Design, synthesis, and SAR of 2-dialkylamino-4-arylpyrimidines as potent and selective corticotropin-releasing factor1 (CRF1) receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.02.053

A series of 2-dialkylamino-4-phenylpyrimidines (7) was designed and synthesized as CRF₁ antagonists. SAR studies of this series resulted in the discovery of potent and selective antagonists 7b and 7n bearing a 4-(2,4,6-trisubstituted-phenyl) ring and a bulky 2-(N-bis(cyclopropane)methyl-N- propyl)amino group.

Full text of DOI:10.1016/j.bmcl.2004.02.053

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2083–2086
Design, synthesis, and SAR of 2-dialkylamino-4-arylpyrimidines
as potent and selective corticotropin-releasing factor1 (CRF1)
receptor antagonists
Charles Q. Huang,* Dimitri E. Grigoriadis, Zhengyu Liu, James R. McCarthy,
John Ramphal, Thomas Webb, Jeffrey P. Whitten, Michael Y. Xie and Chen Chen*
Neurocrine Biosciences, Inc., 10555 Science Centre Drive, San Diego, CA 92121, USA
Received 16 December 2003; accepted 11 February 2004
Abstract—A series of 2-dialkylamino-4-phenylpyrimidines (7) was designed and synthesized as CRF1 antagonists. SAR studies of
this series resulted in the discovery of potent and selective antagonists 7b and 7n bearing a 4-(2,4,6-trisubstituted-phenyl) ring and
a bulky 2-(N-bis(cyclopropane)methyl-N-propyl)amino group.
Ó 2004 Elsevier Ltd. All rights reserved.
Corticotrophin-releasing factor (CRF), a 41 amino acid
peptide, was first isolated, sequenced, and characterized
by Vale and co-workers.1 It is the key regulator of an
organismÕs response to stress and as such, mediates the
endocrine, autonomic, behavioral, and immune
response to stressful stimuli. In addition, this hormone
has been demonstrated to exhibit all of the characteris-
tics of a bona fide neurotransmitter. The binding of
CRF to CRF1 receptors in the pituitary gland is
responsible for the increased release of ACTH and other
peptides.2 On the other hand, prolonged activation of
brain CRF receptors is thought to be related to the
psychological effects of stress leading to anxiety and
depression and blockade of CRF1 receptor activation
has been proposed as a novel approach for the treatment
of these psychiatric disorders.3
results in a group of severely depressed patients.8 While
all these compounds comprise of a core heterocyclic ring
bearing amino, methyl, and substituted aryl function-
alities, the first reported small molecule
5 has
a relatively simple structure.9 To search for novel
heterocyclic templates, 2-dialkylamino-4-phenylpyrim-
idines of the general structure 7 were designed based on
a topographical similarity to previously reported 1-alkyl-
3-amino-5-aryl-1H-[1,2,4]triazole (6)10 and 4-aryl-5-
methyl-2-dialkylaminothiazole (15, SSR125543A).11 In
this paper, we discuss the structure–activity relationship
studies around this nucleus 7 that leads to the discovery
of a potent class of CRF1 receptor antagonists.
