Non-peptide corticotropin-releasing hormone antagonists: Syntheses and structure activity relationships of 2-anilinopyrimidines and -triazines

Article abstract of DOI:10.1021/jm980222w

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [¹²⁵I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist α-helical CRH(9- 41) (K(i) = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH- stimulated adenylate cyclase activity in the same tissue, but it was less potent than α-helical CRH(9-41) (IC₅₀ = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH₁ receptor assay, defined the pharmacophore for optimal binding to hCRH₁ receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH₁ receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH(1)K(1) = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Full text of DOI:10.1021/jm980222w

J . Med. Chem. 1999, 42, 805-818
805
Non -P ep tid e Cor ticotr op in -Relea sin g Hor m on e An ta gon ists: Syn th eses a n d
Str u ctu r e-Activity Rela tion sh ip s of 2-An ilin op yr im id in es a n d -tr ia zin es
Argyrios G. Arvanitis, Paul J . Gilligan,* Robert J . Chorvat, Robert S. Cheeseman, Thomas E. Christos,
Rajagopal Bakthavatchalam, J ames P. Beck, Anthony J . Cocuzza, Frank W. Hobbs, Richard G. Wilde,
Charles Arnold, Dennis Chidester, Matthew Curry, Liqi He, Andrea Hollis, J ohn Klaczkiewicz,
Paul J . Krenitsky,† J oseph P. Rescinito, Everett Scholfield, Steven Culp,‡,3 Errol B. De Souza,§,3
,)
Lawrence Fitzgerald,3 Dimitri Grigoriadis,§,3 S. William Tam,3,| Y. Nancy Wong, Shiew-Mei Huang,X,)
and Helen L. Shen)
Department of Chemical and Physical Sciences, DuPont Pharmaceuticals Company, Experimental Station,
P.O. Box 80500, Wilmington, Delaware 19880-0500
Received September 9, 1998
Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor
assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH
from rat frontal cortex homogenates when compared to the known peptide antagonist R-helical
CRH(9-41) (Ki ) 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated
adenylate cyclase activity in the same tissue, but it was less potent than R-helical CRH(9-41)
(IC50 ) 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the
cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1
receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of
which had superior pharmacokinetic profiles in the rat. This paper describes the structure-
activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic
parameters in the rat. Compound 28-17 (mean hCRH1 Ki ) 32 nM) had a significantly improved
pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog
(20% oral bioavailability at 5 mg/kg) relative to the early lead structures.
In tr od u ction
central nervous system (CNS) has been proposed to
underlie depression and a spectrum of anxiety-related
disorders.5,6 Plasma ACTH and cortisol levels are
elevated in a large segment of the depressed patient
population.1,7 Similarly, levels of CRH in the cerebrospi-
nal fluid (CSF) of depressed patients have been reported
to be higher than those levels in age- and sex-matched
control patients.8 Postmortem studies employing im-
munochemical techniques have shown that CRH-secret-
ing cells are increased 4-fold in the hypothalami of
depressed patients.9 Arguments for a role of CRH in
anxiety disorders have also been advanced.10-14 Intrac-
erebroventricular (icv) injection of CRH in rats elicits
many behavioral and physiological events, which may
be termed “stress-related”.15-17 Moreover, these effects
may be blocked by the peptide antagonist R-helical
CRH(9-41). Chronic restraint stress in rodents or
primates causes long-term activation of the HPA axis.
Transgenic mice, which overexpress CRH, have elevated
plasma ACTH and glucocorticoid levels and manifest
stress-related behaviors.18,19 On the basis of the above
findings, CRH antagonists may be useful for the treat-
ment of depression or anxiety.
Corticotropin-releasing hormone (or factor, CRH or
CRF) is the prime regulator of the hypothalamus-
pituitary-adrenal (HPA) axis, coordinating endocrine,
autonomic, behavioral, and immune responses to
stress.1-3 Synthesis of this 41-amino acid peptide by
corticotrophs in the hypothalamus, followed by trans-
port via the portal system to the anterior pituitary,
stimulates secretion of adrenocorticotropic hormone
(ACTH) from the anterior pituitary into the general
circulation. ACTH stimulates secretion of glucocorticoids
from the adrenal glands. Glucocorticoids exert negative
feedback inhibition on the HPA axis at the levels of the
hypothalamus and pituitary. In CRH knockout mice,
levels of ACTH and corticosterone have been reported
to be only 25% of those concentrations in normal mice,
which testifies to the importance of CRH in regulation
of the HPA axis.4
CRH receptors have become targets for drug design
since dysfunction in the HPA axis has been correlated
with various disorders. Hypersecretion of CRH in the
† Current address: The Scripps Research Institute, La J olla, CA
92037.
There are currently two known subtypes of CRH
receptors, one of which has splice variants.1,20,21 These
subtypes are distinguished by the rank order of poten-
cies of peptide agonists in a coupled adenylate cyclase
assay, as well as their anatomical localization. Rat and
human CRHs have very similar amino acid sequences;
rat and human receptor sequences are highly homolo-
gous. The in vitro pharmacology of rat CRH receptors
was originally studied in brain homogenates; in recent
‡ Current address: Zeneca Inc., 1800 Concord Pike, Wilmington, DE.
§ Current address: Neurocrine Biosciences, 10555 Science Center
Dr, San Diego, CA 92121.
3 Department of CNS Diseases Research.
| Current address: Nitromed Inc., 12 Oak Park Dr, Bedford, MA
01730.
Current address: Cephalon Inc., Brandywine Parkway, West
Chester, PA.
X Current address: Food and Drug Administration, 1451 Rockville
Park, Rockville, MD 20852.
) Stine-Haskell Research Center, Newark, DE 19714.
10.1021/jm980222w CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/18/1999

806 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Arvanitis et al.
Sch em e 1
represented by compound 6 have been described as
CRH1 ligands. Specifically, the binding affinity of com-
pound 6 has been reported (IC50 ) 0.110 µM). We have
reported, in preliminary form, our work on pyrimidine
and triazine CRH antagonists 7 and fused derivatives
thereof.35-39 Finally, others40,41 have reported on related
pyrimidines and triazines of general formula 7, provid-
ing pharmacology data on some derivatives. Compound
7a was reported to have high affinity for hCRH1
receptors (Ki ) 1.7 nM vs 20 nM for D-Phe-CRH(12-
41)). To date, no reports on CRH2-selective ligands have
been published.
Our research focused on the discovery of CRH1
antagonists for the possible treatment of depression or
anxiety disorders. At the time we initiated our program,
the reports cited above had not appeared in print.
Empirical screening of our chemical library using a rat
receptor assay led to the discovery that 15-1 had weakly
displaced [125I]-0-Tyr-oCRH from rat frontal cortex
homogenates when compared to the known peptide
antagonist R-helical CRH(9-41) (Ki ) 5700 nM vs 1
nM). Furthermore, 15-1 weakly inhibited CRH-stimu-
lated adenylate cyclase activity in the same tissue, when
compared to R-helical CRH(9-41) (IC50 ) 20 000 nM
vs 250 nM). We report herein on systematic structure-
activity relationship studies to optimize CRH receptor
binding affinity and pharmacokinetic parameters,
wherein substituents on the heteroaryl and phenyl rings
as well as the central nitrogen were varied. Triazine
variants of 15-1 were also studied. Molecular modeling
studies and research on the effects of restriction of
conformation on biological activity have been reported
separately.37,39,42,43
years, the rodent and human receptors have been cloned
and expressed in stable cell lines for binding assays.
CRH1 receptors are found primarily in the rat cortex,
hypothalamus, amygdala, cerebellum, and pituitary.
The CRH1 receptors substantially outnumber CRH2
sites in the pituitary. The CRH2R receptor is a 411-
amino acid protein, which is similar to the CRH1
subtype (71% overall homology, 83% homology in the
seven-transmembrane regions). CRH2R receptors are
also positively coupled to adenylate cyclase; the rank
order of agonist potencies is sauvagine ) urotensin >
r/hCRH > ovine CRH (oCRH). CRH2R receptors are
primarily found in the rat lateral septum, ventromedial
hypothalamus, amygdala, and entorhinal cortex, while
they are virtually absent in the cerebellum, cortex, and
pituitary. A splice variant, the CRH2â receptor, is a 431-
amino acid protein, which is longer than CRH2R at the
amino terminus and is primarily expressed in rat heart,
lung, and skeletal muscle. However, in human tissues,
the CRH2R subtype is also found in heart, lung, and
skeletal muscle. The functional role of CRH receptor
subtypes in specific diseases is still under investiga-
tion.1,22,23
There are reports in the literature on non-peptide
CRH antagonists, with some biological data on their
affinity for rCRH or hCRH1 receptors. Aminothiazole
derivatives of general formula 1 (Scheme 1) have been
described as CRH antagonists, but no biological data
were provided.24 A series of substituted pyrazoles of
formula 2 and fused and unfused pyrimidines and
pyridines of formulas 3-5 have been reported.25-31 The
pharmacology of one of these compounds, 4a (CP154526-
1, rat CRH Ki ) 5.7 nM vs 33 nM for R-hel-CRH(9-
41)), has been described in detail.32,33 Deazapurines34
Resu lts a n d Discu ssion
Ch em istr y. We sought general routes for the syn-
theses of analogues of 15-1 to enable rapid exploration
of the SAR.
Scheme 2 illustrates a method starting with 1,3-
dicarbonyl compounds or 3-(dimethylamino)propanone
derivatives 8.44 These precursors were readily con-
densed with arylguanidines of formula 9 in refluxing
toluene or N,N-dimethylformamide (DMF) solutions to
afford 2-anilinopyrimidines of formula 10 in 40-80%
yields. Alkylation of the central nitrogen produced the
desired targets 15 in 70-90% yields. Alternatively, the
coupling of various substituted anilines 12 with 2-chloro-
4,6-disubstituted pyrimidines 11 under thermal or base-
mediated conditions generated intermediates 10, which
were then transformed to compounds 15 as described
above.
Scheme 3 depicts variations of the above conversions
where the 4-substituents on the pyrimidine ring were
introduced at later stages in the synthetic sequence.
Condensation of 3-keto esters 16 with arylguanidines
17 under thermal or base-mediated conditions led to the
formation of 2-anilino-4-hydroxypyrimidines 18 in 40-
80% yields. Reaction with POCl3 and subsequent alky-
lation of the central nitrogen generated precursors 14

