Design and synthesis of a series of non-peptide high-affinity human corticotropin-releasing factor1 receptor antagonists

Full text of DOI:10.1021/jm960149e

4358
J . Med. Chem. 1996, 39, 4358-4360
Sch em e 1a
Design a n d Syn th esis of a Ser ies of
Non -P ep tid e High -Affin ity Hu m a n
Cor ticotr op in -Relea sin g F a ctor 1 Recep tor
An ta gon ists
Chen Chen,* Raymond Dagnino, J r.,
Errol B. De Souza, Dimitri E. Grigoriadis,
Charles Q. Huang, Kyung-Il Kim, Zhengyu Liu,
Terry Moran, Thomas R. Webb, J effrey P. Whitten,
Yun Feng Xie, and J ames R. McCarthy*
Neurocrine Biosciences, 3050 Science Park Road,
San Diego, California 92121
Received May 20, 1996
Corticotropin-releasing factor (CRF), a 41 amino acid
peptide of hypothalamic origin, plays a major role in
coordinating the endocrine, behavioral, and autonomic
responses to stressful stimuli.1-3 The actions of CRF
are mediated through high-affinity receptors which are
part of the superfamily of G-protein-coupled seven
transmembrane proteins.4 Recently two members of the
CRF receptor family have been cloned expressed and
characterized. These receptors demonstrate a different
sequence, pharmacology, and regional distribution within
the central nervous system and the periphery. The
CRF1 receptor demonstrates high affinity for CRF and
its related mammalian analogs,4-7 as well as for non-
mammalian related peptides including sauvagine (frog)
and urotensin I (sucker fish).8 The CRF2 family of
receptors, while sharing sequence homology with the
CRF1 receptor subtype, demonstrates low affinity for
CRF itself but retains its high affinity for the nonmam-
malian forms of the peptide.8,9
While there is very little known about the physiology
and function of the CRF2 subfamily of receptors, a
substantial amount of preclinical and clinical data exists
that suggests that CRF and its high-affinity receptor
play a major role in mediating various neurological and
psychological disorders, including major depression,
anxiety, and a variety of stress-related disorders (for
reviews, see refs 2, 3, and 10). Treatments for these
disorders have been hampered by the fact that ligands
for these receptors have thus far been large peptides.11
We report the design and synthesis of high affinity and
selective non-peptide CRF1 receptor antagonists, 4-anili-
no-6-aminopyrimidines (illustrated by 3b), targeted for
the treatment of CRF-mediated disorders.
a
(a) 2,4,6-Cl3C6H2NH2, NaH, THF, RT or reflux; (b) c-C3-
H5CH2NHCH2CH2CH3, 100-190 °C; (c) NBS or NCS, CHCl3,
reflux; (d) NaH, MeI, THF, room temperature.
Ch a r t 1. Sequence Followed in the Design of CRF1
Receptor Antagonists
The reaction sequence for the synthesis of 3a -c, 4,
5, 6, and 9 is illustrated with 3a -c in Scheme 1.
N-Methylation of the anilino nitrogen on 3b with excess
methyl iodide and 1 equiv of sodium hydride in THF
gave 8. Pyrimidines 11 and 12 (see Chart 1 and
Supporting Information for Scheme 2 with synthesis)
were prepared by a similar sequence as that used for
the synthesis of 3a -c starting with commercially avail-
able 2,6-dichloro-6-methylpyrimidine (10) and separat-
ing the anilinopyrimidines 11 and 12 by flash chroma-
tography.
The inhibition of [125I]CRF binding to cells expressing
the human CRF1 receptor was used to identify specific
receptor antagonists in a radioligand binding assay.12
The CRF receptors expressed in these cell lines dem-
onstrated reversible, saturable, high-affinity binding to
CRF with the pharmacological and functional charac-
teristics comparable to those found in a variety of
animal or human tissues.12 CRF receptor binding
assays were carried out essentially as previously de-
scribed.13
S0022-2623(96)00149-5 CCC: $12.00 © 1996 American Chemical Society