The synthesis of the 2-dialkylaminopyrimidines (7) is
outlined in Scheme 1. Condensation of substituted aceto-
phenones (8) with N,N-dimethylformamide dimethyl
acetal or diethyl acetal at reflux gave the corresponding
enamines (9).12 Cyclization of the enamines (9) with a
guanidine hydrochloride in the presence of sodium
methoxide in ethanol at reflux resulted in the production
of the 2-amino-4-arylpyrimidines (10).13 Reductive ami-
nation of the aminopyrimidine (10) with a ketone or
aldehyde offered the 2-alkylaminopyrimidine (11), which
could be further alkylated with an alkyl halide in the
presence of sodium hydride in DMF to give 2-dialkyla-
mino-4-arylpyrimidine (7). The target compounds (7)
could also be synthesized from direct cyclization of the
enamine (9) with an N,N-dialkylguanidine, which is
readily available from bis(benzyloxycarbonyl)-S-meth-
ylthiourea and dialkylamine according to published
Recent discovery of non-peptide antagonists for CRF
receptors has begun a new era of study for this neuro-
transmitter. Several small molecules have been reported
to have good CRF1 receptor antagonistic activity.4 For
example, pyrrolo[2,3-d]pyrimidine (1, CP-154,526),5
anilinopyrimidine (2, NBI 27914),6 and pyrazolo[1,5-
a]pyrimidine (3, DMP904)7 show good profiles in vitro,
as well as oral activity in vivo in several relevant animal
model studies. In a recent human clinical trial, NBI
30775 (4) demonstrated encouraging, albeit anecdotal,
* Corresponding authors. Tel.: +1-858-658-7735; fax: +1-858-658-7601
(C.Q.H.); tel.: +1-858-658-7634; fax: +1-858-658-7619 (C.C.); e-mail
addresses: chuang@neurocrine.com; cchen@neurocrine.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.053

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C. Q. Huang et al. /Bioorg. Med. Chem. Lett. 14 (2004) 2083–2086
N
N
HN
N
N
N
N
N
Cl
N
N
N
N
N
HN
N
Cl
N
N
Cl
N
Cl
O
4 (NBI 30775)
3 (DMP904)
2 (NBI 27914)
1 (CP-154,526)
F
N
N
N
R3
S
6
R4
N 1
N
N
N
N
N
S
O
N
5
O
Cl
2
Cl
O
X
R1
N
3
N
N
N
4
N
O
R2
Cl
15
14
7
6
5
R3
NR1R2
O
O
R4
N
R3C(OMe)2NMe2
r.t. to reflux
R4
H2N
NH HCl
X
R1
HO CH2CH2OH
160 oC
N
N
R4
R3
NMe2
R2
X
X
8
9
7
guanidine HCl
EtONa/EtOH
reflux
R2I/NaH
DMF/r.t.
R3
R3
N
R4
R4
N
N
aldehydes or ketones
X
X
R1
TiCl4/CH2Cl2
N
N
H
NH2
then NaBH4/MeOH
11
10
Scheme 1.
procedures.14 A third method to generate the desired
targets (7) is described in Scheme 2. Thus 2,4-dichloro-
pyrimidine (12) was subjected to a coupling reaction with
phenylboronic acid catalyzed by tetrakis(triphenylphos-
phine)palladium(0) under Suzuki conditions to give the
corresponding 2-chloro-4-phenylpyrimidine (13).15
Replacement of the chlorine on compound 13 with an
amine gave the desired pyrimidine (7).
moderate binding affinity for the CRF1 receptor
(Ki ¼ 240 nM). Replacement of the benzyl with the
2-thiophenylmethyl group (7d, Ki ¼ 250 nM) did not
affect the affinity, while replacement with 3-thio-
phenylmethyl or 3-furanylmethyl group resulted in
reduced activity (7e and 7f, Ki ¼ 2 and 0.7 lM, respec-
tively). However, introduction of a second cyclopropyl
group resulted in an over 15-fold increase in binding
affinity (7b, Ki ¼ 15 nM). On the other hand, replace-
ment of the N-propyl of 7b with an allyl group decreased
the activity almost sevenfold (7a, Ki ¼ 100 nM), while
deletion of the N-propyl group resulted in an inactive
compound (11). These results indicate that the substi-
tution pattern on the 2-amino group is very important
for high CRF1 receptor binding (Table 1).
The CRF1 receptor binding assay was performed on
cloned human CRF1 receptors expressed in CHO cells
using ovine [125I]-CRF as the radiolabeled ligand in a
manner similar to that previously reported6 and the
results are listed in Table 1. The prototypical compound
in this series was the 2-{N-[a-(cyclopropane)benzyl]-
N-propylamino}-4-(2,4,6-trimethoxyphenyl)-pyrimidine
7c, which closely mimics the known thiazole CRF1
antagonist SSR125543A (15),11 and it was found to have
The effect of the substitution on the 4-phenyl ring as well
as the 5- and 6-position of the core pyrimidine was also

C. Q. Huang et al. /Bioorg. Med. Chem. Lett. 14 (2004) 2083–2086
2085
N
N
R1R2NH
heating
N
ArB(OH)2
Pd( PPh3)4
aq. Na2CO3
X
X
R1
N
Cl
N
N
Cl
N
Cl
R2
13
7
12
Scheme 2.