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 807
Sch em e 2a
a
Reactions: (A) DMF or toluene, ∆, 40-80% yields; (B) (CH2OH)2 or dioxane, ∆ (50-60% yields); (C) NaH, THF, DMF or toluene, ∆
(50-70% yields); (D) NaH, RX, THF or DMF, ∆, 70-90% yields.
Sch em e 3a
a
Reactions: (E) neat, DMF or toluene, ∆ or K2CO3, EtOH (40-80% yields); (F) (i) POCl3, ∆, (ii) NaH, RX, DMF (40-80% yields); (G)
(i) neat, ∆, (ii) NaH, RX, DMF, ∆, (iii) POCl3, ∆ (30-70% yields); (H) R4′H, i-Pr2NEt, THF or DMF, ∆ (30-70% yields); (H′) (where R4′ )
NR′R′′) (i) excess R′NH2, ∆, (ii) NaH, R′′X, DMF.
in 40-80% yields. Nucleophilic displacement of the
halogen at the 4-position of the pyrimidine ring gave
the target compounds 15 in 30-80% yields. Alterna-
tively, couplings with 2-(methylthio)-4-hydroxy-6-sub-
stituted pyrimidines 19 under thermal conditions af-
forded 2-anilino-4-hydroxy-6-substituted pyrimidines.
Alkylation of the central nitrogen, followed by treatment
with phosphorus oxychloride (POCl3), gave intermedi-
ates 14, which were then converted to the final products
15 as outlined above. In some cases, it was preferable
to react intermediates 14 with primary amines and then
alkylate the side chain nitrogen in the presence of a base
to give products 15, where R4′ is NR′R′′ (overall yields
range from 40% to 80%).
Scheme 4 illustrates a route to 2-anilinopyrimidines,
which incorporates a novel variation of the Smiles
rearrangement45,46 as one of its key steps. Copper-
mediated reaction of 2-pyrimidinethiols with 2-iodoa-
nilines 12 (R2 ) I) produced 2-(phenylthio)pyrimidines
21. Base- or acid-catalyzed Smiles rearrangement gen-
erated thiols 22 or their disulfide counterparts. Reduc-
tion of the disulfide-thiol mixtures and subsequent
alkylation of the thiols gave intermediates 23. Alkyla-
tion of the central nitrogen proceeded in straightforward
fashion, as described for Scheme 2 above, to provide
compounds 15 (where R2 ) SMe, overall yields range
from 40% to 80%).
2-Anilinotriazines were prepared by the procedures
outlined in Scheme 5.47 Cyanuric chloride (24) was
treated with methylmagnesium chloride to afford the
2,4-dichloro-6-methyltriazine (25) in 60-70% yields.
Condensation of this intermediate with various substi-
tuted anilines generated monochlorotriazines 26 in 50-
70% yields.48 Alkylation of the central nitrogen pro-
ceeded in 80-95% yields, and subsequent nucleophilic
addition of substituted amines in the presence of bases
such as Hunig’s base produced triazine targets 28 in
60-80% yields. In some cases, it was preferable to treat
intermediates 27 with primary amines and then alky-
late the side chain nitrogen to provide compounds 28.
Compounds 25 were also treated with N-alkylanilines
to generate intermediates 27 in 30-70% yields. There
were also instances where it was advantageous to react
intermediates 26 with amines and alkylate these com-

808 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Arvanitis et al.
Sch em e 4a
a
Reactions: (I) Cu, K2CO3, DMF; (J ) HCl, MeOH, Et2O or NaH, xylene or DMF, ∆; (K) (i) NaBH4, EtOH, (ii) MeI; (L) NaH, RX, DMF
(80-90% yields).
Sch em e 5a
a
Reactions: (W) MeMgCl, CH2Cl2, -30 °C to rt (60-70% yields); (X) 12, DMF or dioxane, rt or ∆ (50-70% yields); (Y) NaH, RX, THF
or DMF (80-95% yields); (Y′) i-Pr2NEt, RNHC6H3R2R4R6, THF, ∆, (60-70% yields); (Z) R4′H, i-Pr2NEt, THF, DMSO or DMF (60-80%
yields); (Z′) (for R4′ ) NR′R′′) (i) R′NH2, i-Pr2NEt, THF, DMSO or DMF (60-80% yields), (ii) NaH, DMSO, R′′X, rt (70-90% yields); (Z′′)
(i) R4′H, i-Pr2NEt, THF, DMSO or DMF (55-67% yields), (ii) NaH, THF, RX, rt (80-95% yields).
pounds to produce products 28 where R4′ is NR′R′′
(overall yields range from 40% to 80%).
most promising compounds in terms of affinity were
evaluated in rat or dog pharmacokinetic studies. The
general pharmacology profiles of a few compounds have
been reported elsewhere.49
P h a r m a cology. The screening strategy for our com-
pounds consisted of evaluation of compounds in a
receptor binding assay to assess affinity, followed by
testing select analogues with good binding affinity (Ki
e 50 nM) in a CRH-coupled adenylate cyclase assay to
measure antagonist potency and rat pharmacokinetic
experiments to determine plasma levels after different
routes of administration. We initially employed a rat
receptor binding assay, in which the displacements of
2-An ilin op yr im id in es. Initial SAR studies using the
rat receptor binding assay focused on the effects of
substitution on the phenyl ring of lead structure 15-1
(Table 1). Replacement of bromine at the 2-position with
iodine afforded a modest enhancement in affinity (com-
pare 15-1 vs 15-2, mean Ki ) 5700 nM vs 3655 nM).
Movement of the iodine from the 2- to the 4- position
retained binding affinity, suggesting that modification
of these two positions in tandem might improve affinity
for rCRH receptors (compare 15-2 with 15-3, mean Ki
) 3655 and 3301 nM, respectively). Introduction of
several other ortho substituents destroyed binding
affinity.36 In the 2-bromophenyl subseries, binding
affinity improved as the size of alkyl groups at the
4-position increased from methyl (molar refractivity
(MR) ) 0.56)50,51 to isopropyl (MR ) 1.50) at the
4-position of the phenyl ring. Replacement of the methyl
[
125I]Tyr-o-CRH by our test compounds from rat frontal
cortex homogenates were measured, since only this
source for the rat receptor was readily available at the
time we began our studies. Subsequently, we switched
to a cloned human hCRH1 receptor binding assay. We
report data from the cloned human receptor assay in
Tables 2-9. Antagonist potency for some high-affinity
compounds was assessed in an adenylate cyclase inhibi-
tion assay using either rat frontal cortex homogenates55
or HEK293 cells expressing hCRH1 receptors.56 The

