Design, synthesis, and biological evaluation of 1,2,3,7-tetrahydro-6H- purin-6-one and 3,7-dihydro-1H-purine-2,6-dione derivatives as corticotropin-releasing factor1 receptor antagonists

Article abstract of DOI:10.1021/jm049787k

A growing body of evidence suggests that CRF₁ receptor antagonism offers considerable therapeutic potential in the treatment of diseases resulting from elevated levels of CRF, such as anxiety and depression. A series of novel 1,2,3,7-tetrahydro-6H-purin-6-one and 3,7-dihydro-1H-purine-2, 6-dione derivatives was synthesized and evaluated as corticotropin releasing factor-1 (CRF₁) receptor antagonists. Compounds within this series, represented by compound 12d (IC₅₀ = 5.4 nM), were found to be highly potent CRF₁ receptor antagonists. In addition, compounds 12d and 12j were determined to be selective CRF₁ antagonists. The synthesis, structure-activity relationships and pharmacokinetic properties of compounds within this series is presented.

Full text of DOI:10.1021/jm049787k

J . Med. Chem. 2004, 47, 4741-4754
4741
Design , Syn th esis, a n d Biologica l Eva lu a tion of
1,2,3,7-Tetr a h yd r o-6H-p u r in -6-on e a n d 3,7-Dih yd r o-1H-p u r in e-2,6-d ion e
Der iva tives a s Cor ticotr op in -Relea sin g F a ctor 1 Recep tor An ta gon ists
Richard A. Hartz,*,† Kausik K. Nanda,‡ Charles L. Ingalls,§ Vijay T. Ahuja,† Thaddeus F. Molski, Ge Zhang,
Harvey Wong,| Yong Peng, Michelle Kelley, Nicholas J . Lodge, Robert Zaczek, Paul J . Gilligan,† and
George L. Trainor†
Discovery Chemistry, Neuroscience Biology, and Metabolism and Pharmacokinetics, Bristol-Myers Squibb Company,
Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, Connecticut 06492
Received March 17, 2004
A growing body of evidence suggests that CRF1 receptor antagonism offers considerable
therapeutic potential in the treatment of diseases resulting from elevated levels of CRF, such
as anxiety and depression. A series of novel 1,2,3,7-tetrahydro-6H-purin-6-one and 3,7-dihydro-
1H-purine-2,6-dione derivatives was synthesized and evaluated as corticotropin releasing
factor-1 (CRF1) receptor antagonists. Compounds within this series, represented by compound
12d (IC50 ) 5.4 nM), were found to be highly potent CRF1 receptor antagonists. In addition,
compounds 12d and 12j were determined to be selective CRF1 antagonists. The synthesis,
structure-activity relationships and pharmacokinetic properties of compounds within this series
is presented.
In tr od u ction
receptor subtypes throughout the central and peripheral
nervous system, suggest distinct functional roles for the
Corticotropin releasing factor (CRF) receptors have
become an important target for medicinal chemists, as
CRF is believed to be the primary regulator of the
hypothalamic-pituitary-adrenal (HPA) axis. Evidence
for the existence of CRF was reported in 1955.1,2 It was
not until 1981, however, that CRF was isolated and
characterized from ovine hypothalamus by Vale and co-
workers.3 Rat CRF was subsequently isolated in 19834
and was found to be identical to human CRF.5 CRF is
a 41 amino acid neuropeptide derived from the paraven-
tricular nucleus of the hypothalamus. Upon its release
from the hypothalamus, CRF travels in the bloodstream
to the anterior pituitary, wherein it causes the release
of adrenocorticotropic hormone (ACTH).3 This action
subsequently triggers the release of corticosteroid hor-
mones from the adrenal glands, which cause a variety
of metabolic changes that allow the body to respond to
the stressor. The corticosteroid hormones also provide
negative feedback at various levels in the HPA axis to
turn off the stress response and maintain homeostasis.
CRF mediates its actions through two subtypes of
seven-transmembrane G-protein-coupled CRF receptors,
CRF1 and CRF2.6-10 In addition, a soluble CRF binding
protein has also been found in rats and humans.11
Within the CRF2 receptor subtype class, three splice
variants, R, â and γ have been cloned and sequenced.
It is of interest to note that CRF binds to CRF1 receptors
with at least 10-fold greater affinity than to CRF2
receptors.12 This observation, combined with the het-
erogeneous distribution patterns of CRF1 and the CRF2
receptor subtypes.13,14
Since its isolation and characterization, various stud-
ies have documented the important role of CRF in the
regulation of the HPA axis in response to stress.
Hypersecretion of CRF is associated with various en-
docrine and psychiatric disorders15 including depres-
sion,16 anxiety6,17 and post-traumatic stress disorder.
Elevated levels of CRF in cerebrospinal fluid16,18,19 have
been found in depressed patients along with increased
levels of ACTH and cortisol in plasma.6,17,20 Further-
more, intracerebroventricular administration of CRF to
rats promoted behavioral effects similar to those ob-
served in anxiety and depression.7,21-23 On the other
hand, the peptide-based CRF1 receptor antagonists
R-helical CRF(9-41) and astressin successfully blocked
the effects of CRF.6,24 Various studies have also been
conducted with nonpeptide, small-molecule CRF1 an-
tagonists. Extensive investigations with pyrrolopyrimi-
dine 1 (CP-154,526) demonstrated its effectiveness in
rat models for anxiety and depression.25-27 More re-
cently, the results of the first human open label clinical
study of the small molecule CRF1 antagonist
2
(R-121,919) were reported.19 Among the findings re-
ported were reductions in depression and anxiety scores
using both patient and clinician ratings. Thus, there is
an increasing body of evidence suggesting that CRF1
receptor antagonism offers considerable therapeutic
potential in the treatment of diseases resulting from
elevated levels of CRF.
A variety of nonpeptide CRF1 receptor antagonists
have been reported in the literature in recent years.15,28,29
In general, most of the reported antagonists contain five
essential structural features15 (exemplified by compound
3 and illustrated in more detail in Figure 2). Three
essential substituents are attached to a bicyclic (4) or
monocyclic (5) heterocyclic aromatic scaffold containing
* Corresponding author. Tel.: 203-677-7837; Fax: 203-677-7702;
e-mail: richard.hartz@bms.com.
† Department of Discovery Chemistry.
‡ Present address: Merck Research Laboratories, West Point, PA
19486.
§ Present address: Wyeth, Pearl River, NY 10965.
Department of Neuroscience Biology.
| Department of Metabolism and Pharmacokinetics.
10.1021/jm049787k CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/07/2004

4742 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
synthesis and structure-activity relationship of these
compounds is described below.
Resu lts a n d Discu ssion
Ch em istr y. Synthesis of the 3,7-dihydro-1H-purine-
2,6-dione and 1,2,3,7-tetrahydro-6H-purin-6-one deriva-
tives is illustrated below in Schemes 1-4. Preparation
of 3,7-dihydro-1H-purine-2,6-dione 9 (Scheme 1) was
carried out using the general procedure reported by
Merlos et al.30 with several minor modifications. The
synthesis began with commercially available aryl iso-
cyanates (6) which were treated with methylamine to
afford the corresponding ureas in high yield. For ana-
logues where aryl was 2-Cl-4-i-Pr, the resultant 4-i-Pr-
phenyl urea was treated with N-chlorosuccinimide
resulting in chlorination at the ortho position of the
phenyl group. The ureas were heated with cyanoacetic
acid followed by treatment with aqueous sodium hy-
droxide to afford the corresponding 6-amino uracils 8.31
Subsequent nitrosation and reduction32 produced sub-
stituted 5,6-diaminouracil intermediates, which were
cyclized upon heating in the presence of either triethyl
orthopropionate or triethyl orthoformate to furnish 9.
Compounds 10 were prepared from 9 either by treat-
ment with K2CO3 and an alkylating agent in DMF33 or
treatment with an aliphatic alcohol under Mitsunobu
conditions.34,35 The alkylations under basic conditions
generally proceeded in higher yields.36 For the prepara-
tion of a large number of analogues, the Mitsunobu
conditions were also found to be effective because of the
greater selection of commercially available alcohols.