Communications to the Editor
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 22 4359
preparations of the stable cell lines transfected with the
CRF1 receptor, the CRF1 antagonist activity of the series
was demonstrated by inhibition of CRF-stimulated
cAMP production with 3a , 4, and 7a using D-PheCRF-
(12-41) as the standard (IC50 ) 3700, 250, 100, and
200 nM, respectively). Inhibition of CRF-stimulated
adenylate cyclase activity was performed as previously
described.15 None of the compounds demonstrated any
effects on basal cAMP production (that is in the absence
of CRF or other stimulator), indicating that these
compounds are devoid of agonist activity at this receptor
subtype. In addition, these compounds could inhibit
CRF-stimulated ACTH release from primary rat
anterior pituitary cell cultures (data not shown), further
demonstrating antagonist activity at CRF1 receptors. In
order to test for CRF receptor selectivity, the compounds
synthesized in this series (Table 1) were assessed for
inhibition of cAMP production in cells transfected with
the human CRF2R receptor and were completely devoid
of activity (data not shown).
F igu r e 1. Proposed conformation for anilinopyrimidines to
bind to the CRF1 receptor and comparison with an inactive
conformer.
Ta ble 1. Inhibition (Ki) Values for Receptor Antgonists in
Cells Stably Transfected with Human CRF1 Receptorsa
compd
Ki (nM)
compd
Ki (nM)
1.7
2.0
150
3a
3b
3c
4
5
6
30
2.3
3.8
2.5
253
1390
7a
7b
8
9
>10000
D-PheCRF(12-41)
20
a
In conclusion, we have demonstrated the design and
synthesis of selective, high-affinity non-peptide CRF1
receptor antagonists 3b, 3c, 4, 7a , and 7b with Ki values
in the low nanomolar range. In addition, these com-
pounds demonstrate in vitro inhibition of CRF-stimu-
lated cAMP production in stable lines transfected with
the human CRF1 subtype. These compounds, and
further modifications, will be of particular significance
in establishing the utility and potential of CRF1 receptor
antagonists in the treatment of depression and anxiety-
related disorders.
Compounds were tested at 6-12 doses for their ability to
inhibit [125I]CRF binding as described in text. Data are repre-
sentative of duplicate determinations with the experiments re-
peated two or three times.
The design of 3b starting from triazine 1314 and the
structure-activity relationship of related pyrimidines
11, 3a , and 12 with regard to CRF1 receptor binding
activity is outlined in Chart 1. Removal of the 1- or
5-nitrogen from triazine 13 (Ki ) 57 nM), optimized by
rapid microscale synthesis (RMS),14 gave pyrimidine 11
(Ki ) 70 nM) or 3a (Ki ) 30 nM), respectively. However,
changing the position of the 3-nitrogen (i.e. 12) resulted
in complete loss of activity. This CRF1 receptor binding
data led to a proposed pharmacophore model for binding
to the CRF1 receptor illustrated in Figure 1. In this
model the bound conformation of the molecule requires
the anilino group to be orthogonal to and below the
pyrimidine (or triazine) ring. In addition, the nitrogen
at the 3-position of 3a , 3b, 11, and 13, but absent in
12, provides a critical hydrogen-bonding site. Consis-
tent with the model, addition of a substituent at the
5-position of the pyrimidine ring resulted in a 30-50-
fold increase in activity. Thus, addition of a methyl
group (3b) or halogen (chloro, 7a , or bromo, 7b) provided
compounds with Ki values in the 2 nM range (see Table
1). 2,4,6-Trichloroanilinopyrimidines were synthesized
(3a -c, 4, 6, 8, and 9) on the basis of the 2,4,6-
trisubstituted aryl group required for optimal activity
for triazine 13 (Chart 1), the preference for trichloro over
other substituents in related series (see Chart 1 and 3a
vs 6), and the preference for an orthogonal relationship
between the phenyl and pyrimidine rings with this
substitution pattern. 2,4,5-Trisubstituted aryl com-
pound 5 was over 10-fold less active than the corre-
sponding 2,4,6-trisubstituted aryl compound 3a . Inter-
estingly, addition of a methyl group on the anilino
nitrogen (8) resulted in over 100-fold loss in activity.
As in the triazine 13,14 N-propyl-N-cyclopropylmethyl
was optimal for the N6 amino group, but unlike the
triazine series N,N-dipropyl was equally active (4).
While direct inhibition of binding activity gives a valid
measure of the potency of a compound at a specific
receptor, functional tests must be employed in order to
determine whether the compounds can act as agonists
or antagonists at these receptors. Using membrane
Su p p or tin g In for m a tion Ava ila ble: A brief description
of the biological assays, synthetic procedures for new com-
pounds, and analytical data (8 pages). Ordering information
is given on any current masthead page.
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