Discovery of pyrrolo[2,3-d]pyrimidin-4-ones as corticotropin-releasing factor 1 receptor antagonists with a carbonyl-based hydrogen bonding acceptor

Article abstract of DOI:10.1016/j.bmcl.2011.02.086

A new class of pyrrolo[2,3-d]pyrimidin-4-one corticotropin-releasing factor 1 (CRF₁) receptor antagonists has been designed and synthesized. In general, reported CRF₁ receptor antagonists possess a sp ²-nitrogen atom as hy

Full text of DOI:10.1016/j.bmcl.2011.02.086

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2365–2371
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Discovery of pyrrolo[2,3-d]pyrimidin-4-ones as corticotropin-releasing factor 1
receptor antagonists with a carbonyl-based hydrogen bonding acceptor
Kazuyoshi Aso a, , Katsumi Kobayashi a, Michiyo Mochizuki a, Naoyuki Kanzaki b, Yuu Sako c, Takahiko Yano c
⇑
a Medicinal Chemistry Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
b Discovery Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
c Pharmaceutical Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of pyrrolo[2,3-d]pyrimidin-4-one corticotropin-releasing factor 1 (CRF1) receptor antagonists
has been designed and synthesized. In general, reported CRF1 receptor antagonists possess a sp2-nitrogen
atom as hydrogen bonding acceptor (HBA) on their core scaffolds. We proposed to use a carbonyl group of
pyrrolo[2,3-d]pyrimidin-4-one derivatives as a replacement for the sp2-nitrogen atom as HBA in classical
CRF1 receptor antagonists. As a result, several pyrrolo[2,3-d]pyrimidin-4-one derivatives showed CRF1
receptor binding affinity with IC50 values in the submicromolar range. Ex vivo 125I-sauvagine binding
studies showed that 2-(dipropylamino)-3,7-dimethyl-5-(2,4,6-trimethylphenyl)-3,7-dihydro-4H-pyrrol-
o[2,3-d]pyrimidin-4-one (16b) (30 mg/kg, po) was able to penetrate into the brain and inhibit radioligand
binding to CRF1 receptors (frontal cortex, olfactory bulb, and pituitary) in mice. We identified pyrrolo[2,3-
d]pyrimidin-4-one derivatives as the first CRF1 antagonists with a carbonyl-based HBA.
Received 28 January 2011
Revised 18 February 2011
Accepted 21 February 2011
Available online 26 February 2011
Keywords:
Corticotropin-releasing factor
CRF1 antagonist
Pyrrolo[2,3-d]pyrimidin-4-one
Hydrogen bonding acceptor
Ó 2011 Elsevier Ltd. All rights reserved.
Corticotropin-releasing factor (CRF), a 41-amino acid neuro-
peptide secreted in the hypothalamus, was isolated from ovine
brain and characterized in 1981.1 Over the past 30 years, CRF
has been thought to function as the prime regulator of the hypo-
thalamic-pituitary-adrenocortical (HPA) axis and to play an
important role as a neurotransmitter in the mediation of stress-re-
lated behaviors.2–5 The CRF receptor, a class B G-protein-coupled
receptor, has two subtypes (CRF1 and CRF2).6,7 CRF1 receptor-
knockout mice showed anxiolytic-like behaviors in various tests
and blunted HPA axis responses to stress.8 These findings indi-
cated that CRF1 receptor antagonists would be an important target
for developing novel drugs for stress-related disorders such as
depression and anxiety.
The clinical efficacy of CRF1 receptor antagonists against
depression and anxiety is still unclear, but data from few clinical
studies show that these antagonists tend to be efficacious. In a
small open-label phase IIa clinical trial, R121919 (1) effectively re-
duced the Hamilton depression and anxiety scale (HAM-D and
HAM-A) scores in patients with major depression.9 Subchronic
treatment with NBI-34041 (2) attenuated the neuroendocrine re-
sponse to psychosocial stress in a placebo-controlled clinical
study.10 These data suggested that non-peptide CRF1 receptor
antagonists could be useful therapeutic agents in the treatment
of depression and anxiety. On the other hand, in a double-blind,
placebo-controlled clinical study of CP-316,311 (3), the change in
the HAM-D score from baseline to the final visit was not signifi-
cantly different between the CP-316,311 and placebo groups.11
Pexacerfont (4)12,13 and emicerfont (5)14 also failed in clinical trials
for major depression, general anxiety disorder, and irritable bowel
syndrome. However, GSK561679 (6)15 and SSR125543 (7)16 are
still being tested for the potential treatment of post-traumatic
stress disorder (PTSD) in phase II by the Emory university group17
and for depression and PTSD in phase II by Sanofi-Aventis,18
respectively. In addition, CRF1 receptor antagonists are still re-
garded as a relevant target for diseases resulting from elevated lev-
els of CRF, for example, alcohol abuse, pain, and stress-related
disorders.19,20 Identification of structurally diverse CRF1 receptor
antagonists has been an active area of drug discovery research.