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 809
Ta ble 1. Initial SAR Studies on 2-Anilinopyrimidinesa
Variation of groups at the 4-position of the phenyl ring
was explored next, for a constant substituent at the
2-position. Binding affinity for hCRH1 receptors de-
creased quickly as the size of the 4-substituent increased
beyond a certain value. In the 2-bromophenyl subseries,
the replacement of 4-isopropyl group with butyl or tert-
butyl (MR ) 1.50 vs 1.96 for both)50,51 reduced binding
affinity (compare 15-7 vs 15-11 and 15-12 (hCRH1 Ki )
63 nM vs >100 and >100 nM, respectively)). The
introduction of moieties, which are approximately iso-
steric to or smaller than the isopropyl group, afforded
derivatives with moderate to good binding affinity in
the 2-methylthiophenyl subseries (e.g., NMe2, OMe,
COCH3; MR ) 1.55, 0.79, 1.12, respectively)50,51 (com-
pare 15-15 (hCRH1 Ki ) 36 nM) vs 15-16, 15-17, and
15-18 (hCRH1 Ki ) 20, 57, and 97 nM, respectively)).
Removal of the 4-substituent reduced affinity (cf. 15-9
(hCRH1 Ki > 1000 nM)). Previously we reported that
the introduction of hydroxyl-substituted groups or very
basic amino groups at the 4-position of the phenyl ring
decreased binding affinity in the 2-methylthiophenyl
set.36 These data indicate that there are steric bulk,
polarity, and basicity constraints on substitution at the
4-position of the phenyl ring.
mean rCRH scheme,
example
R
R2 R3 R4
Ki (nM)
reactions mp (°C)
R-hel-CRH
(9-41)
15-1
15-2
15-3
15-4
15-5
15-6
15-7
1
Me Br
H
H
H
H
H
H
H
H
H
H
I
Me
Et
5700
3655
3301
1099
187
2-B,D
2-B,D
2-B,D
2-B,D
2-B,D
2-B,D
2-B,D
2-B,D
165-167b
amorph
Me
Me
I
H
175-177b
oil
Me Br
Me Br
Me Br
Et Br
126-127
163-164b
151-153b
150-153b
i-Pr 96
i-Pr 46 (63(h))
i-Pr 38
15-8
Et
I
a
b
Note: h, human CRH1 binding assay result. HCl salt.
group on the central nitrogen with an ethyl moiety also
improved binding affinity. Compound 15-7 (mean rat
CRH Ki ) 46 nM) was a pure CRH antagonist; it
inhibited CRH-stimulated adenylate cyclase activity
(IC50 ) 180 nM for inhibition vs IC50 ) 200 nM for R-hel-
CRH(9-41)), but this compound had no effect on iso-
proterenol-stimulated or basal adenylate cyclase activity
in rat frontal brain homogenates. Subsequently, we
discovered that 15-7 had moderate affinity for the cloned
hCRH1 receptor (mean Ki ) 63 nM, n ) 3). This
compound had no significant affinity for several other
receptors (e.g., IC50 > 10 000 nM for serotonin receptors
(vs [3H]dLSD), adrenergic R1 or R2 receptors (vs [3H]-
prazosin or [3H]RX781094, respectively), GABAa (vs [3H]-
GABA), and benzodiazepine (vs [3H]flunitrazepam) as
well as serotonin reuptake sites (vs [3H]citalopram)).52
Unfortunately, intraveneous, intraperitoneal, or oral
administration of 15-7 to rats at 12 mg/kg (iv) or 30 mg/
kg (ip, po) failed to generate detectable levels in plasma.
Compound 15-7 became the starting point for studies,
which were aimed at improving pharmacokinetic profile
without reducing CRH binding affinity. Our operating
hypothesis was that the high lipophilicity of 15-7 (HPLC
log P > 6.4) was largely responsible for poor plasma
levels.
The SAR at the 6-position of the phenyl ring was
briefly explored. Replacement of hydrogen at the 6-posi-
tion by methoxy or methylthio groups afforded com-
pounds with good to moderate binding affinity (compare
15-7 vs 15-23 and 15-24 (hCRH1 Ki ) 63 nM vs 26 and
51 nM, respectively).
Variations in substitution on the central nitrogen in
15-7 have been reported previously.36 Allyl or ethyl
substitutions were optimal; replacement of these two
moieties by hydrogen abolished affinity for hCRH1
receptors.
Substitution on the 4- or 6-position of the pyrimidine
ring in 15-7 was evaluated next. Binding affinity for the
CRH receptor drops as the size of both the 4- and
6-substituents increases from methyl to ethyl to pro-
pyl.36,50,51 However, if the substituent at the 6-position
was held constant as a methyl group, then a wide
variety of substituents could be introduced at the
4-position of the pyrimidine ring, generating anilinopy-
rimidines with moderate to high binding affinity. The
4-(3-pentyl) analogue 15-26 (MR for 3-pentyl ) 2.42)
had high affinity (hCRH1 Ki ) 2nM), indicating large
groups may be tolerated at the 4-position in the pyri-
midine ring. Structurally diverse amino substituents at
this position were also very well-tolerated. Many 4-sub-
stituted amino analogues of 15-7 had high binding
affinity; for example, 15-35, 15-36, 15-37, 15-28, 15-29,
and 15-30 had hCRH1 Ki values of 9, 5, 9, 22, 11, and
14 nM, respectively. Therefore, size alone cannot be
considered a critical factor for substitution at the
4-position of the pyrimidine ring. The introduction of
the dibasic amine piperazine reduced binding affinity
(cf. 15-42 (hCRH1 Ki > 1000 nM)) as did the introduction
of acidic moieties (compare 15-38, bearing a tetrazole
ring with 15-39, bearing a N-methyltetrazole ring
(hCRH1 Ki > 1000 nM vs 41 nM)). Restricting the
conformation of the flexible dialkylamino side chains
had minimal effect on receptor affinity; cyclic amino
analogues had comparable or 2-3-fold weaker affinity
Additional investigations of substituent effects on the
phenyl ring, using the cloned human CRH1 receptor
assay, revealed that additional groups at the 2-position
were well-tolerated. A methylthio group could replace
bromine at the 2-position, affording some analogues
with comparable or superior binding affinity (e.g.,
compare 15-7 (hCRH1 Ki ) 63 nM) with 15-15 (hCRH1
Ki ) 36 nM)). Introduction of methyl or trifluoromethyl
groups also provided analogues with good binding
affinity (compare 15-16 (hCRH1 Ki ) 20 nM) with 15-
19 (hCRH1 Ki ) 54 nM) and 15-21 (hCRH Ki ) 68 nM)).
Removal of an ortho substituent greatly reduced recep-
tor binding affinity (cf. 15-13 (hCRH1 Ki > 1000 nM)).

810 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Arvanitis et al.
Ta ble 2. SAR Studies on 2-Anilino-4,6-dimethylpyrimidines: The Phenyl Ring
mean hCRH1
scheme,
example
R2
Br
Br
Br
Br
Br
H
SMe
SMe
SMe
SMe
Br
Me
CF3
Br
R4
R6
Ki (nM)
reactions
mp (°C)
R-hel-CRH (9-41)
15-9
1
>1000
63
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
OMe
OMe
SMe
2-B,D
2-B,D
2-B,C,D
2-B,D
2-B,C
2-B,D
4-I,J ,K,L
4-I,J ,K,L
a
94-95
15-7
i-Pr
OMe
Bu
t-Bu
i-Pr
151-153b
amorph
124-126b
180-185
66-68
15-10
15-11
15-12
15-13
15-15
15-16
15-17
15-18
15-19
15-20
15-21
15-22
15-23
15-24
35
>100
>100
>1000
36
i-Pr
64-66
NMe2
OMe
COCH3
OMe
NMe2
NMe2
NMe2
i-Pr
20
60
90
12
54
68
5
26
119-120
128-130
125-126
113-115
79-81
a
2-B,D
2-B,D
2-B,D
2-B,D
2-C,D
4-I,J ,K,L
121-123
amorph
amorph
117-119b
Br
Br
i-Pr
51
a
b
See Experimental Section. HCl salt.
Ta ble 3. SAR Studies on 2-Anilinopyrimidines: The Pyrimidine Ring
′
′
mean hCRH1
scheme,
example
R4′
R6′
Ki (nM)
reactions
mp (°C)
R-hel-CRH(9-41)
15-7
1
63
>100
Me
Et
3-pentyl
NHCH-3-pentyl
N(Et)CH2CH2OMe
N(Pr)CH2CH2OMe
N(CH2Ph)CH2CH2OMe
N(CH2CH2OMe)2
NEt2
NBuEt
N(Et)CH2Ph
NPr2
NPr(c-C3H5)
NPr(CH2-c-C3H5)
N(Bu)CH2-5-tetrazolyl
N(Bu)CH2-(1-Me-5-tetrazolyl)
morpholino
piperidinyl
piperazine
Me
Et
2-A,D
2-A,D
2-A,D
3-G,H
3-E,F,H′′
3-E,F,H′′
3-E,F,H′′
3-E,F,H′′
3-G,H
151-153
120-121
oil
15-25
15-26
15-27
15-28
15-29
15-30
15-31
15-32
15-33
15-34
15-35
15-36
15-37
15-38
15-39
15-40
15-41
15-42
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
2
44
22
11
14
25
22
19
17
9
5
9
oil
oil
127-129b
oil
oil
150-152c
159-160
oil
3-G,H
3-E,F,H′′
3-E,F,H′′
3-E,F,H′′
3-G,H
3-E,F,H′
a
2-B,D
2-C,D
2-B,D
oil
oil
138-139
oil
>1000
41
22
29
oil
219-222c
211-212c
233-234c
>1000
a
b
See Experimental Section. Fumarate salt. c HCl salt.
than their acyclic counterparts (compare 15-32, the
diethylamino analogue, with 15-41, the piperidyl de-
rivative (hCRH1 Ki ) 22 and 29 nM, respectively)). The
data from the above discussion suggest that increasing
the lipophilicity of the substituted amino groups at the
4-position of the pyrimidine ring may enhance binding
affinity, but increasing the basicity or acidity of these
groups diminishes binding affinity.
The SARs for discrete regions of the 2-anilinopyrimi-
dine core structure were not independent for each
region. High-affinity analogues could be obtained by
simultaneous modification of substituents at both the
pyrimidine and phenyl termini (Tables 4 and 5).
The SARs for the 2-anilinopyrimidines based on data
reported here and previously36 may be summarized as
depicted in Scheme 6. For the phenyl group, medium-
sized lipophilic substituents at the 2-position, preferably
Br, I, or MeS moieties, enhance hCRH1 binding affinity.
In separate reports,39,42 molecular modeling and NMR
studies indicate that the ortho substituent enforces an
approximate orthogonal relationship between the phen-
yl and pyrimidine rings. Medium-sized lipophilic groups
at the 4-position of the phenyl ring, which may or may
not be weak hydrogen bond acceptors, contribute to good
binding affinity (e.g., i-Pr, OMe, NMe2, COCH3). At the
6-position of the phenyl ring, H, OMe, and SMe appear