Selective hydrolysis of the urea carbonyl in 10 was
achieved by treatment with either aqueous sodium
hydroxide37 or lithium aluminum hydride. The resulting
imidazoles 11 were heated in toluene in the presence
of an aldehyde or an aldehyde equivalent to furnish the
desired target compounds 12 in good yield.
To facilitate the preparation of analogues with a
variety of R2 substituents, target compounds were also
prepared using the modified route illustrated in Scheme
2 in which R2 was introduced late in the synthetic route.
Upon completion of the synthesis of the bicyclic, het-
erocyclic core (Scheme 1), the nitrogen at the 7-position
of compound 9 was protected with a benzyloxymethyl
group (Scheme 2). Chemistry described previously was
then used to convert the 3,7-dihydro-1H-purine-2,6-
dione ring system to the 1,2,3,7-tetrahydro-6H-purin-
6-one heterocycle (15). The benzyloxymethyl protecting
group was subsequently removed by heating in trifluo-
roacetic acid to afford compounds 16 in good yield.
Finally, alkylation either under basic conditions or
Mitsunobu conditions as described previously afforded
the desired products 12.
F igu r e 1. Extensively studied compounds.
F igu r e 2. General features of most reported small molecule
CRF1 receptor antagonists.
F igu r e 3. The 1,2,3,7-tetrahydro-6H-purin-6-one and 3,7-
dihydro-1H-purine-2,6-dione ring systems.
an sp2-hybridized basic nitrogen. This nitrogen was
found to be critical for binding and is presumed to be
involved in a hydrogen bonding interaction with the
receptor. The small alkyl substituent is believed to fit
into a small hydrophobic pocket of the receptor. The
larger alkyl substituent appears to fit into a large
hydrophobic pocket which can accommodate a wide
variety of substituents. It was found that optimal
binding occurs when the aryl group has an orthogonal
relationship to the heterocyclic core as aryl groups with
an ortho substituent are significantly more potent than
the corresponding compounds lacking an ortho substitu-
ent.
In our search for novel CRF1 antagonists, we inves-
tigated the SAR of a series of 1,2,3,7-tetrahydro-6H-
purin-6-one and 3,7-dihydro-1H-purine-2,6-dione de-
rivatives. These ring systems, depicted in Figure 3,
appeared to be attractive scaffolds based on their
structural relationship to known CRF1 receptor antago-
nists. The decision to explore the SAR of compounds
containing these heterocyclic ring systems was also
based on our desire to investigate a series of compounds
containing a bicyclic core which was not fully aromatic
(i.e. non 10-π), in contrast to most previously discovered
series of compounds containing a bicyclic core.15 In
addition, selective modification of the core heterocycle
at “A” provided an opportunity to alter the nature of
the heterocyclic ring system. The choice of substituents
was based on prior SAR knowledge (vide supra). The
Interestingly, the nature of the substitution on the
aryl group had a significant effect on the reactivity of
the nitrogen at the 7-position of the 1,2,3,7-tetrahydro-
6H-purin-6-one ring system (16). The presence of elec-
tron-withdrawing groups (e.g. 2,4-dichloro) facilitated
reaction at the nitrogen while electron-donating sub-
stituents retarded the reactivity of this nitrogen. When
the aryl group was 2,4,6-trimethylphenyl, alkylation
under either basic conditions or Mitsunobu conditions
proved to be ineffective. Alkylations with this substrate
had to be carried out on the more electron deficient
dione (9).

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4743
Sch em e 1a
a
Reagents and conditions: (a) (1) NH2Me, THF, 74-96%; (2) (when necessary) N-chlorosuccinimide, MeCN, 76-90%; (b) (1) HO2CCH2CN,
Ac2O, 85 °C; (2) 20% NaOH, 70 °C, 54-89% (two steps); (c) NaNO2, H2O/HOAc, 65 °C, 81-89%; (d) Na2S2O4, 25% aq NH4OH, 50 °C,
86-97%; (e) (MeO)3CR1, EtOH, reflux, 62-92%; (f) R2OMs, K2CO3, DMF, 90 °C, 60-94%; (g) PPh3, R2OH, DEAD, THF, 50 °C, 14-82%;
(h) NaOH, H2O/EtOH/dioxane, 42-61%; (i) LiAlH4, THF, 26-64% 11 and 0-36% 12; (j) (CH2O)n, or (MeO)2CHMe or EtCHO, p-TsOH‚H2O,
toluene, 54-92%. Method A: a, b, c, d, e; Method B: f; Method C: g; Method D: h, j; Method E: i or i, j.
Sch em e 2a
a
Reagents and conditions: (a) BOMCl, Bu4NI, K2CO3, DMF, 65-73%; (b) NaOH, H2O/EtOH/dioxane, 25-53%; (c) LiAlH4, THF, 50%;
(d) (CH2O)n, p-TsOH‚H2O, toluene, 56-58%; (e) TFA, CHCl3, 69-95%; (f) R2OMs, K2CO3, DMF, 90 °C, 18-38%; (g) PPh3, R2OH, DEAD,
THF, 12-61%. Method F: a, b, d, e; Method G: a, c, d, e; Method H: f; Method I: g.
While Scheme 2 allowed for the rapid preparation of
analogues at R2, preparation of analogues of the aryl
group was more cumbersome since the aryl group was
introduced at the start of the synthesis. Within the past
few years numerous reports involving the palladium-
catalyzed coupling of aryl halides38-40 or copper-
promoted coupling of aryl boronic acids41,42 with a
variety of alkylamines or anilines have appeared in the
literature. It was anticipated that one of the above
methods would be applicable to the preparation of aryl
analogues on the 3-position of the 1,2,3,7-tetrahydro-
6H-purin-6-one ring system.
a-f in Scheme 1. Initially the urea carbonyl of com-
pound 17 was selectively reduced with LiAlH4 to give a
mixture of 18 and 19. Imidazole 18 was treated with
paraformaldehyde to form additional 19. Subsequent
removal of the p-methoxybenzyl group afforded com-
pound 20 in high yield. Unfortunately all attempts to
couple this substrate with an aryl bromide or an aryl
boronic acid resulted in no reaction.
The lack of reactivity of the nitrogen on the 1,2,3,7-
tetrahydro-6H-purin-6-one ring system was presumably
due to a suboptimal pKa of this NH group. The coupling
reaction was attempted on the corresponding 3,7-
dihydro-1H-purine-2,6-dione ring system, which is more
acidic. To this end, the p-methoxybenzyl group of 17 was
removed in high yield by heating in trifluoroacetic acid
to furnish 21 (Scheme 4). Attempted coupling of 21 with
A limited investigation was undertaken to introduce
the aryl substituent near the end of the synthesis.
p-Methoxybenzyl-protected 3,7-dihydro-1H-purine-2,6-
dione 17 (Scheme 3) was prepared according to steps

4744 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
Sch em e 3a
oid plexus homogenate and [125I]-sauvagine binding
inhibition to determine binding affinities. The binding
affinities for representatives of this series of 1,2,3,7-
tetrahydro-6H-purin-6-one and 3,7-dihydro-1H-purine-
2,6-dione ligands are shown in Tables 1 and 2.
A comparison of the two heterocyclic cores in Table 1
shows that compounds with the 1,2,3,7-tetrahydro-6H-
purin-6-one core were more potent than the correspond-
ing compounds with a 3,7-dihydro-1H-purine-2,6-dione
core (10a vs 12a , 10b vs 12d , 10c vs 12p , and 10d vs
12v). Presumably, the urea carbonyl may have an
unfavorable steric or electronic interaction with the
receptor resulting in reduced binding affinity.
A variety of alkyl substituents at R2 were prepared
in order to examine the SAR at this position. Previous
SAR investigations suggest that R2 interacts with a
large hydrophobic cavity, which can accommodate a
wide variety of substituents at this position provided
that they are neither strongly acidic nor basic.43 The
starting point for this investigation was a 3-pentyl group
(12a , IC50 ) 112 nM, Table 1). Additional alkyl substit-
uents with longer groups from the point of attachment
were investigated (12b-d ) and the optimal group was
determined to be a 4-heptyl group (12d , IC50 ) 5.4 nM).