A variety of small molecule CRF1 receptor antagonists have
been reported in the literature and patents21 since the disclosure
of CP-154,526 (8) in 1996.22 Most of the reported compounds such
as R121919 (1), pexacerfont (4), emicerfont (5), GSK-561679 (6),
and CP-154,526 (8) have a bicyclic heterocyclic core. Moreover,
potent CRF1 receptor antagonists with a monocyclic core, for
example, CP-316,311 (3) and SSR125543 (7), and those with a tri-
cyclic core, for example, NBI-34041 (2), have been reported. All of
them possess a sp2-nitrogen atom as a hydrogen bonding acceptor
(HBA) on the core scaffold (Fig. 1).
In our investigation of a new class of CRF1 receptor antagonists,
we proposed to use a carbonyl group as a replacement for a
sp2-nitrogen atom as HBA on the reported CRF1 receptor antago-
nists. We anticipated that the unique compounds with a car-
bonyl-based HBA would not only exhibit potent affinity to CRF1
⇑
Corresponding author. Tel.: +81 6 6300 6636; fax: +81 6 6300 6306.
E-mail address: Aso_Kazuyoshi@takeda.co.jp (K. Aso).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.086

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K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
Figure 1. Reported CRF1 antagonists.
Figure 2. (a) Pharmacophore of the reported CRF1 antagonists, (b) design of the carbonyl HBA antagonists and (c) pyrrolo[2,3-d]pyrimidin-4-one with the carbonyl HBA.
receptors due to their electrostatic interaction but also show prop-
erties different from those of the reported antagonists. In this arti-
cle, we describe our successful efforts in discovering a new class of
pyrrolo[2,3-d]pyrimidin-4-one CRF1 receptor antagonists contain-
ing a carbonyl-based HBA, their structure–activity relationships
(SAR), and biological profile.
In an effort to design a new class of CRF1 antagonists, analysis of
a general pharmacophore model constructed from the reported
antagonists was very useful (Fig. 2(a)). The pharmacophore con-
tains common structural features, which include (1) a monocyclic,
a bicyclic, or a tricyclic core with a sp2-nitrogen as HBA; (2) a large
lipophilic group (Ra) attached to the core at the opposite position to
the sp2-nitrogen; (3) a pendant aryl ring minimally substituted in
the ortho and the para positions orthogonal to the core ring; and
(4) a small alkyl group (Rb) at the left side of the sp2-nitrogen.
Whereas SARs have been explored with various core structures,
the sp2-nitrogen as the HBA has never been given attention. We
were interested in the replacement of the sp2-nitrogen atom with
a carbonyl group with negative charge as HBA (Fig. 2(b)). Carbonyl
oxygen with two lone-pairs can provide wider angle for hydrogen-
bonding than aromatic sp2-nitrogen with one-lone pair. Consider-
ing the HBA in this case is affected by the intramolecular steric
Figure 3. Superimposition of CP-154,526 8 (orange) and 16b (white).