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 811
Ta ble 4. SAR Studies on 2-Anilinopyrimidines: Additivity Effects
mean hCRH1
scheme,
example
R
R2
R4
i-Pr
i-Pr
I
R6
Ki (nM)
reactions
mp (°C)
R-hel-CRH(9-41)
15-33
15-40
15-43
15-44
1
22
22
9
14
5
NBuEt
morpholinyl
NEt2
NEt-Bu
NEt2
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
3-G,H
2-B,D
2-C,D
2-C,D
2-C,D
2-C,D
159-160a
219-222b
oil
I
oil
15-46
15-47
COCH3
COCH3
amorph
NEt-Bu
8
118-120a
a
b
Fumarate salt. HCl salt.
Ta ble 5. SAR Studies on 4-(Diethylamino)-2-anilinopyrimidines
mean hCRH1
scheme,
example
R
R2
Br
Cl
CF3
Cl
Br
Br
Cl
Br
Br
Br
Br
R4
R6
Ki (nM)
reactions
mp (°C)
15-46
15-48
15-49
15-50
15-51
15-52
15-53
15-54
15-55
15-56
15-57
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
NEt2
COCH3
OMe
NMe2
OMe
SMe
SO2Me
Br
i-Pr
H
OMe
H
Cl
H
H
Cl
SMe
H
OMe
OMe
5
10
11
15
24
24
24
26
27
31
28
2-C,D
2-B,D
2-B,D
2-C,D
2-C,D
2-C,D
2-C,D
2-C,D
3-G,H
2-C,D
a
amorph
oil
106-107
oil
oil
amorph
oil
amorph
oil
NMe2
Br
COCH3
105-107b
91-94
a
b
See Experimental Section. HCl salt.
Sch em e 6. Structure-Activity Relationships
to be optimal. The minimal studies made at the meta
positions of the phenyl ring do not permit any definitive
conclusions. Small lipophilic alkyl or alkenyl groups on
the central nitrogen contribute to optimal binding
affinity (i.e., ethyl or allyl).36 There is a severe steric
constraint on substitution at the 6-position of the
pyrimidine ring; a methyl group is the optimal substitu-
ent. In contrast, substituents with a wide variety of sizes
and shapes may be introduced at the 4-position of the
pyrimidine ring. Lipophilic substituents, which may be
weakly basic or hydrogen bonding, tend to be favored;
very basic or acidic moieties are not well- tolerated.
Compound 15-19, a silent hCRH1 antagonist from this
series,56 was evaluated in rat pharmacokinetic studies.
Data are presented in Table 9 (doses ) 30 mg/kg (iv,
ip, po)). Intraperitoneal administration afforded higher
plasma levels than did oral administration (mean Cmax
) 1884 nM vs 366 nM). Bioavailability via the ip route
was also superior to that by the oral route (26% vs 4%).
The clearance was unusually high (5.32 L/h/kg). None-

812 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Ta ble 6. SAR Studies on Anilinotriazines versus Pyrimidines
Arvanitis et al.
mean hCRH1
scheme,
example
Z
R2
Br
Br
Br
Br
Br
Br
R4
R6
Ki (nM)
reactions
mp (°C)
28-1
15-55
28-2
15-58
28-3
15-46
28-7
15-49
28-4
N
CH
N
CH
N
CH
N
CH
N
CH
NMe2
NMe2
OMe
H
H
OMe
OMe
H
H
H
H
H
6
27
10
48
17
5
20
11
27
30
7-W,X,Y,Z
3-G,H
7-W,X,Y,Z
2-B,D
7-W,X,Y,Z
2-C,D
7-W,X,Z′′
2-B,D
76-78
oil
122-124
oil
amorph
amorph
oil
106-107
100-101
oil
OMe
COCH3
COCH3
NMe2
NMe2
i-Pr
CF3
CF3
Br
7-W,X,Y,Z
3-G,H
15-59
Br
i-Pr
H
Ta ble 7. SAR Studies on Anilinotriazines versus Pyrimidines
ible with good binding affinity; (2) isopropyl, dimethyl-
amino, cyano, methoxy, trifluoromethoxy, and acetyl
substituents at the 4-position of the phenyl ring con-
tribute to good binding affinity; and (3) hydrogen or a
methoxy group at the 6-position of the phenyl ring
appears to be optimal. All the above data indicate the
SARs for the triazine and pyrimidine series to be
similar.
mean hCRH1 scheme,
example
Z
R4
Ki (nM)
reactions mp (°C)
Compound 28-13 (hCRH1 Ki ) 32 nM, HPLC log P )
3.63) was evaluated in the cloned human CRH1 aden-
ylate cyclase assay and rat and dog pharmacokinetic
studies.53-55 This compound was found to be a silent
hCRH1 antagonist.56 In the first trial, the compound was
administered at 12 mg/kg (iv) and 30 mg/kg (iv, po),
while in a second experiment the compound was given
at 1 mg/kg (iv, po) (Table 9). In the first trial, the
bioavailabilities were 47% (ip) and 19% (po) at 30 mg/
kg and the mean peak plasma levels were 2698 nM (ip)
and 753 nM (po). The differences in bioavailability
between the two routes of administration reflected lower
oral absorption. Furthermore, at 1 mg/kg, the oral
bioavailability was 2%, and the mean peak plasma level
was only 21 nM. Possibly, extensive first-pass metabo-
lism was responsible for the lower plasma levels, but
at the higher dose, saturation of the metabolic enzymes
occurred. Mass spectral studies suggest that one of the
methyl ethers was cleaved, but it was not possible to
assign the metabolite’s structure based on NMR data.
In the dog, 28-13 was evaluated at 5 mg/kg (iv, po). The
oral biovailability was 20% and the mean peak oral
plasma level was 730 nM at 0.5 h. The elimination half-
life after iv administration was 6.0 h. While the rat and
dog pharmacokinetic profiles of this compound were
superior to those for the lead 15-7, members from
another chemical series showed more promising phar-
macokinetic profiles,37,43 and compound 28-13 was not
advanced further.
28-6
N
NBuEt
5
18
18
25
27
30
7-W,X,Y,Z 75-77a
15-33 CH NBuEt
28-7 N(CH2CH2OMe)2
15-31 CH N(CH2CH2OMe)2
28-5 NEt2
3-G,H
159-160b
N
7-W,X,Z′′ oil
3-E,F,H′ oil
7-W,X,Y,Z oil
N
15-32 CH NEt2
3-G,H
150-152a
a
b
Fumarate salt. HCl salt.
theless, these parameters were significantly superior to
those for the lead structure, 15-7. The HPLC log P
values were 4.48 for 15-19 and >6.4 for 15-7. Reduction
in lipophilicity relative to 15-7 improved intraperitoneal
and, to a lesser extent, oral pharmacokinetic profiles in
this case.
2-An ilin otr ia zin es. Anilinotriazines were then ex-
plored in an effort to further reduce lipophilicity and to
improve oral absorption. The replacement of CH by N
in the heterocycle did not adversely affect receptor
binding affinity. The SARs for the triazines were paral-
lel, but not identical, to those for the corresponding
pyrimidines (cf. Tables 6 and 7) when the substitution
on the phenyl ring was varied and the 4-substituent on
the heterocycle was kept constant as a diethylamino
group. The hCRH1 Ki values for pairs of congeners
rarely varied by more than 3-fold. Similarly, when the
phenyl group was kept constant as a 2-bromo-4-isopro-
pylphenyl moiety, the effects of amine substitution at
the 4-position of the triazine ring appeared to be parallel
to that in the pyrimidine series for this limited set of
examples. Table 8 delineates the binding data for
4-substituted triazines with a variety of phenyl ring
substitution patterns. A variety of tertiary amine sub-
stitutions (e.g., cycloalkylamino or methoxyalkylamino
moieties) were well-tolerated at the 4-position of the
triazine ring, which is consistent with the observations
for the pyrimidine series described above. The following
observations were made about the effects of phenyl ring
substituents: (1) bromine, methyl, and trifluoromethyl
groups at the 2-position of the phenyl ring are compat-
Con clu sion
The SARs for 2-anilinopyrimidines and -triazines
have been defined through systematic variation of
substituents in each of the three regions of the core
structures. A weak lead structure 15-1 was transformed
into more potent analogues with significantly improved
binding affinity (>1000-fold), and significant improve-
ments in the pharmacokinetic profiles of some analogues

