rings. Among the modi®ed derivatives 6±9,
purine derivatives 6 exhibited moderately good or high
anity for CRF1 receptors (typical compound 6j: IC50=
20 nM). Interestingly, 5-aryl-1,2,3,6-tetrahydropyridino
derivatives generally exhibited higher anity for CRF1
receptors than 4-aryl-1,2,3,6-tetrahydropyridino deriva-
tives. In this paper, we report the synthesis and SARs of
derivatives 6±9 (Fig. 1).
Results and Discussion
Compounds 6±9 (Fig. 1) were evaluated for cortico-
tropin-releasing factor1 (CRF1) receptor binding anity
in rat frontal cortex against radioligand [125I]-ovine
CRF,30 and the IC50 obtained values are shown in Table
1. Compound 6j did not exhibit corticotropin-releasing
factor2 (CRF2) receptor binding anity in rat heart
against radioligand [125I]-sauvagine.31
Purine derivative 6a substituted with a 4-phenyl-1,2,3,6-
tetrahydropyridino group exhibited moderate anity
for CRF1 receptors (IC50=385 nM), but less than that
of pyrimidine derivative 1a (IC50=66 nM). The intro-
duction of a halogen atom (F or Cl) on the phenyl
group in the Up-Area slightly increased or did not a€ect
CRF1 receptor anity (6a versus 6b, 6c), similar to
pyrimidine derivative 1 (1a versus 1b). However, replacing
the bromine atom of 6b or 6c with a MeS group on the
phenyl group in Down-Area did not a€ect CRF1 receptor
anity (6b, 6c versus 6d±6g), unlike the case for pyrimidine
derivative 1 (1b versus 1c). 4-(2-Methylphenyl)-1,2,3,6-
tetrahydropyridine compound 8h exhibited no anity
for CRF1 receptors (IC50>1000 nM), and this decrease
in anity was larger than that for pyrimidine derivative
1. These ®ndings suggested that a purine moiety was less
desirable for obtaining CRF1 receptor ligands using a
4-aryl-1,2,3,6-tetrahydropyridino group for the Up-Area
Chemistry
The synthesis of derivatives 6±9 is shown in Scheme 1. A
crude 4- or 5-aryl-1,2,3,6-tetrahydropyridine 12, which
was prepared by treatment of compound 10 with
Grignard reagent (ArMg-Br or -Cl) followed by acid in
the same fashion as previously presented,28,29 was coupled
with known chloride, 6-chloro-2-methylpurines 13,22
7-chloro-5-methyl-3H-1,2,3-triazolo[4,5-d]pyrimidines 1422
6-chloro-2,7-dimethylpurin-8-ones 1523 or 4-chloro-2-
methyl-7H-pyrrolo[2,3-d]pyrimidines 16,17 to a€ord novel
4- or 5-aryl-1,2,3,6-tetrahydropyridine derivatives 6±9.
Figure 1.
Scheme 1. Reagents and conditions: (a) ArMg-Br, THF; (b) TFA±CH2Cl2 or concd HCl; (c) isoPr2NEt, EtOH.

T. Kumagai et al. / Bioorg. Med. Chem. 9 (2001) 1357±1363
Table 1. 4- or 5-Aryl-1,2,3,6-tetrahydropyridine derivatives: binding data
1359
Compound no.