K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
2367
Table 1
hCRF1 binding affinities of pyrrolo[2,3-d]pyrimidin-4-one derivatives
R1
N
Me
N
N
O
R2
R3
R4
N
Me
R6
R5
a
Compound
R1
R2
R3
R4
R5
R6
hCRF1 IC50
7.7 (3.7–16.5)
(
lM)
Log Pb
16a
16b
16c
Et
nPr
nBu
Et
nPr
nBu
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
3.45
4.52
5.58
0.12 (0.061–0.23)
0.97 (0.49–1.9)
16d
16e
H
H
Me
Me
Me
Me
Me
Me
0.14 (0.091–0.21)
3.8 (1.8–7.8)
4.19
3.16
21a
21b
29a
29b
29c
29d
CP-316,311 (3)
CP-154,526 (8)
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
nPr
Me
Me
H
H
H
Me
H
H
OMe
OMe
CF3
Me
Me
Me
OMe
OMe
CF3
Me
Me
Me
OMe
H
1.1 (0.67–1.9)
3.3 (0.97–11)
1.6 (1.1–2.2)
3.1 (2.5 -3.8)
3.0 (0.97–9.0)
3.0 (1.8- 4.9)
4.98
4.52
4.05
1.92
2.50
5.36
6.20
6.63
H
H
0.092 (0.079–0.11)
0.011 (0.0059–0.020)
a
IC50 values represent 95% confidence intervals.
Calculated by ACD Physchem Batch (Ver.10).
b
Scheme 1. Reagents: (a) MeNH2, AcOH, 80%; (b) PySO3, Et3N, DMSO, 99%; (c) TMS-Br, DMSO/MeCN; (d) K2CO3, DMSO, 68% in two steps; (e) MeI, NaH, DMF, 62%; (f)
alkylhalide, NaH, DMF, 8–87%.
hindrance from the vicinity aromatic ring, the wide angle of HBA
capability of the carbonyl-based HBA can be the advantage to form
a hydrogen bond feasibly with a hydrogen bond donor of the
receptor. On the basis of these considerations, we thought that a
pyrrolo[2,3-d]pyrimidin-4-one scaffold23,24 could be an appropri-
ate core structure with a carbonyl-based HBA. In addition, each
key feature could be flexibly installed in this scaffold by using
the pharmacophore model (Fig. 2(c)).
Alignment of the carbonyl group in the designed pyrrolo[2,3-
d]pyrimidin-4-one derivative 16b and the sp2-nitrogen atom in
CP-154,526 (8) resulted in good overlap of other moieties
(Fig. 3). In the designed pyrrolo[2,3-d]pyrimidin-4-one derivatives,

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K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
alkyl substituents (R1 and R2) on the 2-amino group would be
important to occupy the large lipophilic pocket on the top region.
The steric repulsion between the ortho substituent on the pendant
aromatic ring and the core ring would also play a significant role to
generate the orthogonal conformation. On the other hand, the sub-
stituent at the 6-position (R3) of the core ring might inhibit binding
to the receptor due to its exterior location. From a different point of
view, several of the reported antagonists, such as 2, 3, 7, and 8,
show high lipophilicity (Log P calculated by ACD Physchem Batch
(Ver.10): 2, 5.75; 3, 6.20; 7, 7.75; 8, 6.63). However, the pyrrol-
o[2,3-d]pyrimidin-4-one scaffold offers the potential to provide
derivatives with Log P values closer to the range of 2–4 which is
preferred for access to the central nervous system (Table 1).25,26
Therefore, we prepared a series of pyrrolo[2,3-d]pyrimidin-4-ones,
which were investigated for their ability to act as CRF1 receptor
antagonists with a carbonyl-based HBA.
an arylboronic acid afforded the desired pyrrolo[2,3-d]pyrimidin-
4-ones 21a and 21b.
Preparation of the unsubstituted derivatives 29a–d at the 6-posi-
tion also required 5-halogenated pyrrolo[2,3-d]pyrimidin-4-one
derivative 28 as a key intermediate (Scheme 3). We prepared the
5-iodopyrrolo[2,3-d]pyrimidin-4-one derivative 26 by using a mod-
ified method of Taylor et al.28 After protection of the 2-amino group
of pyrrolo[2,3-d]pyrimidin-4-one derivative 22, treatment of com-
pound 23 with excess amounts of N-iodosuccinimide (NIS) yielded
the 5,6-diiodo derivative 24. Regioselective deiodination of com-
pound 24 was successfully conducted by treatment with zinc in
acetic acid to afford the 5-monoiodo derivative 25. After removing
the protecting group at the 2-amino group, the target compounds
29a–d were prepared by the method described in Scheme 2.