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 813
Ta ble 8. SAR Studies on 2-Anilinotriazines
mean hCRH1
scheme,
example
R
R2
Br
Br
Br
Br
Br
Br
Br
R4
R6
Ki (nM)
reactions
mp (°C)
90
oil
oil
75-77
oil
75-77
122-124
164-165a
oil
oil
oil
oil
oil
oil
oil
76-78
75-78
100-103
oil
R-hel-CRH(9-41)
28-8
1
33
41
4
5
4
NH-allyl
NH-3-pentyl
N(allyl)2
NBuEt
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
OMe
OMe
Me
H
H
H
H
H
H
OMe
OMe
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
7-W,X,Y,Z
7-W,X,Y′,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z′
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z′
7-W,X,Y,Z
7-W,X,Y,Z′
7-W,X,Y,Z′
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
7-W,X,Y,Z
28-9
28-10
28-6
28-11
28-12
28-2
NPr2
N(allyl)-c-C3H5
NEt2
morpholinyl
N(CH2CH2OMe)Bu
NBuEt
N(CH2CH2OMe)(CH2-c-C3H5)
NEt(CH2CH2OMe)
N(allyl)2
5
10
32
13
23
25
31
4
5
3
5
5
5
5
6
6
7
7
8
8
28-13
28-14
28-15
28-16
28-17
28-18
28-19
28-20
28-1
28-21
28-22
28-23
28-24
28-25
28-26
28-27
28-28
28-29
Br
Me
Me
Me
Me
Br
Br
Br
Br
CF3
CF3
CF3
CF3
CF3
Me
Me
Me
Me
Me
Me
Me
OCF3
OCF3
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NMe2
NBuEt
N(allyl)2
NEt2
NPr(CH2-c-C3H5)
N(CH2-c-C3H5)2
NPr(CH2CH2CN)
NPr2
NBuCH2CN
NPrCH2CH2CN
NPr2
85-87
oil
oil
oil
oil
75-78
NBuCH2CH2CN
NBuCH2CN
a
MeSO3H salt.
Ta ble 9. Rat and Dog Pharmacokinetic Dataa
dose CL t1/2 Cmax Tmax
Proton NMR spectra were recorded on Varian VXR or Unity
300 FT-NMR instruments (300 MHz); chemical shifts were
recorded in ppm (δ) from an internal tetramethylsilane
standard in deuteriochloroform or deuteriodimethyl sulfoxide
as specified below. Coupling constants (J ) were recorded in
hertz (Hz). Mass spectra (MS) were recorded on a Finnegan
MAT 8230 spectrometer or a Hewlett-Packard 5988A model
spectrometer (both using chemical ionization (CI) with NH3
as the carrier gas). Gas chromatography-mass spectrosopy
(GC-MS) was occasionally obtained using the former instru-
ment. Chemical ionization high-resolution mass spectra (CI-
HRMS) were obtained on a VG 7-VSE instrument with NH3
as the carrier gas. Combustion analyses were performed by
Quantitative Technologies Inc., Whitehouse, NJ . Melting
points were measured on a Buchi model 510 melting point
apparatus or a Thomas-Hoover capillary apparatus and are
uncorrected. Boiling points are uncorrected.
example species (mg/kg, route) (L/h/kg) (h) (nM) (h) %F
15-19
28-13
rat
rat
30 (iv)
30 (ip)
30 (po)
12 (iv)
30 (ip)
30 (po)
1 (iv)
5.32
2.9
5.9
1884 0.25 26
366 1.2
4
15.6
2698 0.3
753 1.6
47
19
4.0
1.3
7.6
6.0
1 (po)
5 (iv)
21 0.3
3
dog
5 (po)
730 0.5
20
a
For rat studies, n ) 4; for dog studies, n ) 3. Mean values are
given throughout. 0.5% Methocel-water was the vehicle for oral
and ip administrations; a mixed solvent system was employed for
iv studies (N,N′-dimethylacetamide - ethanol - propylene glycol
- PEG400 - 0.1 N NaOH, 1:1:3:3:2). HPLC detection was used
for all studies except the 1 mg/kg experiment where LC/MS/MS
analysis was utilized.
Reagents were purchased from commercial sources and,
when necessary, purified prior to use according to the general
procedures outlined by Perrin and Armarego.57 Chromatog-
raphy was performed on silica gel using the solvent systems
indicated below. For mixed solvent systems, the volume ratios
are given. Otherwise, parts and percentages are by weight.
All reactions were performed under a nitrogen atmosphere
using magnetic stirring. Reactions requiring anhydrous condi-
tions were performed in glassware, which had been flame-dried
or oven-dried with purging under a nitrogen atmosphere.
Reactions using aqueous media were run under the ambient
atmosphere. Anhydrous magnesium sulfate (MgSO4) was used
routinely to dry the combined organic layers from extractions.
Solvent was routinely removed in vacuo, using a rotary
evaporator, followed by evacuation with vacuum pump.
relative to that for an intermediate lead structure 15-7
were also achieved through these SAR studies. Some
representative compounds had good intraperitoneal, but
modest oral pharmacokinetic profiles in the rat. Com-
pound 28-13, in particular, had a promising profile in
the dog. None of the compounds in this paper were
found to have binding affinity for the cloned hCRH2R
receptors.56 These studies served as the basis for design
of more potent hCRH1 compounds with superior phar-
macokinetic profiles.37,43
Commonly used abbreviations: EtOAc (ethyl acetate), MeOH
(methanol), EtOH (ethanol), DMF (N,N-dimethylformamide),
HOAc (acetic acid), THF (tetrahydrofuran), and TLC (thin-
layer chromatography).
Exp er im en ta l Section
Ch em istr y. Analytical data were recorded for the com-
pounds described below using the following general procedures.

814 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Arvanitis et al.
N-[2-Br om o-4-(1-m eth yleth yl)p h en yl]-N-eth yl-4-m eth -
yl-6-m or p h olin yl-2-p yr im id in a m in e, Hyd r och lor id e Sa lt
(15-40) (Sch em e 2, Step s B, D). A mixture of 2,4-dichloro-
6-methylpyrimidine (4 g, 24.5 mmol), morpholine (2.14 mL,
24.5 mmol), and diisopropylethylamine (4.52 mL, 26.0 mmol)
in ethanol (60 mL) was stirred at 0 °C for 3 h, at 25 °C for 24
h, and at reflux temperature for 1 h. The solvent was then
removed in vacuo, and the residue was taken up in a 1 M
NaOH solution (50 mL) and extracted with EtOAc three times
(200 mL). The combined organic layers were washed with
water and then brine; then they were dried, filtered, and
concentrated in vacuo to afford a solid. Recrystallization from
EtOAc-hexanes gave 2-chloro-4-morpholinyl-6-methylpyrimi-
dine (3.8 g): 1H NMR (CDCl3) δ 6.24 (s, 1H), 3.78-3.74 (m,
2H), 3.64-3.61 (m, 2H), 2.35 (s, 3H).
mixture was partitioned between EtOAc (120 mL) and a 0.5
M NaOH solution (100 mL). The aqueous layer was extracted
with EtOAc (120 mL), and the combined organic layers were
washed with water and then brine. Drying, filtration, and
removal of solvent in vacuo afforded a residue. Column
chromatography (8% EtOAc-hexanes) gave the title product
(2.88 g, 73% yield): 1H NMR (CDCl3) δ 7.50 (d, 1H, J ) 2),
7.19 (dd, 1H, J ) 8, 2), 6.85 (br s, 1H), 6.30 (s, 1H), 4.13-4.10
(m, 1H), 3.82-3.76 (m, 1H), 3.00-2.85 (m, 1H), 2.23 (br s, 6H),
1.28 (d, 6H, J ) 7), 1.19 (t, 3H, J ) 7).
Treatment of the free base with a solution of HCl in ether
(1M, 10 mL, 10 mmol) afforded a solid, which was collected
by filtration. Trituration with ether, filtration, and dyring in
vacuo afforded a powder: mp 151-153 °C. Anal. (C17H22BrN3‚
HCl) C, H, N, Br, Cl.
A mixture of the above intermediate (1 g, 4.7 mmol) and
2-bromo-4-isopropylaniline (1 g, 4.7 mmol) in ethylene glycol
(6 mL) was heated at reflux temperature for 1.5 h. After being