Ar
Y1±Y2
X1
CRF1 receptor IC50 (nM)a
6a
6b
6c
6d
6e
6f
6g
6h
6i
4±Ph
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
NˆC(H)
Br
Br
Br
MeS
MeS
MeS
MeS
MeS
MeS
MeS
390
350
170
110
340
110
150
4-(4-F±Ph)
4-(4-Cl±Ph)
4-(4-F±Ph)
4-(4-Cl±Ph)
4-(3-F±Ph)
4-(3-Cl±Ph)
4-(2-Me±Ph)
5-(4-F±Ph)
5-(2-Me±Ph)
>1000
60
20
6j
7a
7b
7c
4-(4-Cl±Ph)
4-(3-F±Ph)
5-(2-Me±Ph)
NˆN
NˆN
NˆN
Br
MeS
MeS
>1000
154
96
8a
8b
8c
8d
4-(4-Cl±Ph)
4-(3-F±Ph)
5-(4-F±Ph)
5-(2-Me±Ph)
N(Me)±CO
N(Me)±CO
N(Me)±CO
N(Me)±CO
MeS
MeS
MeS
MeS
860
630
860
290
9a
9b
9c
9d
9e
4-(4-Cl±Ph)
4-(3-F±Ph)
5-(2-Me±Ph)
4-(3-F±Ph)
5-(2-Me±Ph)
C(Me)ˆC(Me)
C(Me)ˆC(Me)
C(Me)ˆC(Me)
C(Me)ˆC(H)
C(Me)ˆC(H)
MeS
MeS
MeS
MeS
MeS
>1000
>1000
>1000
>1000
210
1ab
1bb
1cb
1dc
66
22
10
11
aIC50 values from duplicate determination.
bRef 28.
cRef 29.
than a pyrimidine moiety. Notably, however, introduc-
tion of a 5-aryl-1,2,3,6-tetrahydropyridino group onto
the purine ring enhanced CRF1 receptor anity (6i:
IC50=60 nM). 5-(2-Methylphenyl)-1,2,3,6-tetrahydro-
pyridinopurine 6j exhibited high anity for CRF1
receptors (IC50=20 nM). This notable enhancement of
CRF1 receptor anity by replacement of the 4-phenyl
group with 5-(2-methylphenyl) group (6j/6a: 19-fold, 6j/
6f: 5.6-fold) was larger than in the case of pyrimidine
derivative 1 (1d/1a: 6-fold, 1d/1c: 0.9-fold). This di€er-
ence in SARs between aryl-1,2,3,6-tetrahydropyridino-
pyrimidine and aryl-1,2,3,6-tetrahydropyridinopurine
derivatives suggests that the molecular structure of
CRF1 receptor might have a narrow and non-¯exible
space for the phenyl group of derivatives 1 and 6 and
that this space might require steric control of the phenyl
group in the phenyl-1,2,3,6-tetrahydropyridine deriva-
tive to yield high anity for CRF1 receptors.
highest anity for CRF1 receptors in the derivatives 6±9,
respectively. These ®ndings suggest that the substituent
on the phenyl group (Ar) might stereochemically control
the positioning of the phenyl group (Ar) in aryl-1,2,3,6-
tetrahydropyridine derivatives 6±9, and that this stereo-
chemistry might be important for interaction with CRF1
receptors. Furthermore, the decreased CRF1 receptor
anity noted for derivatives 7, 8 and 9 suggests that the
CRF1 receptor anity of the aryl-1,2,3,6-tetrahydro-
pyridine derivative might be more a€ected by the Central-
Area moiety than that of N,N-dialkylamine derivatives16
such as compounds 2±5 (Fig. 1).
Conclusions
The structural similarity of the known CRF1 receptor
antagonists appears to include two alkyl groups of N,N-di-
alkylamino or N-(secondary-alkyl)amino moiety in the Up-
Area.16 In contrast, 4- or 5-aryl-1,2,3,6-terahyropyridine
CRF1 receptor antagonists have unique structures without
two alkyl groups. This present study suggests various pos-
sibilities and limitations of the use of 4- or 5-aryl-1,2,3,6-
terahyropyridino moiety for discovery of CRF1 receptor
antagonists. However, it appears that Up-Area might be
a useful target as the position of chemical modi®cation
and that chemical modi®cation of the Up-Area may be
useful for discovery of CRF1 receptor antagonists.