In order to confirm the function of the carbonyl group as the
HBA, we prepared 4-chloro (30), 4-hydroxy (31), and 4-methoxy
(35) derivatives. Chlorination of pyrrolo[2,3-d]pyrimidin-4-one
derivative 14 with phosphorus oxychloride, followed by alkylation
of 2-amino group provided the 4-chloropyrrolo[2,3-d]pyrimidine
derivative 30. Compound 30 was converted into 4-hydroxy deriv-
ative 31 by treatment with an alkaline solution. We determined
that compound 31 was in the hydroxyl pyrimidine and not in the
pyrimidone form based on the chemical shift of 6.11 ppm assigned
to the 4-hydroxy proton.29 The chemical shift of the amide NH pro-
ton in the pyrimidone form 32 should be found at 8–9 ppm
(Scheme 4). Methylation of compound 14 by dimethyl sulfate
yielded a mixture of 3-O-methyl 33 and 4-N-methyl 34 derivatives
(11% and 32%, respectively). The 4-methoxy derivative 33 was
alkylated by n-propyl iodide to afford the desired 4-methoxypyr-
rolo[2,3-d]pyrimidine 35 (Scheme 5).
Our initial efforts focused on the substituent variation at the 2-
position (R1 and R2) of pyrrolo[2,3-d]pyrimidin-4-one. The desired
pyrrolo[2,3-d]pyrimidin-4-one derivatives 16a–e were synthesized
as illustrated in Scheme 1. 2-Amino-4-hydroxy-6-methylaminopyr-
imidine 10 was prepared from commercially available 2,4-diamino-
6-hydroxypyrimidine
9 and methylamine. 2-(2,4,6-Trimethyl-
phenyl)ethanol 11 was converted to 2,4,6-trimethylphenylacetal-
dehyde 12 under Swern oxidation conditions. Bromination of
compound 12 with bromotrimethylsilane yielded
a-bromoalde-
hyde 13, which was cyclized with compound 10 under basic condi-
tions to afford pyrrolo[2,3-d]pyrimidin-4-one 14. Treatment of
compound 14 with sodium hydride and iodomethane chemoselec-
tively yielded 3-N-methylpyrrolo[2,3-d]pyrimidin-4-one 15. Alkyl-
ation of the 2-amino group of compound 15 by various alkyl halides
afforded the desired pyrrolo[2,3-d]pyrimidin-4-ones 16a–e.27
We established a different synthetic route to prepare variations
of the 5-aromatic group. This route permitted the installation of
the aromatic group at the final step (Scheme 2). The reaction of
2-amino-4-hydroxy-6-methylaminopyromidine 10 and chloroace-
tone in the presence of sodium acetate yielded the 6-methylpyrrol-
o[2,3-d]pyrimidin-4-one derivative 17, whichwas chemoselectively
methylated at the 3-position to yield the 3-methyl derivative 18.
Alkylation of the 2-amino group of compound 18, followed by bro-
mination with N-bromosuccinimide (NBS) provided 5-bromo-2-
(dipropylamino)pyrrolo[2,3-d]pyrimidin-4-one derivative 20 as a
key intermediate to introduce an aromatic ring at the 5-positon.
The palladium-catalyzed coupling reaction of compound 20 with
The synthesized compounds 16a–e, 21a, 21b, 29a–d, 30, 31, and
35 were screened for their inhibitory activities of ovine [125I]CRF
binding to human CRF1 receptors expressed on Chinese hamster
ovary (CHO) cellular membranes (Tables 1 and 2). We found that
the pyrrolo[2,3-d]pyrimidin-4-ones 16a–e, 21a, 21b, and 29a–d
with the carbonyl group exhibited potent CRF1 receptor binding
affinity (IC50 = 0.12–7.7
(IC50 = 0.12 M) in this series was found to be as potent as CP-
316,311 (3) (IC50 = 0.092 M), evaluated in phase II. Unexpectedly,
lM). The most active compound 16b
l
l
the binding affinity of 16b was less than that of the superimposed
CP-154,526 (8). Lone-pairs on the carbonyl oxygen atom of 16b
would be pointed slightly different direction from that of the sp2-
nitrogen of 8. In addition, compound 16b was inactive for CRF2
a
Scheme 2. Reagents: (a) chloroacetone, NaOAc, EtOH/H2O, 44%; (b) MeI, NaH, DNF, 74%; (c) nPrI, NaH, DMF, 42%; (d) NBS, DMF, 54%; (e) ArB(OH)2, Pd(Ph3P)4, Na2CO3, DME,
10–14%.