Incorporation of an ether group into R2 resulted in an
8-fold loss in binding affinity (compare 12d to 12e);
however, a branched alkyl group appeared to result in
a modest improvement in binding affinity (compare 12a
to 12f).
a
Reagents and conditions: (a) LiAlH4, THF 53% 18 and 36%
19; (b) (CH2O)n, p-TsOH‚H2O, toluene, 75%; (c) TFA, 95%.
Sch em e 4a
A series of cycloalkyl-substituted groups at R2 was
investigated (12g-k ). It was found that increasing the
length of the alkyl group from methyl to ethyl to propyl
resulted in a continuous improvement in binding affin-
ity, as one would expect based on the results from the
straight chain alkyl substituents. Interestingly, the
dicyclopropyl-methyl substituent in 12j proved to be the
most potent of the cycloalkyl substituents examined
(IC50 ) 6.0 nM). The cyclobutyl methyl substituent
appears to be somewhat more potent than the corre-
sponding cyclopropyl-methyl group (compare 12k to
12g).
In addition to analogues with branched alkyl groups,
several analogues with aryl or alkyl-aryl groups at R2
were also prepared and tested (15, 12l-o). Compounds
of this type possessed only moderate binding affinity.
The benzyloxymethyl (15), 4-methoxybenzyl (12l) and
2-methylbenzyl (12m ) substituents have IC50’s of 861,
456 and 458 nM, respectively. The compound where R2
is 4-phenylbenzyl (12n , IC50 ) 324 nM) was somewhat
more potent, indicating that both phenyl rings interact
favorably with the receptor. In contrast, the analogue
with a 1-naphthylmethyl substituent (12o) was inactive.
A limited exploration of the SAR of the aryl group was
also undertaken. Among the aryl groups evaluated, the
2,4-dichlorophenyl substituent proved to be the optimal
aryl group in this series. Replacement of the 4-chloro
substituent on the phenyl ring with either a methoxy
or isopropyl group resulted in a 5-10-fold decrease in
binding affinity respectively (compare 12d , IC50 ) 5.4
nM with 12q, IC50 ) 27 nM and 12t, IC50 ) 50 nM).
Compounds with a 2,4,6-trimethylphenyl group were
also prepared. It was found that the 2,4-dichlorophenyl-
substituted aryl group was 4-fold more potent than the
2,4,6-trimethylphenyl group (compare 12d , IC50 ) 5.4
a
Reagents and conditions: (a) TFA, 91%; (b) Cu(OAc)2, pyridine,
6-methoxy-2-methyl-3-pyridinylboronic acid, CH2Cl2, 41% with
52% recovered starting material; (c) LiAlH4, THF, 14% 12v and
46% 22. Method J : a,b; Method K: a, b, c.
an aryl bromide under palladium catalysis conditions
resulted in no reaction; however, coupling of 21 with
6-methoxy-2-methyl-3-pyridinylboronic acid in the pres-
ence of copper(II) acetate and pyridine afforded a 41%
yield of 10d , while the balance of the material was
recovered intact (52% recovered starting material). The
urea carbonyl was subsequently reduced with lithium
aluminum hydride to afford the target compound 12v
along with imidazole 22.
P h a r m a cology. Our compound progression strategy
for this study was as follows. Compounds were initially
screened for binding affinity at CRF1. Selected com-
pounds were screened for binding affinity at CRF2 and
were subsequently examined for their functional affects
in Y79 and rat pituitary cells to assess antagonist
properties. Subsequently, pharmacokinetic properties of
selected compounds were evaluated in an in vitro assay
by incubation with human and rat liver microsomes
followed by in vivo rat pharmacokinetic studies.
Binding affinities were determined in a CRF1 receptor
binding titration assay using rat frontal cortex homo-
genate, in which inhibition of specific binding of [125I]-
ovine-CRF by our test compounds was measured to
determine their receptor binding affinity. The CRF2
receptor binding titration assay employed porcine chor-

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4745
Ta ble 1. Structure Activity Relationships for the 1,2,3,7-Tetrahydro-6H-purin-6-one and 3,7-Dihydro-1H-purine-2,6-dione CRF1
Receptor Antagonists
a
b
All R2 groups which contain a chiral center are racemic. All values are the average of n ) 3 ( standard deviation. c The alkylation
was performed with the corresponding bromide or chloride.
nM to 12p , IC50 ) 22 nM). Replacement of the phenyl
group with a pyridyl group resulted in a significant loss
in binding affinity (12v, IC50 ) 161 nM). An ortho
substituent on the phenyl group is required for good

4746 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
Ta ble 2. Structure Activity Relationships for the
1,2,3,7-Tetrahydro-6H-purin-6-one and
Ta ble 3. cAMP Activity and CRF2 Binding Profiles for
Compounds 12d and 12j
3,7-Dihydro-1H-purine-2,6-dione CRF1 Receptor Antagonists
CRF1 binding IC50 cAMP (IC50, nM) CRF2 IC50
compound
(nM)
Y-79 cells
(nM)
12d
12j
5.4 ( 2.0
6.0 ( 1.2
24.2 ( 5.6
14.4 ( 5.7
>10000
>10000
concentrations up to 10 µM. Similar results were
obtained in an additional study. Both compounds 12d
and 12j also produced a concentration-dependent inhi-
bition of CRF (0.3 nM)-induced ACTH release from rat
pituitary cells and completely suppressed CRF-stimu-
lated ACTH secretion at higher concentrations, indicat-
ing that these compounds behave as antagonists at
pituitary CRF1 receptors (Note: the CRF EC50 in
pituitary cells was 0.25 ( 0.9 nM, n ) 3). To test for
agonist activity, both compounds 12d and 12j were
exposed alone to pituitary cells. Basal secretion of ACTH
from cells was not altered, indicating a lack of agonist
properties. Moreover, neither compound demonstrated
binding affinity to the CRF2 receptor in a binding assay.
Taken together, these compounds are selective CRF1
receptor antagonists as summarized in Table 3.
An initial set of selected compounds from this series
(10b, 12d , 12f, 12g, 12h , 12q, and 12v) was screened
for metabolic stability by incubation with human and
rat liver microsomes. The results from this study
indicated that each of the above compounds was rapidly
metabolized (0.297-0.300 nmol/min/kg, human; 0.298-
0.300 nmol/min/kg, rat). The substitution of the meth-
ylene group at “A” with a carbonyl group appeared to
have no appreciable effect on the rate of metabolism by
liver microsomes.
To assess the pharmacokinetic properties of this
series in more detail, a lead compound in this series,
compound 12d , was included in a rat cassette dose
study. The pharmacokinetic data from this study sug-
gests that 12d is a high clearance compound (Cl ) 4.0
L/h/kg, Vss ) 17.0 L/kg, t1/2 ) 4.4 h, F ) 2%). Mass
spectral data from subsequent human and rat microso-
mal incubations suggest that hydroxylation of the heptyl
substituent was likely the major pathway of metabo-
lism. Compound 12j (IC50 ) 6.0 nM), containing a
dicyclopropyl-methyl substituent in place of the heptyl
substituent, was subsequently prepared and incubated
with human and rat liver microsomes. It was found that
the rate of metabolism was significantly decreased in
human microsomes (0.178 nmol/min/mg) compared to
12d (0.297 nmol/min/mg). The rate of metabolism in rat
liver microsomes was similar to previously tested com-
pounds (0.300 nmol/min/kg); thus, it appears that the
improvement in metabolic stability is species dependent.