3H-1,2,3-triazolo[4,5-d]pyrimidine derivative 7, purin-8-
one derivative 8 and 7H-pyrrolo[2,3-d]pyrimidine deriva-
tive 9 all exhibited lower anities for CRF1 receptors
than purine derivative 6 (6c, 6f, 6j versus 7a±7c; 6e, 6f,
6i, 6j versus 8a±8d; 6e, 6f, 6j versus 9a±9e). In particular,
marked decline in CRF1 receptor anity was noted
with derivative 9. Notably, however, 5-(2-methylphenyl)
derivatives 6j (IC50=20 nM), 7c (IC50=96 nM), 8d
(IC50=287 nM) and 9e (IC50=210 nM) exhibited the

1360
T. Kumagai et al. / Bioorg. Med. Chem. 9 (2001) 1357±1363
Furthermore, 5-aryl-1,2,3,6-tetrahydropyridinopurine
compound 6j (CRA0186), a selective and potent CRF1
receptor antagonist, might be useful not only for
exploring the functions of CRF1 receptor but also in the
treatment of central nervous system disorders such as
depression and/or anxiety-related disorders.
of the reaction mixture under reduced pressure, the
resulting residue was partitioned between CHCl3 and
saturated aqueous NaHCO3, and the separated water
phase was extracted with CHCl3 twice. The combined
organic phase was washed with saturated brine, dried
(Na2SO4), and concentrated under reduced pressure.
Chromatography of the residue on silica gel using a
mixture of AcOEt and hexanes (1:8) followed by recrys-
tallization from AcOEt/hexane a€orded 243 mg (86%) of
6j as a yellow crystal: mp 150±151 ꢀC (AcOEt/hexane); 1H
NMR (CDCl3) d 1.31 (6H, d, J=7.0 Hz), 2.34 (3H, s),
2.38(3H, s), 2.43±2.56 (2H, m), 2.52 (3H, s), 2.99 (1H,
sept, J=7.0Hz), 4.50±4.66 (2H, m), 4.75±4.90 (2H, m),
5.74±5.82 (1H, m), 7.16±7.32 (7H, m), 7.79 (1H, s); MS
(Ion Spray) m/z 470 (M++H); IR (KBr) 2958, 2923,
2836, 1580, 1513cmÀ1. Anal. (C28H31N5S) C, H, N, S.
Experimental
Chemistry
Melting points were determined on a Yanaco MP-500D
melting point apparatus and are uncorrected. Infra-red
(IR) spectra were obtained on a Perkin-Elmer 1760 spectro-
meter. Proton nuclear magnetic resonance (NMR) spectra
were obtained using a Varian VXR-200 spectrometer.
Chemical shifts are reported in parts per million relatives
to tetramethylsilane as an internal standard. Mass
spectra (MS) were obtained on a Shimazu/Kratos HV-
300. Elemental analyses were performed by a Perkin-
Elmer 240C (for carbon, hydrogen and nitrogen) or
Yokokawa-Denki IC7000P (for halogen and sulfur).
Analytical thin-layer chromatography was conducted on
precoated silica gel 60 F254 plates (Merck). Chromato-
graphy was performed on silica gel C-200, 100±200 mesh
(Wako Pure Chemical), using the solvent systems
(volume ratios) indicated below.
Using a corresponding procedure, the following com-
pounds 6a±6i, 7a±7c, 8a±8d and 9a±9e were prepared.
2-Methyl-6-(4-phenyl-1,2,3,6-tetrahydropyridino)-9-(2-
1
bromo-4-isopropylphenyl)purine 6a. Amorphous; H NMR
(CDCl3) d 1.30 (6H, d, J=6.8Hz), 2.56 (3H, s), 2.70±
2.83 (2H, m), 2.98 (1H, sept, J=6.8Hz), 4.50±4.68(2H,
m), 4.83±4.98 (2H, m), 6.19±6.28 (1H, m), 7.20±7.49 (7H,
m), 7.61 (1H, d, J=2.0 Hz), 7.83 (1H, s); MS (FAB) m/z
490 (M++2+H), 488 (M++H); IR (KBr) 3435, 2961,
1581 cmÀ1. Anal. (C26H26BrN5 H2O) C, H, N, Br.