cooled to ambient temperature, it was partitioned between
EtOAc (100 mL) and a 1 M NaOH solution (20 mL). The
organic layer was washed with water and brine, dried, filtered,
and concentrated in vacuo. The crude product was chromato-
graphed (EtOAc-hexanes, 1:3) to give 2-N-[2-bromo-4-(1-
methylethyl)phenyl]-4-morpholinyl-6-methylpyrimidamine-
(1.5 g): 1H NMR (CDCl3) δ 8.34 (d, 1H, J ) 8), 7.38 (d, 1H, J
) 2), 7.20 (s, 1H), 5.92 (s, 1H), 3.80-3.76 (m, 2H), 3.61-3.57
(m, 2H), 2.90-2.78 (m, 1H), 2.30 (s, 3H), 1.23 (d, 6H, J ) 7).
A solution of the above intermediate (1.0 g, 2.56 mmol) in
THF (10 mL) was treated with NaH (60% in oil, 0.15 g, 3.75
mmol) at room temperature for 20 min, and then EtI (0.32
mL, 4 mmol) was added. The mixture was stirred for 24 h and
then at reflux temperature for 5 h and was partitioned between
EtOAc (100 mL) and water (20 mL). The organic extract was
washed with brine, dried, filtered, and concentrated in vacuo.
The crude product was chromatographed (EtOAc-hexanes, 12:
88) to give the title product (0.94 g) as the free base: 1H NMR
(CDCl3) δ 7.48 (s, 1H), 7.16 (s, 1H), 7.16 (s, 1H), 5.75 (s, 1H),
4.2-4.0 (br, 1H), 3.80-3.60 (br, 1H), 3.70-3.60 (br, 2H), 3.50-
3.30 (br, 2H), 2.96-2.80 (m, 1H), 2.2 (br, 3H), 1.26 (d, 6H, J )
7), 1.19 (t, 3H, J ) 7).
N-(2-Br om o-4-m eth oxyph en yl)-N-eth yl-4,6-dim eth ylpy-
r im id in -2-a m in e (15-10) (Sch em e 2, Step s C, D). Sodium
hydride (60% in oil, 554 mg, 13.9 mmol) was washed with
hexanes and decanted twice. Toluene (15 mL) was added,
followed by a solution of 2-bromo-4-methoxyaniline (1.27 g,
6.30 mmol) in toluene (15 mL). After the reaction mixture was
stirred for 15 min, 2-chloro-4,6-dimethylpyrimidine (1.35 g,
9.45 mmol) was added. The reaction was then warmed to reflux
temperature and stirred for 42 h. After the mixture was cooled
to room temperature, water was carefully added to quench the
reaction. Three extractions with EtOAc, drying the combined
organic layers, filtration, and removal of solvent in vacuo gave
a brown solid. Recrystallization from EtOAc-hexanes afforded
N-(2-br om o-4-m et h oxyph en yl)-4,6-dim et h ylpyr im idin -2-
amine (547 mg, 25% yield): mp 118-119 °C; 1H NMR (CDCl3)
δ 8.40 (d, 1H, J ) 9), 7.2 (br s, 1H), 7.1 (d, 1H, J ) 2), 6.9 (dd,
1H, J ) 9, 2), 6.5 (s, 1H), 3.8 (s, 3H), 2.35 (s, 6H); CI-HRMS
m/z calcd 308.0398, found 308.0404 (M + H)+.
Sodium hydride (60% in oil, 56 mg, 1.4 mmol) was washed
with hexanes and decanted twice. Anhydrous DMF (10 mL)
was added with stirring. The above intermediate (357 mg (1.2
mmol) was added, and the reaction mixture was stirred for
30 min. Iodoethane (0.11 mL, 1.4 mmol) was added; the
mixture was then stirred at ambient temperature for 16 h.
Water (10 mL) was carefully added. The resulting mixture was
extracted three times with dichloromethane (20 mL). The
combined organic layers were washed with water (20 mL)
twice, dried, and filtered. Solvent was removed in vacuo.
Column chromatography (EtOAc-hexanes, 1:4) gave the title
product, a tan solid, after removal of solvent in vacuo (293 mg,
75% yield): 1H NMR (CDCl3) δ 7.20 (d, 1H, J ) 2), 7.15 (d,
1H, J ) 7), 6.9 (dd, 1H, J ) 7, 2), 6.3 (s, 1H), 4.25-4.15 (m,
1H), 3.8 (s, 3H), 3.75-3.65 (m, 1H); CI-HRMS m/z calcd
336.1077, found 336.0711 (M + H)+. Anal. (C15H18BrN3O) C,
H, Br, N.
N-(2-Br om o-4-isop r op ylp h en yl)-N-eth yl-4-(d ieth yla m i-
n o)-6-m eth ylp yr im id in -2-a m in e, Hyd r och lor id e Sa lt (15-
32) (Sch em e 3, Step s G, H). A mixture of 6-methyl-2-
(methylthio)-4(3H)-pyrimidinone (20 g, 128 mmol) and 2-bromo-
4-isopropylaniline (36 g, 169 mmol) was heated at 190 °C for
7 h. Vacuum distillation afforded a semisolid residue. Recrys-
tallization from ether provided 2-[(2-bromo-4-isopropylphenyl)-
amino]-6-methyl-4(3H)-pyrimidinone, a colorless white solid
(24.0 g, 48% yield): 1H NMR (CDCl3) δ 8.04 (d, 1H, J ) 8),
7.43 (d, 1H, J ) 2), 7.18 (dd, 1H, J ) 8, 2), 5.91 (s, 1H), 2.87
(m, 1H, J ) 7.0), 2.21 (s, 3H), 1.22 (d, 6H, J ) 7).
A mixture of the above intermediate (23 g, 66.7 mmol) and
POCl3 (70 mL) was stirred at room temperature for 5 h.
Concentration in vacuo gave a thick liquid which was poured
onto ice. After the ice had melted, EtOAc (200 mL) was added,
and the two-phase mixture was stirred for 15 min. The organic
phase was separated and washed with water (50 mL), a
saturated NaHCO3 solution (100 mL), and brine (100 mL).
Drying, filtration, and removal of solvent in vacuo provided
2-[(2-bromo-4-isopropylphenyl)amino]-4-chloro-6-methylpyri-
midine, a thick amber oil (23 g, 95% yield): 1H NMR (CDCl3)
δ 8.34 (d, 1H, J ) 8.4), 7.50 (br s, 1H), 7.42 (d, 1H, J ) 2),
7.20 (dd, 1H, J ) 8, 2), 6.67 (s, 1H), 2.86 (m, 1H, J ) 7), 2.41
(s, 3H), 1.24 (d, 6H, J ) 7).
The free base was dissolved in ether (10 mL) and treated
with a solution of HCl in ether (1 M, 4 mL, 4 mmol). The
precipitate was collected and dried in vacuo (0.94 g, 98%
yield): mp 219-222 °C. Anal. (C20H27N4BrO‚HCl‚0.5H2O) C,
H, N, Br, Cl.
2-N-[2-Br om o-4-(1-m eth yleth yl)p h en yl]-N-eth yl-4,6-d i-
m et h yl-2-p yr im id in a m in e, H yd r och lor id e Sa lt (15-7)
(Sch em e 2, Step s A, D). 2-Bromo-4-isopropylaniline (6 g, 28.2
mmol) and cyanamide (4.7 g, 112.1 mmol) were dissolved in a
mixture of EtOAc (100 mL) and EtOH (13 mL). To this mixture
was added a solution of HCl in ether (1 M, 38 mL 38 mmol).
The reaction mixture was stirred at room temperature for 1
h, and then, the ether was distilled from the reaction mixture.
The remaining solution was heated at reflux for 3 h, cooled to
ambient temperature, and diluted with ether (120 mL). The
precipitate was collected by filtration and dried to give the
crude guanidinium hydrochloride (10.4 g), which was used for
the next reaction without purification.
The crude product from the previous reaction (5 g, 13.5
mmol), K2CO3 (1.86 g, 13.5 mmol), and 2,4-pentanedione (2.8
mL, 27.28 mmol) were mixed in DMF (35 mL) and stirred at
reflux temperature for 24 h. After being cooled to room
temperature, the reaction mixture was partitioned between
EtOAc (120 mL) and a 0.5 M NaOH solution (100 mL). The
aqueous layer was extracted with EtOAc (120 mL), and the
combined organic layers were washed with water and then
brine. Drying, filtration, and removal of solvent in vacuo
afforded a residue. Column chromatography (8% EtOAc-
hexanes) gave 2-N-[2-bromo-4-(1-methylethyl)phenyl]-4,6-di-
methyl-2-pyrimidinamine (3.37 g).
The above intermediate (3 g, 9.37 mmol) was reacted with
NaH (60% in oil, 0.4 g, 10 mmol, prewashed with hexanes)
and EtI (1.7 g, 11 mmol) in THF at ambient temperature for
16 h. After being cooled to room temperature, the reaction