The information from this study further supports the
results of the mass spectroscopy study with 12d . In
addition, it provides us with a better understanding of
the structural features primarily responsible for the
high clearance of compound 12d and can guide further
optimization of the pharmacokinetics for this class of
compounds.
compound
method
R1
A
IC50 (nM)a
12d
10b
12x
12y
12z
11
A,B,D
A,B
A,B,D
A,B,D
A,B,D
A,h
Et
Et
Et
Et
H
CH2
5.4 ( 2.0
75 ( 21
383 ( 227
626 ( 158
898 ( 462
>10000c
CdO
CHMeb
CHEtb
CH2
-
-
a
All values are the average of n ) 3 ( standard deviation unless
indicated otherwise. Compound is racemic. c Value determined
b
by two measurements.
binding affinity as illustrated by compound 12w with
an IC50 ) 173 nM, a 35-fold decrease in binding affinity
from the corresponding ortho-substituted compound 12d
(IC50 ) 5.4 nM). Presumably the ortho substituent is
required to enforce an orthogonal relationship between
the core ring system and the phenyl group.
A limited investigation of the SAR at “A” and R1 was
also conducted and the results are summarized in Table
2. The optimal group at “A” was a methylene group
(12d ). Increasing the size of this group resulted in
progressively diminished binding affinity (compare 12d
to 10b, 12x, and 12y). The data suggests that there is
limited steric tolerance in this region of the CRF1
receptor binding pocket. The current pharmacophore
model for CRF1 receptor antagonists15 suggests that R1
fits into a small hydrophobic pocket. The data from the
compounds in this series also support this model.
Compounds lacking an alkyl group at R1 are over 150-
fold less potent than compounds with an ethyl group
(compare 12z with 12d ). The linker designated as “A”
proved to be critical for obtaining potent binding affinity
to the CRF1 receptor in this chemotype. Removal of this
group resulted in loss of binding affinity to the CRF1
receptor (Compound 11, Table 2), indicating that the
constrained six-membered ring is preferred over the
unconstrained structure. It is likely that the six-
membered ring holds the aryl substituent in the pre-
ferred bioactive conformation.
In cell-based functional assays, the antagonist prop-
erties of compounds 12d and 12j were assessed by
measuring the ability of these compounds to inhibit
CRF-stimulated cAMP production in human Y-79 ret-
inoblastoma cells and ACTH secretion in cultured rat
pituitary cells.44 In parallel, any potential CRF agonist
properties of 12d and 12j were assessed by measuring
the effect of these compounds on basal cAMP production
(i.e. in the absence of CRF) in both Y-79 and rat
pituitary cells. Both compounds 12d and 12j produced
a concentration-dependent inhibition of CRF (1 nM)-
induced cAMP production with IC50 values of 24.2 ( 5.6
and 14.4 ( 5.7 nM, respectively, and completely sup-
pressed CRF-stimulated cAMP production at higher
concentrations, indicating that these compounds behave
as antagonists. No agonist properties were detected at
Con clu sion
In conclusion, a novel series of 1,2,3,7-tetrahydro-6H-
purin-6-one and 3,7-dihydro-1H-purine-2,6-dione de-
rivatives was synthesized and evaluated as CRF1 re-

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4747
Buchner funnel to afford the desired product (13.5 g, 75% yield)
as a colorless solid: mp 184-185 °C; 1H NMR (300 MHz,
CDCl3) δ 7.85 (d, J ) 9.2 Hz, 1H), 7.81 (s, 1H), 7.00 (d, J ) 2.9
Hz, 1H), 6.86 (dd, J ) 9.1, 2.1 Hz, 1H), 6.58-6.57 (m, 1H),
3.72 (s, 3H), 2.63 (d, J ) 4.8 Hz, 3H); LRMS (APCI) m/e 215.0
[(M + H)+, calcd for C9H12N2O2Cl 215.1].
ceptor antagonists. This study demonstrates that potent
CRF1 antagonists can be designed from a bicyclic core
heterocycle which is not fully aromatic (i.e. non-10 π).
SAR exploration of the “A” group of the core heterocycle
indicates that the CRF1 receptor is unable to accom-
modate sp3-hybridized substituents at this site, sug-
gesting that there is limited steric tolerance in this
region of the CRF1 receptor binding pocket. It was also
found that the bicyclic core is critical for good binding
affinity within this chemotype, a monocyclic imidazole-
based analogue (11) was not active. Further evaluation
of compounds 12d and 12j demonstrates that they are
selective CRF1 receptor antagonists. Initial assessment
of the pharmacokinetic properties using compound 12d
indicates that it is a high clearance compound and
further optimization is required. Replacing the heptyl
substituent in 12d with a dicyclopropyl-methyl sub-
stituent (12j) did, in fact, result in an improved in vitro
metabolic stability profile in human liver microsomes.
6-Am in o-1-(2,4-d ich lor op h en yl)-3-m et h yl-2,4(1H ,3H )-
p yr im id in ed ion e (8). A solution of N-(2,4-dichlorophenyl)-
N′-methyl-urea (12.0 g, 54.8 mmol) in acetic anhydride (100
mL) was treated with cyanoacetic acid (5.6 g, 65.8 mmol). The
reaction mixture was heated at 85 °C for 2.5 h. Additional
cyanoacetic acid (0.90 g, 11.0 mmol) was added, and the
reaction mixture was stirred for 45 min. A third portion of
cyanoacetic acid (0.45 g, 5.5 mmol) was added, and the reaction
mixture was stirred for 30 min. Excess acetic anhydride was
distilled off under reduced pressure at a temperature less than
70 °C. The residue was treated with 20% aqueous sodium
hydroxide (95 mL) in portions. During the addition a sponta-
neous increase in temperature (65-70 °C) occurred. The
reaction mixture was heated at 60 °C for 1 h during which
time the product precipitated. The reaction mixture was then
cooled to 0 °C. The precipitate was collected on a Buchner
funnel, washed with cold water and dried at 55 °C under
vacuum. The crude product was triturated in hot toluene
(stirred for 1 h at 110 °C), and the solid was immediately
collected on a Buchner funnel then dried under vacuum to
afford a pale yellow solid (8.46 g, 54% yield): mp 249-251
°C; 1H NMR (300 MHz, DMSO-d6) δ 7.89 (d, J ) 1.8 Hz, 1H),
7.59 (d, J ) 1.8 Hz, 2H), 6.44 (s br, 2H), 4.79 (s, 1H), 3.09 (s,
3H); LRMS (APCI) m/e 286.0 [(M + H)+, calcd for C11H10N3O2-
Cl2, 286.0].
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-3,7-d ih yd r o-
1H-p u r in e-2,6-d ion e (9). To a suspension of 6-amino-1-(2,4-
dichlorophenyl)-3-methyl-2,4(1H,3H)-pyrimidinedione 8 (8.0 g,
28.0 mmol) in H2O (65 mL) and acetic acid (3.5 mL) was added
NaNO2 (2.12 g, 30.8 mmol) in portions. The reaction mixture
was heated at 50 °C for 2 h during which time a purple color
formed indicating formation of the nitroso derivative. Ad-
ditional NaNO2 (2.12 g, 30.8 mmol) was added, and the
reaction mixture was stirred at 65 °C for an additional 2 h.
Additional NaNO2 (300 mg, 4.35 mmol) was added, and the
reaction mixture was heated at 75 °C for another 1 h. The
suspension was then cooled to 0 °C. The solid was collected
on a Buchner funnel, washed with cold water and dried under
vacuum at 60 °C overnight to afford the nitroso derivative as
a purple solid (7.78 g, 88% yield) which was used directly in
the next step without further purification: mp 230-232 °C;
1H NMR (300 MHz, DMSO-d6) δ 12.62 (s, 1H), 8.70 (s, 1H),
7.98 (s, 1H), 7.68 (s, 2H), 3.32 (s, 3H); LRMS (APCI) m/e 314.9
[(M + H)+, calcd for C11H9N4O3Cl2, 315.0].
Exp er im en ta l Section
Ch em istr y. All procedures were carried out under
a
nitrogen atmosphere unless otherwise indicated using anhy-
drous solvents purchased from commercial sources without
further purification. Reactions requiring anhydrous conditions
were performed in glassware, which was flame-dried or oven-
dried and placed under a nitrogen atmosphere. Chromatog-
raphy was performed on silica gel using the solvent systems
indicated. Solvent systems are reported as v/v percent ratios.
All reactions were monitored by TLC using EM Science, 0.25
mm, precoated silica gel plates or by low resolution APCI mass
spectrometry. Yields refer to chromatographically and spec-
troscopically pure compounds, except as otherwise indicted.