ꢁ
N-tert-Butoxycarbonyl-3-hydroxy-3-(2-methylphenyl)piper-
idine 11a. To a solution of 2-methylphenyl magnesium
bromide in tetrahydrofuran (THF) prepared from 2-bro-
motoluene (18.03 g, 105 mmol) and magnesium
(2.35 g, 96.7 mmol) in THF (120 mL) was added a solu-
tion of N-tert-butoxycarbonyl-3-piperidone (17.50 g,
87.8 mmol) in THF (60mL) with ice cooling. After stirring
for 1.5 h with ice cooling, saturated aqueous NH4Cl was
added to the reaction mixture and extracttion was per-
formed with AcOEt three times. The combined extract
was washed with saturated brine, dried (Na2SO4), and
concentrated under reduced pressure. Chromatography
of the residue on silica gel using a mixture of AcOEt and
hexanes (1:5) a€orded 9.68g (38%) of N-tert-butoxy-
carbonyl-3-hydroxy-3-(2-methylphenyl)piperidine 11a as
2-Methyl-6-(4-(4-¯uorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-bromo-4-isopropylphenyl)purine 6b. Mp 166±167 ꢀC
(CHCl3-hexane); 1H NMR (CDCl3) d 1.31 (6H, d,
J=7.0 Hz), 2.56 (3H, s), 2.66±2.78(2H, m), 2.98(1H,
sept, J=7.0 Hz), 4.51±4.68(2H, m), 4.83±4.98(2H, m),
6.13±6.23 (1H, m), 7.00±7.09 (2H, m), 7.32±7.44 (4H,
m), 7.62 (1H, d, J=2.0 Hz), 7.83 (1H, s); MS (ion
spray) m/z 470 (M++H); IR (KBr) 2964, 2920, 1601,
1574 cmÀ1. Anal. (C26H25BrFN5) C, H, N, Br, F.
2-Methyl-6-(4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6c. Mp 159±
ꢀ
1
160 C (CHCl3/hexane); H NMR (CDCl3) d 1.30 (6H,
d, J=6.8Hz), 2.56 (3H, s), 2.62±2.78(2H, m), 2.98(1H,
sept, J=6.8Hz), 4.50±4.63 (2H, m), 4.82±4.96 (2H, m),
6.17±6.27 (1H, m), 7.29±7.43 (6H, m), 7.61 (1H, d, J=
1.8Hz), 7.83 (1H, s); MS (APCI) m/z 526 (M++4+H),
524 (M++2+H), 522 (M++H); IR (KBr) 2962, 2924,
1572, 1516 cmÀ1. Anal. (C26H25BrClN5) C, H, N, Br, Cl.
1
a light yellow oil: H NMR (CDCl3) d 1.49 (9 H, s),
1.54±1.75 (1H, m), 1.88±2.28 (4H, m), 2.66 (3H, s),
2.75±2.98(1H, m), 3.20 (1H, d, J=13.7 Hz), 4.00±4.22
(1H, m), 4.27 (1H, d, J=13.7 Hz), 7.13±7.43 (5H, m,
ArH); MS (ESI, Pos.) m/z 314 (M++Na).
2-Methyl-6-(4-(4-¯uorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6d. Mp 162±
163 ꢀC (AcOEt); 1H NMR (CDCl3) d 1.31 (6H, d,
J=7.0 Hz), 2.35 (3H, s), 2.55 (3H, s), 2.65±2.77 (2H, m),
2.99 (1H, sept, J=7.0 Hz), 4.50±4.67 (2H, m), 4.83±4.98
(2H, m), 6.12±6.23 (1H, m), 6.98±7.10 (2H, m), 7.16±
7.45 (5H, m), 7.81 (1H, s); MS (ion spray) m/z 474
2-Methyl-6-(5-(2-methylphenyl)-1,2,3,6-tetrahydropyri-
dino)-9-(2-methylthio-4-isopropylphenyl)purine 6j. To a
solution of 11a (590 mg, 2.02 mmol) in dioxane
(0.84 mL) was added concentrated HCl (8.4 mL), and
the resulting mixture was stirred at room temperature
for 30 min and then heated at re¯ux for 3 h. The mixture
was concentrated and the resulting solid was dried
under reduced pressure.