K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
2369
Scheme 3. Reagents: (a) chloroacetaldehyde, K2CO3, MeOH, 22%; (b) (i) tBuCOCl, pyridine; (ii) aq NH3, 75%; (c) NIS, DMF, 70%; (d) Zn, AcOH, H2O, 78%; (e) NaOH, THF/H2O,
91%; (f) MeI, NaH, DNF, 55%; (g) nPrI, NaH, DMF, 73%; (h) ArB(OH)2, Pd(Ph3P)4, Na2CO3, DME, 24–44%.
We attempted to investigate the primary SAR for these unique
CRF1 receptor antagonists with the carbonyl-based HBA. We ini-
tially determined the effect of the 2-amino group on CRF1 receptor
binding affinity (Table 1). The N-di-n-propylamino 16b and N-di-
(cyclopropylmethyl)amino 16d derivatives displayed high binding
affinity (IC50 = 0.12 and 0.14
di-n-propylamino group with the N-diethylamino moiety resulted
in a 70-fold lower binding affinity (16a, IC50 = 7.7 M). N-Di-n-
butylamino derivative 16c (IC50 = 0.97 M) with longer alkyl
lM, respectively). Replacement of the
l
l
chains was also ninefold less active than the N-di-n-propylamino
derivative 16b. The cyclic tertiary amine 16e displayed diminished
affinity (IC50 = 3.8 lM). These data suggested that the 2 alkyls of C3
chain length (i.e., n-propyl and cyclopropyl) as 2-amino substitu-
ents adequately occupied the large lipophilic pocket on the top re-
gion in Figure 2(b).
Substituent effects at the 6-position orthogonal to the 5-aryl
group to the pyrrolo[2,3-d]pyrimidine core ring were also studied.
Introduction of a methyl group into compound 16b at the 6-position
resulted in a 10-fold lower binding affinity (21a, IC50 = 1.1 lM).
Comparison of 21b versus 29a of the 5-(2,4-dimethylphenyl) deriv-
atives also showed that the 6-methyl analogue 21b was less active
than the 6-unsubstituted analogue 29a. These results suggested
that the substituent at the 6-position of the core ring inhibited bind-
ing to the receptor due to its exterior location as expected from the
superimposed forms of 16b and 8.
Scheme 4. Reagents: (a) (i) POCl3, PhNEt2; (ii) nPrI, NaH, DMF, 31%; (b) 1 N NaOH in
H2O, 81%.
We also briefly investigated the variation in substituents of the
5-phenyl group on binding affinity. The 2,4,6-trimethylphenyl
derivative 16b exhibited more potent binding affinity than the
and CRF2b receptors (IC50, >10
human CRF-stimulated cAMP accumulation with IC50 value of
1.2 M (0.83–1.7 M; 95% confidential interval) in CHO cells
lM). Compound 16b also inhibited
l
l
2,4-dimethylphenyl derivative 21b (16b, IC50 = 0.12
lM and 21b,
expressing human CRF1 receptor, indicating that this compound
functioned as CRF1 receptor antagonist. The non-carbonyl deriva-
tives, 4-chloro (30), 4-hydroxy (31), and 4-methoxy (35) analogues,
IC50 = 3.3 M). The orthogonal conformation of the aromatic ring
could be supported by the two ortho substituents. Introduction
of electron donating groups, that is, methoxy groups, led to dimin-
l
were significantly less active (IC50, >10
lM) (Table 2). These data
ished binding affinity (29b, IC50 = 3.1 lM and 29c, IC50 = 3.0 lM).
indicated that the carbonyl group of the pyrrolo[2,3-d]pyrimidine
derivatives functioned as HBA to bind selectively to the CRF1 recep-
tor. Our hypothesis of the carbonyl-based HBA could be supported
by these results.
Compounds with electron withdrawing groups, that is, trifluoro-
methyl groups, also did not show improved binding affinity (com-
pare 29c vs 29d). These results suggested that the electron effects
on the 5-phenyl group were independent of the binding activity.