Melting points were obtained on a Laboratory Devices, Inc.
Mel Temp 3.0 melting point apparatus and are uncorrected.
Proton NMR spectra were recorded on either a Varian (Palo
Alto, CA) Inova 300, 400, or 500 MHz or Brucker 400 or 500
MHz NMR spectrometer. Chemical shifts are reported in δ
values relative to tetramethylsilane. Atmosphere pressure
chemical ionization (APCI) low-resolution mass spectra were
obtained on a Finnigan Navigator LC/MS single quadrupole
mass spectrometer. Electrospray ionization (ESI) high-resolu-
tion mass spectra were obtained on a Finnigan MAT95S
reverse geometry sector instrument. Microanalyses were
performed by Quantitative Technologies, Inc., Whitehouse, NJ .
HPLC purity was measured by using an analytical C18
reversed phase column (method A) or an analytical silica gel
normal phase column (method B). Method A: 0.1% TFA in
water-0.1% TFA in acetonitrile gradient (15%-95% acetoni-
trile over 30 min); method B: hexanes-ethyl acetate with 3%
methanol in the indicated ratio.
To a suspension of finely ground 6-amino-1-(2,4-dichlo-
rophenyl)-3-methyl-5-nitroso-2,4(1H,3H)-pyrimidinedione (7.32
g, 23.2 mmol) in 25% NH4OH (60 mL) was added in portions
Na2S2O4 (20.0 g, 116 mmol). After the addition was complete,
the reaction mixture was heated at 50 °C for 2.5 h. The purple
color gradually disappeared. The reaction mixture was cooled
to 0 °C. The solid was collected on a Buchner funnel, washed
with cold water, then dried under vacuum overnight at 60 °C
to afford a pale green solid (6.40 g, 92% yield) which was used
directly in the next step without further purification: mp 170-
172.5 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.88 (d, J ) 2.2 Hz,
1H), 7.61-7.53 (m, 2H), 5.73 (s, 2H), 3.34 (s, 2H), 3.15 (s, 3H);
LRMS (APCI) m/e 300.9 [(M + H)+, calcd for C11H11H4O2Cl2,
301.0].
N-(2,4-Dich lor op h en yl)-N′-m eth yl-u r ea (7). A cooled (0
°C) solution of methylamine in EtOH (50 mL, 400 mmol, 8.0
M) was dissolved in anhydrous THF (300 mL) and was treated
with 2,4-dichlorophenyl isocyanate (25.0 g, 133 mmol). The
cooling bath was removed, and the mixture was warmed to
65 °C for 20 min. The reaction mixture was then cooled to 0
°C. The solid was collected on a Buchner funnel, washed with
cold ether and dried under vacuum to afford N-(2,4-dichlo-
rophenyl)-N′-methyl-urea (21.7 g, 74% yield) as a colorless
1
solid: mp 213.5-214.5 °C; H NMR (300 MHz, DMSO-d6) δ
8.17 (d, J ) 9.2 Hz, 1H), 8.11 (s, 1H), 7.55 (d, J ) 2.5 Hz, 1H),
7.32 (dd, J ) 8.8, 2.6 Hz, 1H), 6.91 (d, J ) 4.8 Hz, 1H), 2.65
(d, J ) 4.4 Hz, 3H); LRMS (APCI) m/e 259.9 [(M + H + CH3-
CN)+, calcd for C10H12N3OCl2, 260.0].
N-(2-Ch lor o-4-m et h oxyp h en yl)-N′-m et h yl-u r ea (7). A
suspension of N-(4-methoxyphenyl)-N′-methyl-urea (15.00 g,
83.24 mmol) in CH3CN (120 mL) was warmed to 65 °C.
N-Chlorosuccinimide (12.22 g, 91.56 mmol) was added, and
the reaction mixture was heated at 90 °C for 40 min. The
mixture was cooled to 0 °C, and the solid was collected on a
A suspension of 5,6-diamino-1-(2,4-dichlorophenyl)-3-meth-
yl-2,4(1H,3H)-pyrimidinedione (4.0 g, 13.3 mmol) in EtOH (32
mL) was treated with triethylorthopropionate (12.8 mL, 63.7
mmol). The reaction mixture was heated at reflux for 6 h
during which time the product precipitated from the reaction
mixture. The reaction mixture was then cooled to 0 °C to
achieve complete precipitation. The solid was collected on a
Buchner funnel, washed with cold ether, and dried under
vacuum to give a colorless solid (2.97 g). The filtrate was
concentrated and the residue was purified by column chro-

4748 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
matography on silica gel (5% MeOH in CH2Cl2) to afford an
additional 0.87 g of desired product. The total yield of desired
product 9 was 3.84 g (85% yield) as a colorless solid: mp
274.5-275.5 °C; 1H NMR (300 MHz, CDCl3) δ 12.53 (s br, 1H),
7.61 (d, J ) 1.8 Hz, 1H), 7.45-7.38 (m, 2H), 3.52 (s, 3H), 2.87
(q, J ) 7.3 Hz, 2H), 1.38 (t, J ) 7.7 Hz, 3H); LRMS (APCI)
m/e 339.0 [(M + H)+, calcd for C14H13N4O2Cl2, 339.0].
J ) 8.7, 2.5 Hz, 1H), 6.91 (s br, 1H), 6.86 (d, J ) 8.8 Hz, 1H),
6.20 (s, 1H), 2.82 (d, J ) 4.7 Hz, 3H), 2.74 (q, J ) 7.4 Hz, 2H),
1.98-1.86 (m, 4H), 1.33 (t, J ) 7.7 Hz, 3H), 0.86 (t, J ) 7.7
Hz, 6H); HRMS (ESI) m/e 383.1423 [(M + H)+; calcd for
C
18H25N4OCl2 383.1405].
2-Eth yl-1-(4-h eptyl)-N-m eth yl-4-[(2,4,6-tr im eth ylph en yl)-
a m in o]-1H-im id a zole-5-ca r boxa m id e (11). (Method E) A
solution of 8-ethyl-7-(4-heptyl)-1-methyl-3-(2,4,6-trimethyl-
phenyl)-3,7-dihydro-1H-purine-2,6-dione 10 (50 mg, 0.122
mmol) was cooled to -10 °C. LiAlH4 (0.24 mL, 0.244 mmol, 1
M in THF) was added dropwise, and the reaction mixture was
stirred at -10 °C for 1.5 h. An additional portion of LiAlH4
(0.24 mL, 0.244 mmol, 1 M in THF) was added dropwise, and
the cooling bath was removed and the reaction mixture was
allowed to stir at room temperature for 30 min. The reaction
was quenched by the dropwise addition of absolute ethanol
followed by saturated aqueous NaHCO3. The precipitate was
removed by filtration, and the filtrate was poured into a
separatory funnel containing saturated aqueous NaHCO3 (10
mL). The aqueous layer was extracted with EtOAc (3 × 10
mL). The combined organic layers were washed with brine,
dried over MgSO4, filtered and concentrated. The residue was
purified by preparative TLC (1000 µM silica gel plate, 30%
ethyl acetate in hexanes with 2% methanol) to afford the
desired product (30 mg, 64% yield) as a tan solid: mp 135-
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-7-(3-p en t yl)-
3,7-dih ydr o-1H-pu r in e-2,6-dion e (10a). (prepared by Method
B) A solution of 3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-3,7-
dihydro-1H-purine-2,6-dione 9 (544 mg, 1.60 mmol) in anhy-
drous DMF (8 mL) was treated with finely ground K2CO3 (662
mg, 4.80 mmol). After stirring 5 min at room temperature,
3-methanesulfonylpentane (682 µL, 4.48 mmol) was added via
syringe and the reaction mixture was heated at 80 °C for 2 h.