(M++H); IR (KBr) 2963, 2918, 2866, 1578, 1509 cmÀ1
Anal. (C27H28FN5S) C, H, N, S, F.
.
A mixture of the above solid, 2-methyl-6-chloro-9-(2-
2-Methyl-6-(4-(4-chlorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6e. Mp 165±
166 ꢀC (AcOEt); 1H NMR (CDCl3) d 1.31 (6H, d,
J=6.8Hz), 2.35 (3H, s), 2.55 (3H, s), 2.64±2.77 (2H, m),
methylthio-4-isopropylphenyl)purine
0.60 mmol) and diisopropylethylamine
13a
(200 mg,
(5.0 mL,
28.7 mmol) was stirred at re¯ux for 1 h. After concentration

T. Kumagai et al. / Bioorg. Med. Chem. 9 (2001) 1357±1363
1361
2.99 (1H, sept, J=6.8Hz), 4.50±4.67 (2H, m), 4.84±4.97
(2H, m), 6.18±6.26 (1H, m), 7.19 (1H, dd, J=1.9, 8.0 Hz),
7.27±7.39 (6H, m), 7.81 (1H, s); MS (ion spray) m/z 492
(M++2+H), 490 (M++H); IR (KBr) 2953, 2917,
1578cmÀ1. Anal. (C27H28ClN5Sꢁ 0.2H2O) C, H, N, S, Cl.
J=7.0 Hz), 4.29±4.81 (2H, m), 4.82±5.42 (2H, m), 6.21±
6.34 (1H, m), 6.93±7.40 (7H, m); MS (FAB) m/z 475
(M++H); IR (KBr) 2962, 2928, 1594, 1564 cmÀ1. Anal.
(C26H27FN6Sꢁ 0.2H2O) C, H, N, S, F.
5-Methyl-7-(5-(2-methylphenyl)-1,2,3,6-tetrahydropyridino)-
3(-2-methylthio-4-isopropylphenyl)-3H-1,2,3-triazolo[4,5-d]
2-Methyl-6-(4-(3-¯uorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6f. Mp 168±
169 ꢀC (AcOEt); 1H NMR (CDCl3) d 1.31 (6H, d,
J=6.8Hz), 2.35 (3H, s), 2.55 (3H, s), 2.65±2.78(2H, m),
2.99 (1H, sept, J=6.8Hz), 4.50±4.67 (2H, m), 4.85±4.99
(2H, m), 6.24±6.33 (1H, m), 6.90±7.02 (1H, m), 7.08±7.37
(6H, m), 7.82 (1H, s); MS (ion spray) m/z 474 (M++H); IR
(KBr) 2960, 2918, 2865, 1578 cmÀ1. Anal. (C27H28FN5S)
C, H, N, S, F.
pyrimidine 7c. Mp 81±83 ꢀC (AcOEt/hexane); H NMR
1
(CDCl3) d 1.32 (6H, d, J=6.8Hz), 2.38 (3H, s), 2.40
(3H, s), 2.44±2.68(5H, m), 3.01 (1H, sept, J=6.8Hz),
4.20±4.73 (2H, m), 4.80±5.33 (2H, m), 5.78±5.89 (1H,
m), 7.16±7.28(5H, m), 7.32±7.40 (2H, m); MS (FAB) m/
z 471 (M++H); IR (KBr) 2956, 2925, 1594, 1563 cmÀ1
Anal. (C27H30N6Sꢁ 0.2H2O) C, H, N, S.
.