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 815
Sodium hydride (100%, 240 mg, 10 mmol) was added to a
solution of the above intermediate (2.5 g, 6.9 mmol) in
anhydrous DMSO (10 mL). After 5 min, iodoethane (2.5 g, 16
mmol) was added, and the reaction mixture was stirred at
ambient temperature for 1 h. The reaction mixture was
partitioned between ethyl acetate and water, and the organic
phase was washed with brine, dried, and evaporated to give
N-(2-bromo-4-isopropylphenyl)-N-ethyl-4-chloro-6-methylpyri-
midin-2-amine, a thick amber oil (2.05 g, 76.4% yield): 1H
NMR (CDCl3) δ 7.51 (d, 1H, J ) 2), 7.20 (m, 2H), 5.28 (s, 1H),
4.18 (m, 1H), 3.69 (m, 1H, J ) 7), 2.92 (m, 1H, J ) 7), 2.21 (br
s, 3H), 1.28 (d, 6H, J ) 7), 1.20 (t, 3H, J ) 7); MS (CI) m/z
368, 370 (M + H)+.
A solution of the above intermediate (900 mg, 2.31 mmol)
in a 1:1 mixture of DMSO and diethylamine (5 mL) was stirred
at reflux temperature for 4 h. Additional diethylamine (2.5 mL)
was added at 0.5-h intervals. The cooled reaction mixture was
poured onto brine and extracted twice with ethyl acetate. The
combined extracts were dried and evaporated in vacuo.
Column chromatography (EtOAc-hexanes, 15:85, then 30:70,
then 1:1) gave the title compound, an oil (860 mg, 92% yield):
1H NMR (CDCl3) δ 7.48 (s, 1H), 7.15 (s, 2H), 5.65 (s, 1H), 4.15
(br s, 1H), 3.75 (br s, 1H), 3.21 (br s, 2H), 2.90 (m, 1H J ) 7),
2.21 (br s, 3H), 1.25 (d, 6H, J ) 7), 1.20 (t, 3H, J ) 7), 0.97 (br
s, 6H); MS (CI) m/z 405, 407 (M + H)+. Anal. (C20H29N4Br) C,
H, N.
(MgSO4), and concentrated in vacuo. Column chromatography
(20% EtOAc-hexanes) gave ethyl 4-amino-3-iodobenzoate
(7.92 g, 90% yield): 1H NMR (CDCl3) δ 8.33 (d, 1H J ) 2),
7.82 (dd, 1H, J ) 8, 2), 6.70 (d, 1H, J ) 8), 4.32 (q, 2H, J ) 7),
4.50-4.00 (br s, 2H), 1.36 (t, 3H, J ) 7).
Ethyl 4-amino-3-iodobenzoate (4 g, 13.7 mmol), 4,6-dimeth-
yl-2-mercaptopyrimidine (2.32 g, 16.5 mmol), K2CO3 (2.37 g,
17.00 mmol), and Cu powder (0.52 g, 8.46 mmol) were mixed
in DMF (30 mL) and stirred at reflux temperature for 30 min.
After being cooled to ambient temperature, the mixture was
diluted with EtOAc (60 mL) and the solids were collected by
filtration. The filtrate was partitioned between EtOAc (100
mL) and water (100 mL). The organic extract was washed
twice with water (100 mL). The combined aqueous layers were
extracted with EtOAc (50 mL). The combined organic extracts
were washed with brine (50 mL), dried, and concentrated in
vacuo. The crude product, S-(2-amino-5-carbethoxyphenyl)-4,6-
dimethyl-2-thiopyrimidine (3.95 g, 95%), was used without
further purification: 1H NMR (CDCl3) δ 8.19 (d, 1H, J ) 2),
7.92 (dd, 1H, J ) 8, 2), 6.77 (d, 1H, J ) 8), 6.70 (s, 1H), 4.70
(br s, 2H), 4.31 (q, 2H, J ) 7), 2.33 (s, 3H), 1.36 (t, 3H, J ) 7).
The above intermediate (2.95 g, 7.72 mmol) was dissolved
in MeOH (40 mL) and treated with a solution of HCl in ether
(1 M, 10 mL, 10 mmol) at room temperature for 60 h. The
solvent was concentrated in vacuo. The residue was dissolved
in EtOH (70 mL), and the resulting solution was cooled to 0
°C. NaBH4 (603 mg, 16 mmol) was added in portions, and the
mixture was allowed to warm to room temperature with
stirring over 1 h. Iodomethane (0.6 mL, 9.6 mmol) was added;
then the reaction mixture was stirred for 16 h. EtOAc (50 mL)
was added, and the precipitate was removed by filtration. The
filtrate was concentrated in vacuo. Column chromatography
(20% EtOAc-hexanes) gave N-[2-(methylthio)-4-carbethoxy-
phenyl]-4,6-dimethyl-2-pyrimidinamine (1.45 g, 45% overall
yield): 1H NMR(CDCl3) δ 8.80 (d, 1H, J ) 9 Hz), 8.47 (br s,
1H), 8.22 (d, 1H, J ) 2), 8.00 (dd, 1H, J ) 9, 2), 6.60 (s, 1H),
4.36 (q, 2H, J ) 7), 2.42 (s, 3H), 2.41 (s, 3H), 1.39 (t, 3H, J )
7).
The HCl salt was prepared by treatment with a solution of
HCl in ether (1 M, 3.0 mL, 3.0 mmol), followed by recrystal-
lization from EtOAc-ether: mp 150-152 °C; CI-MS m/z 405,
407 (M + H)+.
N-(2-Iod o-4-isop r op ylp h en yl)-4-ch lor o-6-m et h ylp yr i-
m id in -2-a m in e (Sch em e 3, Step s E, F ). A mixture of 2-iodo-
4-isopropylphenylguanidine (12.0 g, 39.5 mmol), ethyl ace-
toacetate (15 mL, 118 mmol), and K2CO3 (2 g, 14.50 mmol) in
ethanol (120 mL) was stirred at reflux temperature for 100 h.
The reaction mixture was cooled to ambient temperature; the
solvent was removed in vacuo. The residue was chromato-
graphed (EtOAc-hexanes, 4:6) to give 2-N-[2-iodo-4-(1-meth-
ylethyl)phenyl]-4-hydroxy-6-methyl-2-pyrimidinamine (4.0 g,
27% yield): 1H NMR (CDCl3) δ 7.74 (d, 1H, J ) 8), 7.70 (s,
1H), 7.23 (d, 1H, J ) 8), 5.88 (s, 1H), 2.92-2.8 (m, 1H), 2.18
(s, 3H), 1.24 (d, 6H, J ) 7); CI-MS m/z 370 (M + H)+.
The above intermediate (2.5 g, 6.7 mmol) was dissolved in
POCl3 (20 mL), and the reaction mixture was stirred for 4 h.
The mixture was then poured into ice water. The aqueous mix
was stirred for 30 min and then extracted twice with EtOAc
(200 mL). The combined organic extracts was washed with
brine, dried, and concentrated in vacuo. The residue was
chromatographed (EtOAc-hexanes, 1:4) to give 2-N-[2-iodo-
4-(1-m et h ylet hyl)ph enyl]-4-ch loro-6-m et h yl-2-pyrim idin-
amine (1.64 g, 63% yield): 1H NMR (CDCl3) δ 8.17 (d, 1H, J
) 9), 7.66 (d, 1H, J ) 2), 7.27 (br s, 1H), 7.23 (dd, 1H, J ) 9,
2), 2.89-2.90 (m, 1H), 2.40 (s, 3H), 1.23 (d, 6H, J ) 7).
The above intermediate (1.6 g, 4.2 mmol) was reacted with
NaH (60% in oil, prewashed with hexanes, 190 mg, 5.0 mmol)
and iodoethane (1.0 g, 5.5 mmol) in DMF (25 mL) for 24 h.
The reaction mixture was poured onto water (200 mL). The
aqueous mix was extracted three times with EtOAc. The
combined organic layers were dried, filtered, and concentrated
in vacuo. Flash chromatography (EtOAc-hexanes, 1:6) af-
forded the title product (1.2 g, 70% yield): 1H NMR(CDCl3) δ
7.77 (d, 1H, J ) 2), 7.25 (dd, 1H, J ) 8, 2), 7.09 (d, 1H, J ) 8),
4.30-4.16 (m, 1H), 3.68-3.52 (m, 1H), 2.96-2.84 (m, 1H),
2.42-2.10 (m, 3H), 1.27 (d, 6H, J ) 7), 1.22 (t, 3H, J ) 7);
CI-MS m/z 416, 418 (M + H)+.
N-[2-(Methylthio)-4-carbethoxyphenyl]-N-ethyl-4,6-dimethyl-
2-pyrimidinamine (Scheme 4, Steps I-L, Acid-Catalyzed
Smiles Rearrangement). A solution of ethyl 4-aminobenzoate
(5 g, 30.3 mmol) in CH2Cl2 (12 mL) was added to a solution of
NaHCO3 (4.2 g, 50 mmol) in water (12 mL). Iodine (9.25 g,
39.2 mmol) was added, and then the mixture was stirred at
room temperature for 20 h. The reaction mixture was parti-
tioned between EtOAc (100 mL) and a saturated Na2SO3
solution. The organic extract was washed with brine, dried
The above intermediate (1.2 g, 3.8 mmol) was dissolved in
dry DMF (12 mL) and treated with NaH (60% in oil, 278 mg,
6.88 mmol) at room temperature for 15 min. EtI (0.64 mL, 8
mmol) was added, the reaction mixture was stirred for 16 h,
and then it was partitioned between EtOAc (100 mL) and
water (30 mL). The organic extract was washed twice with
water (30 mL) and then brine (30 mL), dried, filtered, and
concentrated in vacuo. Column chromatography (20% EtOAc-
hexanes) provided the title product (1.2 g, 92% yield): mp 99-
1
100 °C; H NMR (CDCl3) δ 7.96 (d, 1H, J ) 2), 7.85 (dd, 1H,
J ) 8, 2), 7.21 (d, 1H, J ) 8), 4.40 (q, 2H, J ) 7), 4.05-3.90
(br s, 2H), 2.42 (s, 3H), 2.22 (s, 6H), 1.41 (t, 3H, J ) 7), 1.19 (t,
3H, J ) 7). Anal. (C18H23N3O2S) C, H, N, S.
N-(4-Acetyl-2-br om o-6-m eth oxyp h en yl)-N-eth yl-4-(d i-
eth yla m in o)-6-m eth ylp yr im id in a m in e (15-57). A mixture
of N-(2,4-dibromo-6-methoxyphenyl)-N-ethyl-4-(diethylamino)-
6-methylpyrimidinamine (2.0 g, 4 mmol), ethoxyvinyltri-n-
butyltin (1.4 mL, 4.15 mmol), Pd(PPh3)2Cl2 (70 mg, 0.1 mmol),
and Pd(PPh3)4 (100 mg, 0.1 mmol) in dry toluene (15 mL) was
heated at reflux temperature for 20 h. After being cooled to
room temperature, the reaction mixture was concentrated in
vacuo and the residue was taken up in a 10% HCl solution
(75 mL) and stirred for 1 h. The aqueous solution was washed
twice with ether (30 mL) and then basified with a 50% NaOH
solution. The basic solution was extracted twice with EtOAc
(50 mL), and the combined organic extracts were washed with
brine (30 mL), dried, filtered, and concentrated in vacuo. The
residue was chromatographed (20% EtOAc-hexanes) to give
the title product (890 mg, 51% yield): mp 91-94 °C; 1H NMR
(CDCl3) δ 7.80 (d, 1H, J ) 2), 7.51-7.46 (m, 1H), 5.72-5.65
(m, 1H), 4.05-3.80 (m, 2H), 3.81 (s, 3H), 3.55-3.45 and 3.15-
3.05 (2m, 4H), 2.61 (s, 3H), 2.29, 2.02 (2s, 3H), 1.10-1.24 and
0.82-0.94 (2m, 6H), 1.16 (t, 3H, J ) 7). Anal. (C20H27BrN4O2)
C, H, N, Br.
2-N-[4-Meth oxy-2-(m eth ylth io)p h en yl]-N-eth yl-4,6-d i-
m eth yl-2-p yr im id in a m in e (15-17). A mixture of 2-N-[4-