The mixture was cooled to room temperature and transferred
to a separatory funnel containing ether (120 mL). The organic
layer was washed with water (4 × 15 mL), brine (15 mL), dried
over MgSO4, filtered and concentrated. The solid residue was
crystallized from hexane/ethyl acetate and collected on a
Buchner funnel to give a colorless solid (508 mg). The filtrate
was concentrated and the residue was purified by column
chromatography on silica gel (25% ethyl acetate in hexanes)
to give an additional 105 mg of product. The total yield of
desired product was 613 mg (94% yield) as a colorless solid:
mp 186-187 °C; 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J ) 2.2
Hz, 1H), 7.44-7.36 (m, 2H), 4.00-3.90 (m, 1H), 3.45 (s, 3H),
2.74 (q, J ) 7.7 Hz, 2H), 2.39-2.26 (m, 2H), 2.05-1.92 (m,
2H), 1.24 (t, J ) 7.3 Hz, 3H), 0.83 (t, J ) 7.3 Hz, 6H); HRMS
(ESI) m/e 409.1212 [(M + H)+, calcd for C19H23N4O2Cl2,
409.1198]. Anal. (C19H22N4O2Cl2) C, H, N.
1
136 °C; H NMR (300 MHz, CDCl3) δ 7.18 (s br 1H), 6.84 (s,
2H), 5.31 (s br, 1H), 4.02 (s br, 1H), 2.78 (d, J ) 4.8 Hz, 3H),
2.68 (q, J ) 7.7 Hz, 2H), 2.24 (s, 3H), 2.11 (s, 6H), 1.92-1.62
(s br, 3H), 1.41-1.01 (m, 5H), 1.21 (t, J ) 7.3 Hz, 3H) 0.89 (t,
7.3 Hz, 6H); LRMS (APCI) m/e 385.1 [(M + H)+, calcd for
C
23H37N4O, 385.3].
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-7-(3-p en t yl)-
3,7-dih ydr o-1H-pu r in e-2,6-dion e (10a). (prepared by Method
C) A solution of 3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-3,7-
dihydro-1H-purine-2,6-dione 9 (100 mg, 0.295 mmol) in an-
hydrous THF (1.2 mL) was heated to 50 °C. PPh3 (120 mg,
0.594 mmol) and 3-pentanol (96 µL, 0.889 mmol) were added.
After stirring 5 min, DEAD (102 µL, 0.648 mmol) was added
rapidly via syringe and the reaction mixture was stirred for
15 min at 50 °C. The mixture was cooled to room temperature
and was poured into a separatory funnel containing saturated
aqueous NaHCO3 (15 mL). The aqueous layer was extracted
with EtOAc (3 × 15 mL). The combined organic layers were
washed with brine, dried over MgSO4, filtered and concen-
trated. The residue was purified by preparative TLC (two 1000
µM silica gel plates, 60% ethyl acetate/40% hexanes) to afford
the desired product (88 mg, 64% yield) as a colorless solid. The
spectral data is identical to the material prepared via method
B.
3-(2,4-Dich lor op h en yl)-8-et h yl-7-(4-h ep t yl)-1-m et h yl-
3,7-d ih yd r o-1H-p u r in e-2,6-d ion e (10b). Colorless solid; mp
185-186 °C; 1H NMR (300 MHz, DMSO-d6) δ 7.73 (d, J ) 2.5
Hz, 1H), 7.38 (dd, J ) 8.8, 2.6 Hz, 1H), 6.94 (d, J ) 8.4 Hz,
1H), 4.96 (q, J ) 6.2 Hz, 1H), 4.20-3.95 (m br, 1H), 2.82 (s,
3H), 2.74-2.59 (m, 2H), 2.50-2.30 (m br, 1H), 2.15-2.05 (m,
1H), 1.90-1.70 (m, 2H), 1.42 (d, J ) 5.8 Hz, 3H), 1.15 (t, J )
7.7 Hz, 3H), 0.77 (t, J ) 7.7 Hz, 3H), 0.72-0.68 (m, 3H); HRMS
(ESI) m/e 437.1512 [(M + H)+, calcd for C21H27N4O2Cl2
437.1511]. Anal. (C21H26N4O2Cl2) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-2-eth yl-N-m eth yl-1-(3-
p en t yl)-1H -im id a zole-5-ca r b oxa m id e (11). (Method D)
3-(2,4-Dichlorophenyl)-8-ethyl-1-methyl-7-(3-pentyl)-3,7-dihy-
dro-1H-purine-2,6-dione 10a (268 mg, 0.655 mmol) was dis-
solved in dioxane (1 mL). EtOH (1 mL) and aqueous 3 N NaOH
(2 mL) were added, and the reaction mixture was heated at
reflux for 3.5 h. The mixture was cooled to room temperature
and transferred to a separatory funnel containing saturated
aqueous NaHCO3 (15 mL). The aqueous layer was extracted
with EtOAc (3 × 15 mL). The combined organic layers were
washed with brine, dried over MgSO4, filtered and concen-
trated. The residue was purified by column chromatography
on silica gel (30% ethyl acetate in hexanes with 0.2% MeOH)
to afford a colorless amorphous solid (120 mg, 48% yield): 1H
NMR (300 MHz, CDCl3) δ 7.33 (d, J ) 2.2 Hz, 1H), 7.07 (dd,
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-7-(3-p en t yl)-
1,2,3,7-t et r a h yd r o-6H -p u r in -6-on e (12a ). 4-[(2,4-Dichlo-
rophenyl)amino]-2-ethyl-N-methyl-1-(3-pentyl)-1H-imidazole-
5-carboxamide 11 (35 mg, 0.091 mmol) was dissolved in
toluene (5 mL) and was treated with paraformaldehyde (150
mg) and p-TsOH‚H2O (10 mg, 0.053 mmol). The mixture was
heated at reflux for 1.25 h in a flask equipped with a Dean-
Stark trap. The mixture was cooled to room temperature and
transferred to a separatory funnel containing saturated aque-
ous NaHCO3 (15 mL). The aqueous layer was extracted with
EtOAc (3 × 15 mL). The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated. The
residue was purified by preparative TLC (1000 µM silica gel
plate, 30% ethyl acetate in hexanes) to afford 3-(2,4-dichlo-
rophenyl)-8-ethyl-7-(3-pentyl)-1-methyl-1,2,3,7-tetrahydro-6H-
purin-6-one 12a (29 mg, 81% yield) as a colorless solid: mp
132-133 °C; 1H NMR (500 MHz, DMSO-d6, 100 °C) δ 7.61 (d,
J ) 2.2 Hz, 1H), 7.37 (dd, J ) 8.4, 2.1 Hz, 1H), 7.19 (d, J )
8.8 Hz, 1H), 4.81 (s, 2H), 4.35-4.25 (m, 1H), 2.86 (s, 3H), 2.67
(q, J ) 7.3 Hz, 2H), 2.23-2.12 (m, 2H), 1.93-1.84 (m, 2H),
1.19 (t, J ) 7.4 Hz, 3H), 0.80 (t, J ) 7.3 Hz, 6H); HRMS (ESI)
m/e 395.1406 [(M + H)+, calcd for C19H25N4OCl2 395.1405].
Anal. (C19H24N4OCl2) C, H, N.
7-[(Ben zyloxym et h yl]-3-(2,4-d ich lor op h en yl)-8-et h yl-
1-m et h yl-3,7-d ih yd r o-1H -p u r in e-2,6-d ion e (13). 3-(2,4-
Dichlorophenyl)-8-ethyl-1-methyl-3,7-dihydro-1H-purine-2,6-
dione 9 (6.00 g, 17.69 mmol) was dissolved in anhydrous DMF
(80 mL). K2CO3 (6.11 g, 44.23 mmol), Bu4NI (1.31 g, 3.54 mmol)
and BOMCl (4.4 mL, 31.84 mmol) were added, and the reaction
mixture was heated at 45 °C for 50 min. The reaction mixture
was cooled to room temperature and was stirred with satu-
rated aqueous NaHCO3 (180 mL) for 30 min. The mixture was
poured into a separatory funnel containing ether (600 mL).