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K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
Scheme 5. Reagents: (a) Me2SO4, K2CO3, acetone; (b) nPrI, NaH, DMF, 4%.
Table 2
Table 3
hCRF1 binding affinities of pyrrolo[2,3-d]pyrimidines
Ex vivo studies of 16b at 30 mg/kg (po) in mice
125I-sauvagine binding inhibition (%)
Me
Frontal cortex
35
Olfactory bulb
22
Pituitary
53
Me
N
N
N
Me
N
Me
The representative compound 16b in this series showed potent
and selective CRF1 receptor binding affinity in in vitro and ex vivo
tests. Compound 16b was also well absorbed orally and penetrated
into the brain in mice. Further optimization studies of the pyrrol-
o[2,3-d]pyrimidin-4-one derivatives with a carbonyl-based HBA
will be reported in due course.
R7
Me
Me
Compound
R7
Cl
OH
OMe
hCRF1 IC50 (lM)
30
31
35
>10
>10
>10
Acknowledgments
We thank Dr. Takanobu Kuroita and Dr. Masaki Tomimoto for
helpful discussions during the preparation of the manuscript. We
also thank Dr. Yoshio Yamamoto for the superimposition study,
and Kenichi Kuroshima and Yuji Shimizu for performing the
in vitro CRF1 binding assay. We extend our gratitude to Dr. Jim
Blake, Dr. Kevin Condroski, Dr. Albert Gyorkos, Dr. Tim Turner,
Dr. Chris Corrette, Dr. Suk Young Cho and Dr. Scott Pratt (Array Bio-
Pharma, Boulder, CO) for their valuable discussions.
The penetration into the brain and the CRF1 receptor binding of
the most active compound 16b in the unique carbonyl-based HBA
series was evaluated by ex vivo 125I-sauvagine (CRF1 receptor li-
gand) binding in mice (Table 3).30 Compound 16b showed dis-
placement of 125I-sauvagine in the frontal cortex, olfactory bulb,
and pituitary gland in mice at 30 mg/kg (po). The inhibitory rate
of 125I-sauvagine binding to the pituitary gland (53%) with regard
to peripheral CRF1 receptor binding was higher than that to the
cortex (35%) and olfactory bulb (22%) in the brain. The relative
brain/plasma ratio (0.42–0.66) estimated by ex vivo binding stud-
ies was sufficiently acceptable for a CNS drug. These data indicated
that compound 16b penetrated into the brain after oral absorption
and bound to CRF1 receptors in the CNS.
In conclusion, we identified a new class of pyrrolo[2,3-d]pyrim-
idin-4-one derivatives as CRF1 receptor antagonists with a carbonyl-
based HBA. Eleven pyrrolo[2,3-d]pyrimidin-4-one derivatives,
prepared by efficient synthetic routes, showed CRF1 receptor bind-
ing affinity, and their carbonyl group on the pyrrolo[2,3-d]pyrimi-
dine core would function as HBA to bind CRF1 receptors.
Meanwhile, the non-carbonyl derivatives, 4-chloro (30), 4-hydroxy
(31), and 4-methoxy (35) analogues, were significantly less active
References and notes
1. Vale, W.; Spiess, J.; River, C.; River, J. Science 1981, 213, 1394.
2. Dzierba, C. D.; Hartz, R. A.; Bronson, J. J. Annu. Rep. Med. Chem. 2008, 43, 3.
3. Owens, M. J.; Nemeroff, C. B. Pharmacol. Rev. 1991, 43, 425.
4. Holsboer, F. J. Psychiatry Res. 1999, 33, 181.
5. Grigoriadis, D. E.; Haddach, M.; Ling, N.; Saunders, J. Curr. Med. Chem. 2001, 1,
63.
6. Perrin, M. H.; Grace, C. R.; Riek, R.; Vale, W. W. Ann. N.Y. Acad. Sci. 2006, 1070,
105.
7. Spiess, J.; Dautzenberg, F. M.; Sydow, S.; Hauger, R. L.; Ruhmann, A.; Blank, T.;
Radulovic, J. Trends Endocrinol. Metab. 1998, 9, 140.
