3-Aryl pyrazolo[4,3-d]pyrimidine derivatives: Nonpeptide CRF-1 antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00358-X

The synthesis of a series of 3-aryl pyrazolo[4,3-d]pyrimidines as potential corticotropin-releasing factor (CRF-1) antagonists is described. The effects of substitution on the aromatic ring, the amino group and the pyrazolo ring on CRF-1 receptor binding

Full text of DOI:10.1016/S0960-894X(02)00358-X

Bioorganic & Medicinal ChemistryLetters 12 (2002) 2133–2136
3-Aryl Pyrazolo[4,3-d]pyrimidine Derivatives:
Nonpeptide CRF-1 Antagonists
Jun Yuan, Michael Gulianello, Stephane De Lombaert, Robbin Brodbeck,
Andrzej Kieltyka and Kevin J. Hodgetts*
Neurogen Corporation, 35 Northeast Industrial Road, Branford, CT 06405, USA
Received 26 March 2002; accepted 6 May2002
Abstract—The synthesis of a series of 3-aryl pyrazolo[4,3-d]pyrimidines as potential corticotropin-releasing factor (CRF-1)
antagonists is described. The effects of substitution on the aromatic ring, the amino group and the pyrazolo ring on CRF-1 receptor
binding were investigated. # 2002 Elsevier Science Ltd. All rights reserved.
Corticotropin releasing factor (CRF) is a 41-amino acid
peptide that acts as a modulator of the body’s responses
to stress.1 Compelling clinical evidence exists for the
hypothesis that over-stimulation of CRF may underlie the
pathologyof several diverse neuropscyhiatric diseases
including depression, anxiety, and stress-related disorders.2
Until now, two G-protein coupled receptors for CRF
(CRF-1 and CRF-2) have been identified in mammals.
Theyare encoded bytwo separate genes and display
distinct pharmacologyand distribution within the CNS
and periphery.3,4 Although the therapeutic benefits of
blocking the CRF-2 receptor remain to be established,
evidence exists that antagonism of the CRF-1 receptor
produces anxiolytic and antidepressant effects in animals.
In an attempt to produce clinicallyuseful therapeutic
agents, several series of small molecule CRF-1 receptor
antagonists have been reported, as exemplified bypyrrolo-
pyrimidine 1 (CP154526),5 triazolopyrimidine 26 and pyr-
azolo[1,5-a]pyrimidines 3 (NBI 30775/R121919) (Fig. 1).7À9
optimization of a new series of CRF-1 antagonists, the
3-aryl pyrazolo[4,3-d]pyrimidines 4.
The compounds described in this studyare listed in
Tables 1–3, and the methods used for their synthesis are
outlined in Schemes 1 and 2. Reaction of the appro-
priatelyaryl substituted acetophenone 6 with sodium
hydride and diethyl oxalate gave the pyruvate 7 in good
yield (CAUTION: Careful heating of this reaction is
necessaryas an exotherm at 75 ꢀC was observed).
Treatment of 7 with dinitrogen trioxide (generated from
sodium nitrite and HCl) in ethanol gave the oxime 8.13
Cyclization of 8 with methyl hydrazine gave a mixture
of the nitroso pyrazoles 9 and 10, which were separated
byflash chromatograph.y Reduction of the nitroso
group with sodium dithionite in aqueous THF gave the
respective amino-pyrazoles 11 and 12.
Treatment of the pyrazole 11 with benzyl thioacetimidate
hydrobromide14 in refluxing pyridine afforded the
pyrazolopyrimidinone 13 in moderate yield. Heating
13 with phosphorus oxychloride in the presence of
N,N-diethyl aniline gave the corresponding chloride 14
in high yield. Displacement of the chloride of 14 with the
appropriate amine afforded the target 7-amino substituted
pyrazolo[4,3-d]pyrimidines 15.
A large bodyof evidence gathered with the antagonist 1
(Ki=2.7 nM), and others,10 supports the CRF concept
preclinically.11 Indeed, recent clinical results with
R121919 3 (Ki=5 nM) have turned out to be very
encouraging, despite an open label design.12 Further
development of R121919 was eventuallydiscontinued
due to clinical signs of hepatotoxicityand the search for
new chemical matter is still ongoing. In this paper, we
describe the synthesis and structure–activity based
Pyrazole 12 was subjected to the same sequence of
reactions outlined in Scheme 1 and gave the corresponding
1-methyl analogues. The unsubstituted, N-ethyl and N-
propyl substituted pyrazolo[4,3-d]pyrimidines (Table 1)
were prepared employing analogous chemistry to that
outlined in Schemes 1 and 2 but using hydrazine, ethyl
*Corresponding author. Tel.: +1-203-488-8201; fax: +1-203-483-7027;
e-mail: khodgetts@nrgn.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00358-X