Table 1. Effects of 2-amino substituents on binding to CRF1 receptor
OMe
between the 4-phenyl and the core pyrimidine is critical
for high affinity binding.6;16
N
Because of the importance of the combination of 4-
ortho-substituted phenyl ring and 5-substituted pyrim-
idine, we further screened different ortho-substituted
2,4-dimethoxyphenyl groups. Introduction of a fluorine
at the 6-position of the 4-phenyl group increased almost
eightfold the binding affinity (7m, Ki ¼ 63 nM), while a
chlorine gave much a better analogue (7n, Ki ¼ 11 nM).
Introduction of a methyl group at the 5-position of 7b
actually did not affect the binding affinity (7o,
Ki ¼ 20 nM). Similarly, incorporation of a methyl group
at the ortho-position of the 4-(4-bromophenyl) ring of 7p
had hardly any effect in binding (7p, Ki ¼ 49 nM, vs 7q,
Ki ¼ 63 nM). These results again emphasize the
orthogonal relation between the 4-phenyl ring and the
pyrimidine core. Finally, the fact that introduction of a
methyl group at the 6-position of the pyrimidine core of
7p abolished binding affinity (7r) implies this region is
very sensitive to stereo effect.
R1
N
N
R2
MeO
OMe
Compound R1
R2
Ki (nM)a
10
11
7a
7b
7c
7d
7e
7f
H
H
Inactive
Inactive
100
Bis(cyclopropane)methyl
Bis(cyclopropane)methyl
Bis(cyclopropane)methyl
a-(Cyclopropane)benzyl
1-(2-Thiophenyl)cyclopropyl
1-(3-Thiophenyl)cyclopropyl
1-(3-Furyl)cyclopropyl
1-Naphthyl
H
Allyl
Pr
Pr
Pr
Pr
Pr
Pr
Pr
15
240
250
2,000
700
7g
7h
Inactive
2,200
Cyclopentyl
a Ki values are means of two or more determinations.
Table 2. Effects of 4-phenyl substituents on binding to CRF1 receptor
Selected compounds were tested to determine their
functional activity at the CRF1 receptor. Functional
antagonism was assessed in the same cell line as that
used in the binding studies above however utilizing a live
whole cell preparation to measure intracellular cAMP
accumulation. All compounds were tested in the pres-
ence or absence of peptide agonist in order to determine
if any of the non-peptides exhibited any intrinsic agonist
activity. Compounds 7b and 7n dose-dependently
inhibited CRF-stimulated cAMP production in CHO
cells expressing the CRF1 receptor with EC50 values of
990 nM and 1.6 lM, respectively.17 In addition to the
functional cAMP assay, compounds 7b and 7n were also
tested and found to functionally antagonize CRF-stim-
ulated ACTH release from primary rat anterior pituitary
cell cultures.18 Neither compounds 7b and 7n nor any
other non-peptide molecules examined had any func-
tional intrinsic activity for cAMP or ACTH release
suggesting that these molecules are indeed functional
CRF1 receptor antagonists. Table 3 summarizes all these
results.
R3
R4
N
N
N
R
X
Compound
X
R
R4
R3
Ki (nM)a
7i
2,4-MeO
2,4-MeO
2,4-MeO
2,4-MeO
2,4-MeO
2,4-MeO
2,4-MeO
2,4-MeO
4-Br
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
Me
460
19
7j
Me
Et
7k
7l
860
Inactive
63
COOMe
H
7m
7n
7b
7o
7p
7q
7r
Cl
H
11
15
MeO
MeO
H
H
Me
Me
Me
Me
20
49
4-Br
4-Br
Me
H
63
Inactive
a Ki values are means of two or more determinations.