8. Timpl, P.; Spanagel, R.; Sillabar, I.; Kresse, A.; Reul, J. M.; Stalla, G. K.; Blanquet,
V.; Steckler, T.; Holsboer, F.; Wurst, W. Nat. Genet. 1998, 19, 162.
9. Zobel, A. W.; Nickel, T.; Kunzel, H. E.; Ackl, N.; Sonntag, A.; Ising, M.; Holsboer,
F. J. Psychiatry Res. 2000, 309, 171.
10. Ising, M.; Zimmermann, U. S.; Kunzel, H. E.; Uhr, M.; Foster, A. C.; Learned-
Coughlin, S. M.; Holsboer, F.; Grigoriadis, D. E. Neuropsychopharmacology 2007,
32, 1941.
(IC50, >10 lM). These findings would provide support for the
hypothesis that the carbonyl group acted as a replacement for the
11. Binneman, B.; Feltner, D.; Kolluri, S.; Shi, Y.; Qiu, R.; Stiger, T. Am. J. Psychiatry
2008, 165, 617.
sp2-nitrogen atom as HBA in classical CRF1 receptor antagonists.

K. Aso et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2365–2371
2371
12. Coric, V.; Feldman, H. H.; Oren, D. A.; Shekhar, A.; Pultz, J.; Dockens, R. C.; Wu,
X.; Gentile, K. A.; Huang, S.-P.; Emison, E.; Delmonte, T.; D’Souza, B. B.;
Zimbroff, D. L.; Grebb, J. A.; Goddard, A. W.; Stock, E. G. Depress. Anxiety 2010,
27, 417.
13. Sweetser, S. R.; Linker Nord, S. J.; Burton, D. D.; Grudell, A.; Eckert, D. J.; Manini,
M. L.; Busciglio, I.; Tong, G.; Dockens, R. C.; Zinsmeister, A. R.; Camilleri, M.
Gastroenterology 2008, 134, Abstr T1405.
14. Thoua, N. M.; Hobson, A. R.; Dukes, G. E.; Kelleher, D. L.; Keller, D. L.; Hicks, K. J.;
Boardley, R. L.; Raeburn, A. J.; Emmanuel, A. V. Gastro 2009, London, UK,
November 2009, OP097.
15. Tellew, J. E.; Lanier, M.; Moorjani, M.; Lin, E.; Luo, Z.; Slee, D. H.; Zhang, X.;
Hoare, S. R. J.; Grigoriadis, D. E.; Denis, Y. S.; Romano Di Fabio, R. D.; Modugno,
E. D.; Saunders, J.; Williams, J. P. Bioorg. Med. Chem. Lett. 2010, 20, 7259.
16. Gully, D.; Geslin, M.; Serva, L.; Fontaine, E.; Roger, P.; Lair, C.; Darre, V.; Marcy,
C.; Pierre-Eric Rouby, P.-E.; Simiand, J.; Guitard, J.; Gout, G.; Steinberg, R.;
Rodier, D.; Griebel, G.; Philippe Soubrie, P.; Pascal, M.; Pruss, R.; Scatton, B.;
Maffrand, J.-P.; Fur, G. L. J. Pharmacol. Expert Ther. 2002, 301, 322.
17. ClinicalTraials.gov (A service of the U.S. National Institutes of Health; http://
clinicaltrial.gov), October 23, 2010.
18. Sanofi-Aventis company report (http://en.sanofi-aventis.com), R&D Portfolio as
of February 9, 2011.
19. Gehlert, D. R.; Cippitelli, A.; Thorsell, A.; Lê, A. D.; Hipskind, P. A.; Hamdouchi,
C.; Lu, J.; Hembre, E. J.; Cramer, J.; Song, M.; McKinzie, D.; Morin, M.;
Ciccocioppo, R.; Heilig, M. J. Neurosci. 2007, 27, 2718.
25. Hitchcock, S. A.; Pennington, L. D. J. Med. Chem. 2006, 49, 7559.
26. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. W.; Verhoest, P. R.;
Villalobos, A.; Will, Y. A. C. S. Chem. Neurosci. 2010, 1, 420.