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J. Yuan et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2133–2136
Table 2. Effects of the 3-aryl substituent on CRF-1 binding
Compd
Ar
Ki, nM
17a
18a
18b
18c
18d
18e
18f
18g
2,4,6-Trimethyl
Ph
2-Chloro
3Æ1
>5000
765Æ93
>5000
>5000
20Æ4
2Æ1
3-Chloro
4-Chloro
2,6-Dichloro
2,4-Dichloro
2,4-Methoxy10
2-Methyl-4-chloro
—
Æ3
18h
CP154526 (1)
4Æ2
2.7
Figure 1. Examples of small molecule CRF-1 receptor antagonists.
Ki values are the mean of three independent experiments.
Table 3. Effect of the 7-amino substituent on CRF-1 binding
Table 1. Effects of 1- and 2-pyrazolo[4,3-d]pyrimidine substituents
on CRF-1 binding
Compd
R
R1
Ki, nM
Compd
R
Ki, nM
19a
19b
19c
19d
19e
19f
19g
19h
19i
18f
19j
19k
19l
19m
19n
Methyl
Ethyl
nPropyl
nButyl
nPentyl
nHexyl
Cyclopentyl
Cyclohexyl
CH(Et)2
nPropyl
(CH2)2OMe
Ethyl
Ethyl
Ethyl
nPropyl
nPropyl
nPropyl
—
H
H
H
H
H
H
H
H
H
>5000
192Æ82
24Æ10
25Æ9
16a
16b
16c
16d
17a
17b
17c
CP154526 (1)
H
Me
Et
Pr
Me
Et
16Æ2
1Æ0.4
15Æ3
62Æ15
3Æ1
38Æ17
112Æ31
17Æ4
3Æ1
Pr
—
11Æ3
2.7
174Æ39
16Æ8
nPropyl
(CH2)2OMe
nButyl
nPropyl
nHexyl
(CH2)2NMe2
(CH2)2N(CH2)4
(CH2)2N(CH2)5
—
2Æ1
Ki values are the mean of three independent experiments.
3Æ2
2Æ1
3Æ2
29Æ15
145Æ71
115Æ33
561Æ212
2.7
hydrazine and propyl hydrazine respectively in the
cyclization step.
19o
19p
CP154526 (1)
The affinityof the compounds (Tables 1–3) for the
CRF-1 receptor were determined byusing a modified
version of the assaydescribed byGrigoriadis and De
Souza15 byexamining their displacement of 125I-sauva-
gine from CRF-1 receptors endogenouslyexpressed in
IMR-32 human neuroblastoma cells.
Ki values are the mean of three independent experiments.
affinity( Ki=1 nM). Increasing the chain length (ethyl
16c and propyl 16d) resulted in a significant loss in affinity.
A similar, but less pronounced trend was observed for
the 2-alkyl substituted analogues 17a–17c. The 2-methyl
17a and 2-ethyl 17b analogues were of similar affinityto
16b and while the affinityof the corresponding 2-propyl
17c analogue was reduced the magnitude of the loss was
greatlyreduced. Compounds 16b, 17a, and 17b all
displayed binding affinity comparable to that reported
for CP154526 1.5b
The effects of alkyl substituents at the 1- and 2-position
of the pyrazolo[4,3-d]pyrimidine on CRF-1 receptor
binding were examined first (Table 1). The 3-mesityl and
7-dipropylamine substituents were kept constant
throughout. The unsubstituted analogue 16a was found
to have good affinityfor the CRF-1 receptor ( Ki=16
nM). Introduction of a methyl group to the 1-position
gave compound 16b and a large increase in binding