The organic layer was washed with H2O (4 × 80 mL), brine,
dried over MgSO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel (40% ethyl
acetate in hexanes) to give 7-benzyloxymethyl-3-(2,4-dichlo-
rophenyl)-8-ethyl-1-methyl-3,7-dihydro-1H-purine-2,6-dione 13
(5.28 g, 65% yield) as an amorphous solid: 1H NMR (300 MHz,
CDCl3) δ 7.59 (d, J ) 2.2 Hz, 1H), 7.42 (dd, J ) 8.4, 2.2 Hz,
1H), 7.37-7.26 (m, 6H), 5.86 (ABq, J AB ) 11.0, ∆υ ) 39.7 Hz,
2H), 4.72 (s, 2H), 3.46 (s, 3H), 2.79 (q, J ) 7.7 Hz, 2H), 1.25 (t,

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4749
J ) 7.6 Hz, 3H); LRMS (APCI) m/e 459.0 [(M + H)+, calcd for
min at 50 °C. The mixture was cooled to room temperature
and was poured into a separatory funnel containing saturated
aqueous NaHCO3 (15 mL). The aqueous layer was extracted
with EtOAc (3 × 15 mL). The combined organic layers were
washed with brine, dried over MgSO4, filtered and concen-
trated. Purification of the residue by preparative TLC (two
1000 µM silica gel plates, 50% ethyl acetate/50% hexanes)
followed by further purification by reverse phase HPLC
furnished 3-(2,4-dichlorophenyl)-8-ethyl-7-(1-methoxymethyl-
butyl)-1-methyl-1,2,3,7-tetrahydro-6H-purin-6-one 12e (59 mg,
56% yield) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ
7.45 (d, J ) 2.2 Hz, 1H), 7.18 (dd, J ) 8.5, 2.2 Hz, 1H), 7.07
(d, J ) 8.4 Hz, 1H), 4.84 (ABq, J ) 11.4, ∆υ ) 46.9 Hz, 2H),
4.21 (q, J ) 7.4 Hz, 2H), 3.76 (dd, J ) 9.9, 4.4 Hz, 1H), 3.32
(s, 3H), 2.96 (s, 3H), 2.75 (q, J ) 7.7 Hz, 2H), 2.21-2.05 (m,
1H), 1.95-1.81 (m, 1H), 1.30-1.20 (m, 5H), 0.92 (t, J ) 7.0
Hz, 3H); HRMS (ESI) m/e 425.1519 [(M + H)+, calcd for
C
22H21N4O3Cl2 459.1].
1-Ben zyloxym et h yl-4-[(2,4-d ich lor op h en yl)a m in o]-2-
eth yl-N-m eth yl-1H-im id a zole-5-ca r boxa m id e (14). 7-Ben-
zyloxymethyl-3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-3,7-di-
hydro-1H-purine-2,6-dione 13 (5.20 g, 11.32 mmol) was dissolved
in dioxane (25 mL), and the mixture was treated with EtOH
(25 mL) and aqueous 3 N NaOH (50 mL). The reaction mixture
was heated at 60 °C for 2 h. The mixture was then cooled to
room temperature and was transferred to a separatory funnel
containing saturated aqueous NaHCO3 (100 mL). The aqueous
layer was extracted with EtOAc (3 × 100 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (40% ethyl acetate in hexanes
f 40% ethyl acetate in hexanes with 0.2% MeOH) to afford
1-benzyloxymethyl-4-[(2,4-dichlorophenyl)amino]-2-ethyl-N-
methyl-1H-imidazole-5-carboxamide 14 (2.90 g, 59% yield) as
a light yellow amorphous solid: 1H NMR (300 MHz, CDCl3) δ
9.35 (s, 1H), 8.41 (d, J ) 8.8 Hz, 1H), 7.44-7.29 (m, 7H), 7.16
(dd, J ) 9.1, 2.5 Hz, 1H), 5.45 (s, 2H), 4.63 (s, 2H), 2.89 (d, J
) 4.8 Hz, 3H), 2.65 (q, J ) 7.7 Hz, 2H), 1.35 (t, J ) 7.7 Hz,
3H); LRMS (APCI) m/e 433.1 [(M + H)+, calcd for C21H23N4O2-
Cl2 433.1].
7-Ben zyloxym et h yl-3-(2,4-d ich lor op h en yl)-8-et h yl-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (15). A solution
of 1-benzyloxymethyl-4-[(2,4-dichlorophenyl)amino]-2-ethyl-N-
methyl-1H-imidazole-5-carboxamide 14 (2.70 g, 6.23 mmol)
dissolved in toluene (100 mL) was treated with paraformal-
dehyde (8.0 g) and p-TsOH‚H2O (0.67 g, 3.52 mmol). The
reaction mixture was heated at reflux for 15 min. The mixture
was cooled to room temperature and transferred to a separa-
tory funnel containing saturated aqueous NaHCO3 (150 mL).
The aqueous layer was extracted with EtOAc (3 × 100 mL).
The combined organic layers were washed with brine, dried
over MgSO4, filtered and concentrated. The residue was
purified by column chromatography on silica gel (40% ethyl
acetate in hexanes) to afford 7-benzyloxymethyl-3-(2,4-di-
chlorophenyl)-8-ethyl-1-methyl-1,2,3,7-tetrahydro-6H-purin-
6-one 15 (1.6 g, 58% yield) as a colorless amorphous solid: 1H
NMR (300 MHz, CDCl3) δ 7.46 (d, J ) 2.2 Hz, 1H), 7.34-7.29
(m, 5H), 7.19 (dd, J ) 8.4, 2.2 Hz, 1H), 7.03 (d, J ) 8.8 Hz,
1H), 5.84 (s, 2H), 4.85 (s, 2H), 4.71 (s, 2H), 2.97 (s, 3H), 2.77
(q, J ) 7.7 Hz, 2H), 1.28 (t, J ) 7.7 Hz, 3H); HRMS (ESI) m/e
445.1212 [(M + H)+, calcd for C22H23N4O2Cl2 445.1198]. Anal.
(C22H22N4O2Cl2) C, H, N.
3-(2,4-Dich lor oph en yl)-8-eth yl-1-m eth yl-1,2,3,7-tetr ah y-
d r o-6H-p u r in -6-on e (16). A solution of 7-benzyloxymethyl-
3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-1,2,3,7-tetrahydro-6H-
purin-6-one 15 (1.3 g, 2.92 mmol) in CHCl3 (2 mL) was treated
with trifluoroacetic acid (15 mL). The solution was heated at
80 °C in a pressure tube for 5 h. The mixture was then cooled
to room temperature and concentrated. The residue was
concentrated from heptane (2×), transferred into a separatory
funnel containing saturated aqueous NaHCO3 (75 mL), and
the aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with brine, dried over
MgSO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (3% methanol in CH2-
Cl2) to furnish 3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-1,2,3,7-
tetrahydro-6H-purin-6-one 16 (900 mg, 95% yield) as a color-
less solid: mp 200-201 °C; 1H NMR (300 MHz, CDCl3) δ 12.05
(s, 1H), 7.47 (d, J ) 2.2 Hz, 1H), 7.20 (dd, J ) 8.8, 2.6 Hz,
1H), 7.12 (d, J ) 8.4 Hz, 1H), 4.90 (s, 2H), 3.02 (s, 3H), 2.79
(q, J ) 7.7 Hz, 2H), 1.34 (t, J ) 7.7 Hz, 3H); LRMS (APCI)
m/e 325.0 [(M + H)+, calcd for C14H15N4OCl2 325.1].
C
20H27N4O2Cl2 425.1511]. HPLC: (a) 98.2%; (b) 95.8% (75%
hexanes-25% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-eth yl-7-(1-eth yl-2-m eth ylp r o-
p yl)-1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12f). A
solution of 3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-1,2,3,7-tet-
rahydro-6H-purin-6-one 16 (50 mg, 0.154 mmol) in anhydrous
DMF (1.0 mL) was treated with finely ground K2CO3 (64 mg,
0.462 mmol). After stirring 5 min at room temperature,
3-methanesulfonyl-2-methylpentane (75 µL, 0.431 mmol) was
added via syringe and the reaction mixture was heated at 90
°C for 4 h. The mixture was cooled to room temperature and
transferred to a separatory funnel containing ethyl acetate (50
mL). The organic layer was washed with water (3 × 10 mL),
brine (15 mL), dried over MgSO4, filtered and concentrated.