This series of compounds was also tested on the CRF2a
receptor binding assay using cloned human CRF2a
examined (Table 2). The 2,4-dimethoxy analogue 7i was
found to have a Ki value of 460 nM, which is much less
active than the 2,4,6-trimethoxy analogue 7b. However,
introduction of a methyl substituent on the 5-position of
pyrimidine re-gained the binding affinity (7j, Ki ¼
19 nM). Replacement of the methyl with a larger ethyl
group resulted in over 40-fold loss of binding (7k,
Ki ¼ 860 nM), while the bulky and polar methoxycar-
bonyl group at this position abolished the activity
completely (7l). These results suggest the dihedral angle
Table 3. In vitro activity profiles of compounds 7b and 7n
Compound Ki CRF1 Ki CRF2a EC50 cAMP EC50 ACTH
(nM)
(nM)
CRF1 (nM)a CRF1 (nM)a
7b
7n
15
11
Inactive
Inactive
990
1600
970
1100
a EC50 values are means of two or more determinations.

2086
C. Q. Huang et al. /Bioorg. Med. Chem. Lett. 14 (2004) 2083–2086
receptors as previously described19 to access the selec-
tivity of this series of compounds. Compounds 7b and
7n, which exhibited Ki values of 16 and 11 nM, respec-
tively, at the CRF1 receptor, showed no activity (less
than 40% inhibition at a concentration of 10 lM) at the
CRF2a receptor. Thus, compound 7b exhibited good
selectivity profile. In a NovaScreene performed on 66
neurotransmitter receptors (such as D1, GABAa, H1,
and 5HT), brain/gut peptides (such as bradykinin,
CGRP, NPY, and somatostatin), and growth factors
and ion channels (Ca2þ and Naþ), 7b showed no sig-
nificant affinity at 5 lM.
2. Dieterich, K. D.; Lehnert, H.; De Souza, E. B. Exp. Clin.
Endocrinol Diabetes 1997, 105, 65.
3. Heit, S. O.; Owens, M. J.; Plotsky, P.; Nemeroff, C. B.
Neuroscientist 1997, 3, 186.
4. (a) Grigoriadis, D. E.; Haddach, M.; Ling, N.; Saunders,
J. Curr. Med. Chem. 2001, 1, 63; (b) Gilligan, P. J.;
Robertson, D. W.; Zaczek, R. J. Med. Chem. 2000, 43,
1641.
5. Chen, Y. L.; Mansbach, R. S.; Winter, S. M.; Brooks, E.;
Collins, J.; Corman, M. L.; Dunaiskis, A. R.; Faraci,
W. S.; Gallaschun, R. J.; Schmidt, A.; Schulz, D. W.
J. Med. Chem. 1997, 40, 1749.
6. Chen, C.; Dagnino, R., Jr.; De Souza, E. B.; Grigoriadis,
D. E.; Huang, C. Q.; Kim, K. I.; Liu, Z.; Moran, T.;
Webb, T. R.; Whitten, J. P.; Xie, Y. F.; McCarthy, J. R.
J. Med. Chem. 1996, 39, 4358.
7. Gilligan, P. J.; Baldauf, C.; Cocuzza, A.; Chidester, D.;
Zaczek, R.; Fitzgerald, L. W.; McElroy, J.; Smith, M. A.;
Shen, H.-S. L.; Saye, J. A.; Christ, D.; Trainor, G.;
Robertson, D. W.; Hartig, P. Bioorg. Med. Chem. 2000, 8,
181.
8. Zobel, A. W.; Nickel, T.; Kunzel, H. E.; Ackl, N.;
Sonntag, A.; Ising, M.; Holsboer, F. J. Psychiatr. Res.
2000, 34, 171.
9. Abreu, M. E.; Rzeszotarski, W.; Kyle, D. J.; Hiner, R. N.;
Elliott, R. L. U.S. Patent 5063245, 1991.
The basic pharmacophore for this series of compounds
is a six-membered heterocyclic ring supporting a lipo-
philic dialkylamino group and a substituted aryl ring,
which needs to be orthogonal to the pyrimidine core.
This pharmacophore seems to be different from other
more popular CRF1 antagonists such as 1–4, in which a
nitrogen served as hydrogen-bond acceptor and a
methyl group are critical for high affinity binding to the
CRF1 receptor, and the lipophilic side chain is relative
small.16 However, for 7,
a
bulky bis(cyclopro-
pane)methyl group on the 2-amino was very important
for high binding affinity, and the 2-nitrogen may serve as
a hydrogen-bonding acceptor. A recent publication
disclosed a series of arylpyrimidinones such as 14, which
also showed SAR different from traditional bicyclic
cores.20 This difference in pharmacophore requirements
may offer the advantage to design potent CRF1 antag-
onists with different structural features.