J. Yuan et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2133–2136
2135
the propyl and butyl analogues 19c and 19d proving the
most active. The activitydecreased as the alkyl chain
was increased to pentyl and hexyl (19e and 19f). The
cyclopentyl analogue 19g had good affinity( Ki=17
nM), but the cyclohexyl analogue 19h was less well tol-
erated. The open chain analogue of 19g, the 3-pentyl
amino derivative 19i, was of similar affinity. The tertiary
amines 18f and 19j–19l provided active compounds
(Ki’s<5 nM) so long as the steric requirement was not
exceeded (19m, Ki=29 nM). In an attempt to increase the
solubilityof the series, several basic amines were incorpo-
rated into the side chain (19n–19p), however, a substantial
loss in affinitywas the result of this modification.
Scheme 1. Reagents and conditions: (i) NaH, diethyl oxalate, PhMe,
.
reflux, (71–89%); (ii) N2O3, EtOH, (85–94%); (iii) MeNHNH2 HCl,
MeOH–H2O (69–78%); (iv) Na2S2O4, THF–H2O, (59–68%).
It next became important to determine the functional
activityof these compounds at the CRF-1 receptor.
Compounds 16b and 17a were shown to inhibit sauvagine
stimulated cAMP accumulation in AtT20 cells expressing
the CRF-1 receptor with Ki’s of 5 and 22 nM, respec-
tively.17 This data confirms that 16b and 17a are functional
antagonists at the CRF-1 receptor.
In summary, a series of 3-aryl pyrazolo[4,3-d]pyr-
imidines has been described having affinityfor the CRF-1
receptor. Small alkyl substituents were well tolerated at
both the 1- and 2-position of the pyrazolopyrimidine.
An ortho substituent on the 3-aryl group was essential
for binding and a second substituent at the 4-position
gave a further increase in binding affinity. Tertiary
amines at the 7-position were optimal as long as the
steric requirement was not exceeded. Seven of the com-
pounds described had affinities comparable (Ki<3 nM)
to that reported for CP154526 1.5b
.
Scheme 2. Reagents and conditions: (i) benzylthioacetimidate HBr,
pyridine, reflux, (47–55%); (ii) POCl3, N,N-diethylaniline, reflux, (89–
93%); (iii) NHR1R2, ethanol, reflux (68–89%).
Acknowledgements
The effects of the substituents on the 3-aryl group were
also investigated (Table 2). The 7-dipropyl amine and
the 2-methyl substituent were kept constant in the pyr-
azolopyrimidine. For optimal CRF-1 binding, previous
studies have suggested that the aryl ring lies out of the
plane of the core heterocycle and our results are in
accord with that model.16 The unsubstituted phenyl
analogue 18a and the 3- and 4-chloro analogues (18c
and 18d) were all inactive. An ortho substituent, however,
on the aromatic ring enforces an active conformation
and the 2-chloro analogue 18b showed moderate affinity
(Ki=765 nM). A large increase in affinitywas observed
byaddition of a second ortho substiutent (18e, Ki=20
nM). The 2,4,6-trimethylphenyl analogue 17a described
earlier gave a further increase in affinity. A para sub-
stituent in combination with one ortho substituent also
gave compounds of a similar affinity( 18f, Ki=2 nM,
18g, Ki=10 nM, and 18h, Ki=4 nM).
The authors gratefullythank Dr. George Maynard and
Dr. Dario Doller for helpful discussions.
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