816 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5
Arvanitis et al.
bromo-2-(methylthio)phenyl]-N-ethyl-4,6-dimethyl-2-pyrimi-
dinamine (352 mg, 1 mmol), CuBr (14.3 mg, 0.1 mmol), a
solution of MeONa in MeOH (25% w/w, 0.5 mL, 2.5 mmol),
and dry DMF (5 mL) was heated to reflux temperature and
stirred for 1.5 h. After being cooled to ambient temperature,
the reaction mixture was partitioned between EtOAc (100 mL)
and water (30 mL). The organic layer was washed twice with
water (30 mL) and once with brine, dried, and concentrated
in vacuo. The residue was chromatographed (20% EtOAc-
hexanes) to give the title product (210 mg, 69% yield): mp
N-(2-Br om o-4-isopr opylph en yl)-N-eth yl-4-(dipr opylam i-
n o)-6-m et h yl-1,3,5-t r ia zin -4-a m in e (28-11) (Sch em e 5,
Step s Y′, Z). A solution of 2,4-dichloro-6-methyl-1,3,5-triazine
(1.3 g, 7.9 mmol), diisopropylamine (3.0 mL, 17.4 mmol), and
N-ethyl-2-bromo-4-isopropylaniline (1.92 g, 7.9 mmol) in THF
(65 mL) was heated at reflux temperature for 2 h. The cooled
reaction mixture was poured onto water, and the aqueous
mixture was extracted twice with EtOAc. The combined
extracts were washed with brine, dried, and evaporated in
vacuo. Column chromatography (EtOAc-hexanes, 1:19, then
1:9) provided N-(2-bromo-4-isopropylphenyl)-N-ethyl-2-chloro-
6-methyl-1,3,5-triazin-4-amine, an oil (1.96 g, 67% yield): 1H
NMR (CDCl3) (this spectrum is consistent for approximately
equal populations of two rotamers) δ 7.53 (s, 1H), 7.25 (d, 1H,
J ) 8), 7.10 (d, 1H, J ) 8), 4.19 (m, 1H), 3.67 (m, 1H), 2.94
(m, 1H), 2.51 (s, 1.5H), 2.28 (s, 1.5H), 1.29 (d, 3H, J ) 7), 1.28
(d, 3H, J ) 7), 1.23 (t, 1.5H, J ) 7), 1.22 (t, 1.5H, J ) 7).
A solution of the above intermediate (1.2 g, 3.2 mmol),
dipropylamine (2.6 g, 3.5 mL, 25.2 mmol), and diisopropyl-
ethylamine (0.65 mL, 3.7 mmol) in dry DMSO (20 mL) was
heated at 150 °C for 1 h. The cooled mixture was poured onto
water (150 mL) and extracted twice with ethyl acetate (75 mL).
The combined extracts were washed with brine, dried, and
evaporated in vacuo. Column chromatography (CH2Cl2-
MeOH, 98:2) afforded the title product, an oil (1.16 g, 94%
yield). Anal. (C17H32N5Br) C, H, N, Br.
2-N -[4-Ace t yl-2-(m e t h ylt h io)p h e n yl]-N -e t h yl-4,6-d i-
m eth yl-2-p yr im id in a m in e (15-18). 2-N-[4-Cyano-2-(meth-
ylthio)phenyl]-N-ethyl-4,6-dimethyl-2-pyrimidinamine (0.5 g,
1.68 mmol) was dissolved in dry C6H6 (10 mL). A solution of
CH3MgI in ether (3 M, 1.1 mL, 3.3 mmol) was added. The
mixture was stirred for 2 h and at reflux temperature for 1 h.
After the mixture cooled to room temperature, water (5 mL)
and a 10% HCl solution (5 mL) were added. Stirring was
continued for 15 min. A 1 M NaOH solution was added until
the reaction mixture was alkaline. Three extractions with
EtOAc, drying the combined organic layers, filtration, and
removal of solvent in vacuo afforded a residue. Column
chromatography (20% EtOAc-hexanes) provided the title
product (370 mg, 70% yield): mp 125-126 °C; 1H NMR (CDCl3)
δ 7.89 (d, 1H, J ) 2), 7.75 (dd, 1H, J ) 8, 2), 7.25 (d, 1H, J )
8), 6.33 (s, 1H), 4.05-3.90 (m, 2H), 2.64 (s, 3H), 2.43 (s, 3H),
2.23 (s, 6H), 1.20 (t, 3H, J ) 7). Anal. (C17H21N3OS) C, H, N,
S.
1
128-130 °C; H NMR (CDCl3) δ 7.04 (d, 1H, J ) 9), 6.80 (d,
1H, J ) 3), 7.70 (dd, 1H, J ) 9, 3), 6.28 (s, 1H), 4.35-4.20 (m,
1H), 3.85 (s, 3H), 3.65-3.50 (m, 1H), 2.35 (s, 3H), 2.22 (s, 6H),
1.18 (t, 3H, J ) 7). Anal. (C16H21N3OS‚0.25H2O) C, H, N, S.
N-[2-Br om o-4-(d im et h yla m in o)p h en yl]-N-et h yl-4-(d i-
eth ylam in o)-6-m eth yl-1,3,5-tr iazin -2-am in e (28-1) (Sch em e
5, Step s W-Z). A solution of methylmagnesium bromide in
ether (3 M, 100 mL, 300 mmol) was added dropwise to a stirred
solution of cyanuric chloride (13.0 g, 70.5 mmol) in anhydrous
dichloromethane (300 mL) at -20 °C. After the addition was
complete, the reaction mixture was stirred at -20 °C for 4 h,
after which time water (38 mL, 2.1 mol) was added dropwise
at a rate such that the temperature of the reaction stayed
below 10 °C. After warming to room temperature, the reaction
mixture was diluted with additional water and methylene
chloride and passed through a pad of filter-aid. The organic
layer was dried and evaporated to give 2,4-dichloro-6-methyl-
1,3,5-triazine, a yellow solid (9.0 g, 77.8%): mp 170 °C dec;
1H NMR (CDCl3) δ 2.71 (3H, s).
A mixture of 2,4-dichloro-6-methyltriazine (1.78 g, 10.8
mmol), 2-bromo-4-(dimethylamino)aniline (2.33 g, 10.8 mmol),
diisopropylethylamine (4.19 g, 32.5 mmol), and dioxane (20
mL) was stirred at room temperature for 16 h; then it was
poured onto water (200 mL). Three extractions with EtOAc
(75 mL), two washings of the combined organic layers with
water (50 mL), drying the combined organic layers, filtration,
and removal of solvent in vacuo provided a solid.
Column chromatography (EtOAc-hexanes, 1:4) gave one
fraction after removal of solvent in vacuo: N-[2-bromo-4-
(dimethylamino)phenyl]-4-chloro-6-methyltriazin-2-amine (2.13
g, 57% yield): mp 183-184 °C; NMR (CDCl3) δ 7.90-7.80 (m,
1H), 7.45-7.28 (m, 1H), 6.90 (d, 1H, J ) 3), 6.70 (dd, 1H, J )
9, 3), 2.48 (s, 3H), 2.96 (s, 6H).
Sodium hydride (60% in oil, 200 mg, 5.0 mmol) was washed
with hexanes and decanted twice. Anhydrous DMF (7 mL) was
added, followed by a solution of the above intermediate (1.63
g, 4.8 mmol) in DMF (6 mL). The reaction mixture was stirred
until gas evolution stopped. Iodoethane (780 mg, 5.00 mmol)
was added. The reaction mixture was stirred for 60 h at room
temperature; then it was poured onto water (100 mL). Three
extractions with EtOAc (75 mL), two washings of the combined
organic layers with water (50 mL), drying the combined
organic layers, filtration, and removal of solvent in vacuo
provided a solid. Column chromatography (EtOAc-hexanes,
1:4) gave N-[2-bromo-4-(dimethylamino)phenyl]-N-ethyl-4-
chloro-6-methyltriazin-2-amine, a white solid (Rf ) 0.3, 1.45
g, 82% yield), after removal of solvent in vacuo: 1H NMR
(CDCl3) δ 7.00 (d, 1H, J ) 9), 6.95 (d, 1H, J ) 3), 6.69-6.64
(m, 1H), 4.26-4.12 (m, 1H), 3.69-3.61 (m, 1H), 3.00, 2.99 (2s,
6H), 2.49, 2.28 (2s, 3H), 1.21 (t, 3H, J ) 7).
Biology. Ra t CRH Recep tor Bin d in g Assa y: This assay
was performed essentially as described in the literature53 with
minor modifications. Mean Ki values from two determinations
each are reported.
Clon ed Hu m a n CRH1 Bin d in g Assa y: The following is a
description of the isolation of cell membranes containing cloned
human CRF-R1 receptors for use in the standard binding assay
as well as a description of the assay itself.
Messenger RNA was isolated from human hippocampus.
The mRNA was reverse-transcribed using oligo (dt) 12-18,
and the coding region was amplified by PCR from start to stop
codons. The resulting PCR fragment was cloned into the
EcoRV site of pGEMV, from whence the insert was reclaimed
using XhoI + XbaI and cloned into the XhoI + XbaI sites of
vector pm3ar (which contains a CMV promoter, the SV40 't′
splice and early poly(A) signals, an Epstein-Barr viral origin
of replication, and a hygromycin selectable marker). The
resulting expression vector, called phchCRFR, was transfected
in 293EBNA cells, and cells retaining the episome were
selected in the presence of 400 µM hygromycin. Cells surviving
4 weeks of selection in hygromycin were pooled, adapted to
growth in suspension, and used to generate membranes for
the binding assay described below. Individual aliquots con-
taining approximately 1 × 108 of the suspended cells were then
centrifuged to form a pellet and frozen.
A mixture of the above intermediate (400 mg, 1.1 mmol),
diethylamine (788 mg, 10.8 mmol), and diisopropylethylamine
(0.42 g, 3.24 mmol) was stirred at room temperature for 16 h.
The reaction mixture was diluted with water (10 mL) and
extracted three times with EtOAc (10 mL). The combined
organic layers were dried, filtered, and concentrated in vacuo.
Flash chromatography (CH2Cl2-MeOH, 98.5:1.5) afforded the
title product, a solid (Rf ) 0.35, 440 mg, 100% yield), after
1
removal of solvent in vacuo: mp 76-78 °C; H NMR (CDCl3)
δ 7.05-7.03 (m, 1H), 6.97 (d, 1H, J ) 3), 6.67 (d, 1H, J ) 9),
4.30-4.15 (m, 1H), 3.80-3.25 (m, 4H), 3.25-3.00 (m, 1H), 2.97
(s, 6H), 2.35, 2.15 (2s, 3H), 1.25-1.00 (m, 8H), 0.95-0.8 (br s,
1H); CI-HRMS m/z calcd for C18H27BrN6 407.1559, found
407.1578 (M + H)+. Anal. (C18H27BrN6) C, H, N.
For the binding assay a frozen pellet described above
containing 293EBNA cells transfected with hCRFR1 receptors
was homogenized in 10 mL of ice-cold tissue buffer (50 mM
HEPES buffer, pH 7.0, containing 10 mM MgCl2, 2 mM EGTA,
1 µg/L aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin).

Non-Peptide CRH Antagonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 5 817
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The homogenate was centrifuged at 40000g for 12 min and
the resulting pellet rehomogenized in 10 mL of tissue buffer.
After another centrifugation at 40000g for 12 min, the pellet
was resuspended to a protein concentration of 360 µg/mL to
be used in the assay. Protein determinations were performed
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concentration 150 pM), and 150 µL of the cell homogenate
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P h a r m a cok in etic Stu d ies: Pharmacokinetic parameters
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specified in the text. At 15 min (iv only), 30 min, and 1, 2, 4,
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times and trunk blood samples were collected into tubes
containing EDTA. Dogs (n ) 3) were given 5 mg/kg of
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72 h after dosing. Compounds were extracted from plasma by
simple liquid-liquid extraction. The liquid chromatography
consisted of a Perkin-Elmer series 200 solvent delivery system
(Norwalk, CT), a Perkin-Elmer ISS 200 autoinjector, and a
Waters Symmetry octyl minibore column (2.1 × 50 mm).
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Ack n ow led gm en t. The authors gratefully acknowl-
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