10. Chen, C.; Dagnino, R., Jr.; Huang, C. Q.; McCarthy, J.
R.; Grigoriadis, D. E. Bioorg. Med. Chem. Lett. 2001, 11,
3165.
11. Gully, D.; Geslin, M.; Serva, L.; Fontaine, E.; Roger, P.;
Lair, C.; Darre, V.; Marcy, C.; Rouby, P.-E.; Simiand, J.;
Guitard, J.; Gout, G.; Steinberg, R.; Rodier, D.; Griebel,
G.; Soubrie, P.; Pascal, M.; Pruss, R.; Scatton, B.;
Maffrand, J.-P.; Le Fur, G. J. Pharmacol. Exp. Ther.
2002, 301, 322.
12. (a) Lin, Y.-I.; Lang, S. A., Jr. J. Org. Chem. 1980, 45,
4857; (b) Bredereck, H.; Effenberger, F.; Botsch, H. Chem.
Ber. 1964, 97, 3397.
13. Tanaka, A.; Motoyama, Y.; Takasugi, H. Chem. Pharm.
Bull. 1994, 42, 1828.
In summary, a series of 2-dialkyl-4-arylpyrimidines
exemplified by 7b and 7n was discovered as potent and
selective CRF1 receptor antagonists. SAR studies sug-
gest that the bis(cyclopropane)methyl group on the
2-amino functionality and the 2,4,6-trisubstituted aryl
or 2,4-disubstituted aryl with a small alkyl group at the
5-position of the pyrimidine core are required for opti-
mum CRF1 receptor binding affinity. From this study a
number of 2-dialkyl-4-arylpyrimidines with high binding
affinity for the CRF1 receptor have been characterized.
In addition, these compounds demonstrated in vitro
inhibition of CRF-stimulated cAMP production in sta-
ble lines transfected with human CRF1 receptor subtype
and ACTH release from primary rat anterior pituitary
cell cultures. The different SAR from the previously
reported series should provide us with a different venue
to search for novel CRF1 receptor antagonists.
14. Levy, O. E.; Semple, J. E.; Lim, M. L.; Reiner, J.; Rote,
W. E.; Dempsey, E.; Richard, B. M.; Zhang, E.; Tulinsky,
A.; Ripka, W. C.; Nutt, R. F. J. Med. Chem. 1996, 39,
4527.
15. Solberg, J.; Undheim, K. Acta Chem. Scand. 1989, 43, 62.
16. Arvanitis, A. G.; Gilligan, P. J.; Chorvat, R. J.; Cheese-
man, R. S.; Christos, T. E.; Bakthavatchalam, R.; Beck, J.
P.; Cocuzza, A. J.; Hobbs, F. W.; Wilde, R. G.; Arnold,
C.; Chidester, D.; Curry, M.; He, L.; Hollis, A.;
Klaczkiewicz, J.; Krentitsky, P. J.; Rescinito, J. P.;
Scholfield, E.; Culp, S.; De Souza, E. B.; Fitzgerald, L.;
Grigoriadis, D.; Tam, S. W.; Wong, Y. N.; Huang, S.-M.;
Shen, H. L. J. Med. Chem. 1999, 42, 805.
17. Battaglia, G.; Webster, E. L.; De Souza, E. B. Synapse
1987, 1, 572.
18. De Souza, E. B. J. Neurosci. 1987, 7, 88.
19. For a CRF2 binding assay see: Grigoriadis, D. E.; Liu,
X. J.; Vaughn, J.; Palmer, S. F.; True, C. D.; Vale, W. W.;
Ling, N.; De Souza, E. B. Mol. Pharmacol. 1996, 50, 679.
20. Hodgetts, K. J.; Yoon, T.; Huang, J.; Gulianello, M.;
Kieltyka, A.; Primus, R.; Brodbeck, R.; De Lombaert, S.;
Doller, D. Bioorg. Med. Chem. Lett. 2003, 13, 2497.
References and notes
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1394.