27. All new compounds gave satisfactory analytical data. For 16b: 1H NMR (CDCl3)
d 0.90 (6H, t, J = 7.5 Hz), 1.61 (4H, m), 2.11 (6H, s), 2.29 (3H, s), 3.11 (4H, t,
J = 7.5 Hz), 3.47 (3H, s), 3.70 (3H, s), 6.43 (1H, s), 6.90 (2H, s). MS calculated:
380. Found: 381 (M+H). Mp: 100–102 °C. Anal. Calcd for C23H32N4O: C, 72.60;
H, 8.48; N, 14.72. Found: C, 72.64; H, 8.66; N, 14.36. For 16d: 1H NMR (CDCl3) d
0.12–0.17 (4H, m), 0.45–0.52 (4H, m), 1.01–1.10 (2H, m), 2.11 (6H, s), 2.29 (3H,
s), 3.10 (4H, d, J = 6.6 Hz), 3.52 (3H, s), 3.72 (3H, s), 6.45 (1H, s), 6.90 (2H, s). MS
calcd: 404; found: 405 (M+H). Mp: 218–221 °C. Anal. Calcd for C25H32N4O: C,
74.22; H, 7.97; N, 13.85. Found: C, 74.20; H, 7.89; N, 13.83.
28. Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G. B.; Barredo, J.;
Jannatipour, M.; Moran, R. G. J. Med. Chem. 1992, 35, 4450.
29. For 31: 1H NMR (CDCl3) d 0.94 (6H, t, J = 7.5 Hz), 1.67 (4H, m), 2.06 (6H, s), 2.30
(3H, s), 3.57 (4H, t, J = 7.5 Hz), 3.69 (3H, s), 6.11 (1H, s), 6.48 (1H, s), 6.90 (2H, s).
MS calcd: 366; found: 367 (M+H).
30. Ex vivo binding assay in mice: The test compound or the corresponding vehicle
was administered po to mice (10 per group) at 30 mg/kg, 60 min before mouse
decapitation and organ removal (frontal cortex, olfactory bulb, and pituitary).
Tissues were homogenized in lysis buffer (50 mM Tris–HCl, 10 mM MgCl2,
2 mM ethylenediaminetetraacetic acid (EDTA), 100 KU/mL aprotinin) and
diluted (final protein concentration: 5 mg/mL). [125I]-sauvagine binding was
performed using membrane homogenates in the presence of 100 pM of [125I]-
sauvagine in lysis buffer containing 0.1% bovine serum albumin (BSA), 0.5%
20. Hummel, M.; Cummons, T.; Lu, P.; Mark, L.; Harrison, J. E.; Kennedy, J. D.;
Whiteside, G. T. Neuropharmacology 2010, 59, 160.
dimethyl
sulfoxide
(DMSO),
and
0.05%
3-[(3-cholamido-propy1)-
L.
21. Tellew, J. E.; Luo, Z. Curr. Top. Med. Chem. 2008, 8, 506.
22. Schulz, D. W.; Mansbach, R. S.; Sprouse, J.; Braselton, J. P.; Collins, J.; Corman,
M.; Dunaiskis, A.; Fraci, S.; Schmidt, A. W.; Seeger, T.; Seymour, P.; Tingley, F.
D., III; Winston, E. N.; Chen, Y. L.; Heym, J. Proc. Natl. Acad. Sci. U.S.A. 1996,
10477.
dimethylammonio]-I-propanesulfonate (CHAPS) in a final volume of 200
l
After incubation at room temperature for 2 h, the incubation mixture was
filtered through Whatman GF/C filter presoaked in 0.3% polyethyleneimine.
The filters were washed six times with ice-cold wash buffer (phosphate
buffered saline (PBS) containing 0.05% CHAPS and 0.01% Triton X-100) and
dried. The radioactivity was determined using a gamma scintillation counter.
23. Aso, K.; Hitaka, T.; Yukishige, K.; Ootsu, K.; Akimoto, H. Chem. Pharm. Bull. 1995,
43, 256.
Nonspecific binding was determined using 1 lM unlabeled selective CRF1
24. Aso, K.; Imai, Y.; Yukishige, K.; Ootsu, K.; Akimoto, H. Chem. Pharm. Bull. 2001,
49, 1280.
antagonist R121919. The mean value of the 2 independent experiments was
expressed as inhibitory rate of [125I]-sauvagine binding.