2,7-Dimethyl-6-(4-(4-chlorophenyl)-1,2,3,6-tetrahydro-
pyridino)-9-(2-methylthio-4-isopropylphenyl)purin-8-one
2-Methyl-6-(4-(3-chlorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6g. Mp 155±
156 ꢀC (AcOEt); 1H NMR (CDCl3) d 1.31 (6H, d,
J=6.8Hz), 2.35 (3H, s), 2.55 (3H, s), 2.65±2.78(2H, m),
2.99 (1H, sept, J=6.8Hz), 4.50±4.68(2H, m), 4.73±4.98
(2H, m), 6.21±6.30 (1H, m), 7.16±7.32 (6H, m), 7.42
(1H, dd, J=1.4, 2.7 Hz), 7.82 (1H, s); MS (ion spray) m/z
492 (M++2+H), 490 (M++H); IR (KBr) 2959,
1578cm À1. Anal. (C27H28ClN5Sꢁ 0.2H2O) C, H, N, S, Cl.
8a. Mp 161±163 ꢀC (AcOEt/hexane); H NMR (CDCl3)
1
d 1.30 (6H, d, J=6.8Hz), 2.42 (3H, s), 2.51 (3H, s),
2.67±2.80 (2H, m), 2.97 (1H, sept, J=6.8Hz), 3.50±3.74
(2H, m), 3.67 (3H, s), 4.04±4.13 (2H, m), 6.20±6.28(1H,
m), 7.15±7.41 (7H, m); MS (ion spray) m/z 522
(M++2+H), 520 (M++H); IR (KBr) 2961, 2923,
1731, 1599 cmÀ1. Anal. (C28H30ClN5OS) C, H, N, S, Cl.
2,7-Dimethyl-6-(4-(3-¯uorophenyl)-1,2,3,6-tetrahydropyri-
dino)-9-(2-methylthio-4-isopropylphenyl)purin-8-one 8b.
2-Methyl-6-(4-(2-methylphenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6h. Mp 182±
183 ꢀC (AcOEt); 1H NMR (CDCl3) d 1.31 (6H, d,
J=6.8Hz), 2.33 (3H, s), 2.36 (3H, s), 2.50±2.64 (2H, m),
2.56 (3H, s), 2.99 (1H, sept, J=6.8Hz), 4.50±4.66 (2H,
m), 4.80±4.93 (2H, m), 5.72±5.77 (1H, m), 7.10±7.24
(5H, m), 7.26±7.33 (2H, m), 7.81 (1H, s); MS (ion spray)
Mp 156±157 ꢀC (AcOEt/hexane); H NMR (CDCl3) d
1
1.30 (6H, d, J=6.8Hz), 2.42 (3H, s), 2.51 (3H, s), 2.70±
2.81 (2H, m), 2.98 (1H, sept, J=6.8Hz), 3.50±3.77 (2H,
m), 3.66 (3H, s), 4.06±4.14 (2H, m), 6.23±6.33 (1H, m),
6.92±7.04 (1H, m), 7.12±7.38(6H, m); MS (ion spray) m/
z 504 (M++H); IR (KBr) 2957, 2925, 1727, 1603 cmÀ1
.
m/z 470 (M++H); IR (KBr) 2959, 1576, 1515 cmÀ1
.
Anal. (C28H30FN5OS) C, H, N, S, F.
Anal. (C28H31N5S) C, H, N, S.
2,7-Dimethyl-6-(5-(3-¯uorophenyl)-1,2,3,6-tetrahydropyr-
idino)-9-(2-methylthio-4-isopropylphenyl)purin-8-one 8c.
2-Methyl-6-(5-(4-¯uorophenyl)-1,2,3,6-tetrahydropyridino)-
9-(2-methylthio-4-isopropylphenyl)purine 6i. Mp 135±
Mp 131±132 ꢀC (AcOEt/hexane); H NMR (CDCl3) d
1
ꢀ
1
136 C (AcOEt/hexane); H NMR (CDCl3) d 1.31 (6H,
d, J=7.0 Hz), 2.35 (3H, s), 2.45±2.57 (2H, m), 2.55 (3H,
s), 2.99 (1H, sept, J=7.0 Hz), 4.43±4.58(2H, m), 5.04±
5.19 (2H, m), 6.16±6.27 (1H, m), 7.01±7.13 (2H, m), 7.19
(1H, dd, J=1.8, 8.1 Hz), 7.28 (1H, d, J=1.8Hz), 7.30
(1H, d, J=8.1Hz), 7.43±7.55 (2H, m), 7.81 (1H, s); MS
(ion spray) m/z 474 (M++H); IR (KBr) 2960, 2922, 2837,
1579cmÀ1. Anal. (C27H28FN5Sꢁ 0.5H2O) C, H, N, S, F.
1.30 (6H, d, J=6.8Hz), 2.42 (3H, s), 2.47±2.61 (2H, m),
2.51 (3H, s), 2.98(1H, sept, J=6.8Hz), 3.43±3.62 (2H,
m), 3.64 (3H, s), 4.16±4.23 (2H, m), 6.13±6.24 (1H, m),
6.99±7.12 (2H, m), 7.15±7.31 (3H, m), 7.35±7.43 (2H,
m); MS (ion spray) m/z 504 (M++H); IR (KBr) 2961,
2923,
1727,
1600,
(C28H30FN5OS) C, H, N, S, F.
1510,
1500 cmÀ1
.
Anal.
2,7-Dimethyl-6-(5-(2-methylphenyl)-1,2,3,6-tetrahydro-
pyridino)-9-(2-methylthio-4-isopropylphenyl)purin-8-one
8d. Mp 164±165 ꢀC (AcOEt/hexane); 1H NMR
(CDCl3) d 1.30 (6H, d, J=6.8Hz), 2.35 (3H, s), 2.41
(3H, s), 2.44±2.56 (2H, m), 2.48(3H, s), 2.97 (1H, sept,
J=6.8Hz), 3.50±3.63 (2H, m), 3.65 (3H, s), 4.00±4.06
(2H, m), 5.70±5.79 (1H, m), 7.14±7.33 (7H, m); MS (ion
spray) m/z 500 (M++H); IR (KBr) 2962, 2925, 2886,
1727, 1601 cmÀ1. Anal. (C29H33N5OS) C, H, N, S.
3-(2-Bromo-4-isopropylphenyl)-7-(4-(4-chlorophenyl)-
1,2,3,6-tetrahydropyridino)-5-methyl-3H-1,2,3-triazolo-
[4,5-d]pyrimidine 7a. Mp 162±163 ꢀC (CHCl3/hexane);
1H NMR (CDCl3) d 1.31 (6H, d, J=6.8Hz), 2.58
(3H, s), 2.63±2.88 (2H, m), 3.00 (1H, sept, J=6.8Hz),
4.30±4.79 (2H, m), 4.87±5.36 (2H, m), 6.18±6.28 (1H, m),
7.31±7.45 (6H, m), 7.65 (1H, d, J=1.8Hz); MS (FAB) m/z
527 (M++4+H), 525 (M++2+H), 523 (M++H); IR
(KBr) 3436, 2963, 2916, 1610, 1593, 1570cmÀ1. Anal.
(C25H24BrClN6) C, H, N, Br, Cl.
4-(4-(4-Chlorophenyl)-1,2,3,6-tetrahydropyridino)-7-(2-
methylthio-4-isopropylphenyl)-2,5,6-trimethyl-7H-pyrrolo
[2,3-d]pyrimidine 9a. Mp 173±174 ꢀC (AcOEt/hexane);
1H NMR (CDCl3) d 1.33 (6H, d, J=7.0 Hz), 2.05 (3H,
s), 2.32 (3H, s), 2.42 (3H, s), 2.52 (3H, s), 2.70±2.83 (2H,
m), 3.00 (1H, sept, J=7.0 Hz), 3.64±3.93 (2H, m), 4.17±
4.26 (2H, m), 6.19±6.28(1H, m), 7.0±87.22 (3H, m),
7 - (4 - (3 - Fluorophenyl) - 1,2,3,6 - tetrahydropyridino) - 5 -
methyl-3(-2-methylthio-4-isopropylphenyl)-3H-1,2,3-tria-
zolo[4,5-d]pyrimidine 7b. Mp 115±116 ꢀC (AcOEt/hex-
1
ane); H NMR (CDCl3) d 1.32 (6H, d, J=7.0 Hz), 2.40
(3H, s), 2.59 (3H, s), 2.66Ð2.89 (2H, m), 3.01 (1H, sept,

1362
T. Kumagai et al. / Bioorg. Med. Chem. 9 (2001) 1357±1363
7.29±7.43 (4H, m); MS (FAB) m/z 519 (M++2+H), 517
with slight modi®cations. The reaction was initiated by
incubating 0.5 mL of membrane preparation with
0.2 nM [125I]-ovine CRF or 0.2 nM [125I]-sauvagine. The
reaction mixture was incubated for 2 h at 25 ꢀC (for
(M++H); IR (KBr) 2945, 2920, 1582, 1557, 1546 cmÀ1
Anal. (C30H33ClN4S) C, H, N, S, Cl.
.
4-(4-(3-Fluorophenyl)-1,2,3,6-tetrahydropyridino)-7-(2-
methylthio-4-isopropylphenyl)-2,5,6-trimethyl-7H-pyrrolo
[
125I]-ovine CRF binding) or at 23 ꢀC (for [125I]-sauva-
gine binding), and reaction was terminated by rapid ®l-
tration through Whatman GF/C glass ®ber ®lters
presoaked with 0.3% polyethyleneimine, after which the
®lters were washed three times with 3 mL of phosphate-
bu€ered saline (PBS) containing 0.01% Triton X-100.
Radioactivity was quanti®ed in a gamma-counter.
Nonspeci®c binding was determined in the presence of
unlabeled 1 mM ovine CRF (for [125I]-ovine CRF binding)
or 1 mM sauvagine (for [125I]-sauvagine binding). Speci®c
binding was determined by subtracting nonspeci®c
binding from total binding. In the competition-binding
assay, the concentration of the test compound that
caused 50% inhibition of speci®c radiolabeled ligand
binding (IC50 values) was determined from each con-
centration±response curve.
1
[2,3-d]pyrimidine 9b. Amorphous; H NMR (CDCl3) d
1.32 (6H, d, J=6.8Hz), 2.05 (3H, s), 2.32 (3H, s), 2.42
(3H, s), 2.53 (3H, s), 2.70±2.85 (2H, m), 2.99 (1H, sept,
J=6.8Hz), 3.63±3.93 (2H, m), 4.16±4.28(2H, m), 6.24±
6.34 (1H, m), 6.89±7.40 (7H, m); MS (FAB) m/z 501
(M++H); IR (KBr) 2960, 2921, 1610, 1583, 1547 cmÀ1
Anal. (C30H33FN4Sꢁ 0.2H2Oꢁ 0.2CHCl3) C, H, N, S, F.
.
4-(5-(2-Methylphenyl)-1,2,3,6-tetrahydropyridino)-7-(2-
methylthio-4-isopropylphenyl)-2,5,6-trimethyl-7H-pyrrolo
[2,3-d]pyrimidine 9c. Mp 133±134 ꢀC (AcOEt/hexane);
1H NMR (CDCl3) d 1.32 (6H, d, J=6.8Hz), 2.04 (3H,
s), 2.31 (3H, s), 2.35 (3H, s), 2.40 (3H, s), 2.45±2.59 (2H,
m), 2.49 (3H, s), 2.99 (1H, sept, J=6.8Hz), 3.62±3.83
(2H, m), 4.11±4.20 (2H, m), 5.70±5.79 (1H, m), 7.08±
7.25 (7H, m); MS (FAB) m/z 497 (M++H); IR (KBr)
2961, 2922, 2867, 2831, 1593, 1585, 1556 cmÀ1. Anal.
(C31H36N4S) C, H, N, S.
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Rats were decapitated and the frontal cortex and heart
were rapidly dissected. The frontal cortex or the heart
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