Purification of the residue by medium-pressure liquid chro-
matography (10% f 20% ethyl acetate in hexanes) afforded
3-(2,4-dichlorophenyl)-8-ethyl-7-(1-ethyl-2-methylpropyl)-1-
methyl-1,2,3,7-tetrahydro-6H-purin-6-one 12f (14 mg, 22%
yield) as an amber solid: mp 100-101 °C; 1H NMR (400 MHz,
CDCl3) δ 7.44 (d, J ) 2.2 Hz, 1H), 7.15 (dd, J ) 8.5, 2.2 Hz,
1H), 6.95 (d, J ) 8.5 Hz, 1H), 4.83 (ABq, J AB ) 11.5, ∆υ )
25.3 Hz, 2H), 3.55-3.49 (m, 1H), 2.93 (s, 3H), 2.83-2.75 (m,
1H), 2.70 (q, J ) 7.5 Hz, 2H), 2.47-2.38 (m, 1H), 1.98-1.88
(m, 1H), 1.27 (t, J ) 7.3 Hz, 3H), 1.10-1.05 (m, 3H), 0.89-
0.70 (m, 6H); HRMS (ESI) m/e 409.1562 [(M + H)+, calcd for
C
20H27N4OCl2 409.1562]. HPLC: (a) 100%; (b) 100% (75%
hexanes-25% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-7-(2-p en t yl)-
1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12b). colorless oil; 1H
NMR (300 MHz, CDCl3) δ 7.45 (d, J ) 2.5 Hz, 1H), 7.17 (dd,
J ) 8.4, 2.2 Hz, 1H), 7.04 (d, J ) 8.5 Hz, 1H), 4.84 (ABq, J AB
) 11.4, ∆υ ) 26.2 Hz, 2H), 4.21-4.17 (m, 1H), 2.96 (s, 3H),
2.73 (q, J ) 7.7 Hz, 2H), 2.17-2.12 (m, 1H), 1.97-1.92 (m,
1H), 1.64 (d, J ) 6.6 Hz, 3H), 1.36-1.20 (m, 5H), 0.93 (t, J )
7.3 Hz, 3H); HRMS (ESI) m/e 395.1368 [(M + H)+, calcd for
C
19H25N4OCl2 395.1405]. HPLC: (a) 97.3%; (b) 99.5% (65%
hexanes-35% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-et h yl-7-(3-h ep t yl)-1-m et h yl-
1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12c). colorless solid; mp
94-95 °C; 1H NMR (300 MHz, DMSO-d6, 80 °C) δ 7.64 (d, J )
2.6 Hz, 1H), 7.39 (dd, J ) 5.8, 2.2 Hz, 1H), 7.17 (d, J ) 8.8 Hz,
1H), 4.80 (s, 2H), 4.40-4.30 (m, 1H), 2.86 (s, 3H), 2.66 (q, J )
7.4 Hz, 2H), 2.22-2.12 (m, 2H), 1.90-178 (m, 2H), 1.33-1.07
(m, 4H), 1.17 (t, J ) 7.3 Hz, 3H), 0.85 (t, J ) 6.6 Hz, 3H),
0.878 (t, J ) 7.3 Hz, 3H); HRMS (ESI) m/e 423.1723 [(M +
H)+, calcd for C21H29N4OCl2 423.1718]. Anal. (C21H28N4OCl2)
C, H, N.
3-(2,4-Dich lor op h en yl)-8-et h yl-7-(4-h ep t yl)-1-m et h yl-
1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12d ). colorless solid:
mp 117-118 °C; 1H NMR (300 MHz, CDCl3) δ 7.45 (d, J ) 2.2
Hz, 1H), 7.17 (dd, J ) 8.4, 2.2 Hz, 1H), 7.00 (d, J ) 8.8 Hz,
1H), 4.84 (s, 2H), 4.10-4.00 (m, 1H), 2.95 (s, 3H), 2.71 (q, J )
7.3 Hz, 2H), 2.30-1.80 (m, 4H), 1.40-1.15 (m, 4H), 1.27 (t, J
) 7.7 Hz, 3H), 0.92 (t, J ) 7.3 Hz, 6H); HRMS (ESI) m/e
423.1736 [(M + H)+, calcd for C21H29N4OCl2 423.1718]. Anal.
(C21H28N4OCl2) C, H, N.
3-(2,4-Dich lor op h en yl)-8-eth yl-7-(1-m eth oxym eth ylbu -
tyl)-1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12e). A
solution of 3-(2,4-dichlorophenyl)-8-ethyl-1-methyl-1,2,3,7-tet-
rahydro-6H-purin-6-one 16 (80 mg, 0.246 mmol) in anhydrous
THF (1.2 mL) was heated to 50 °C. PPh3 (100 mg, 0.492 mmol)
and 1-methoxy-2-pentanol (87 mg, 0.738 mmol) were added.
After stirring 5 min, DEAD (85 µL, 0.541 mmol) was added
rapidly via syringe and the reaction mixture was stirred 25

4750 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
7-(1-Cyclop r op yleth yl)-3-(2,4-d ich lor op h en yl)-8-eth yl-
1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12 g). color-
less oil; 1H NMR (300 MHz, CDCl3) δ 7.51 (d, J ) 2.2 Hz, 1H),
7.28 (dd, J ) 8.8, 2.6 Hz, 1H), 7.19 (d, J ) 8.4 Hz, 1H), 4.92
(ABq, J AB ) 11.0, ∆υ ) 41.3 Hz, 2H), 4.05-3.95 (m, 1H), 3.02
(s, 3H), 2.96-2.88 (m, 2H), 1.85-1.75 (m, 1H), 1.78 (d, J )
7.0 Hz, 3H), 1.31 (t, J ) 7.7 Hz, 3H), 0.83-0.79 (m, 1H), 0.78-
0.59 (m, 1H), 0.57-0.40 (m, 1H), 0.39-0.21 (m, 1H); HRMS
(ESI) m/e 393.1244 [(M + H)+, calcd for C19H23N4OCl2 393.1249].
Anal. (C19H22N4OCl2) C, H, N; calcd C, 58.02; found C, 57.35.
7-(1-Cyclop r op ylp r op yl)-3-(2,4-d ich lor op h en yl)-8-eth -
yl-1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12h ). Col-
3H); HRMS (ESI) m/e 491.1399 [(M + H)+, calcd for C27H25N4-
OCl2 491.1405]. HPLC: (a) 98.1%; (b) 97.9% (60% hexanes-
40% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-eth yl-1-m eth yl-7-(1-n a p h th yl-
m eth yl)-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12o). Colorless
oil; 1H NMR (300 MHz, CDCl3) δ 8.06 (d, J ) 8.4 Hz, 1H),
7.90 (d, J ) 6.9 Hz, 1H), 7.78 (d, J ) 8.1 Hz, 1H), 7.63-7.52
(m, 2H), 7.49 (d, J ) 2.6 Hz, 1H), 7.38 (t, J ) 7.3 Hz, 1H),
7.23 (dd, J ) 8.4, 2.2 Hz, 1H), 7.15 (d, J ) 8.4 Hz, 1H), 6.68
(d, J ) 6.6 Hz, 1H), 6.11 (s, 2H), 4.92 (s, 2H), 2.91 (s, 3H),
2.56 (q, J ) 7.3 Hz, 2H), 1.16 (t, J ) 7.7 Hz, 3H); HRMS (ESI)
m/e 465.1248 [(M + H)+, calcd for C22H23N4O2Cl2 465.1249].
Anal. (C22H22N4O2Cl2) C, H, N.
1
orless oil; H NMR (500 MHz, DMSO-d6, 100 °C) δ 7.62 (d, J
) 2.2 Hz, 1H), 7.38 (dd, J ) 8.6, 2.5 Hz, 1H), 7.22 (d, J ) 8.6
Hz, 1H), 4.82 (s, 2H), 3.80-3.70 (m, 1H), 2.87 (s, 3H), 2.68-
2.64 (m, 2H), 2.55-2.40 (m, 1H), 2.20-2.05 (m, 1H), 1.85-
1.77 (m, 1H), 1.18 (t, J ) 7.3 Hz, 3H), 0.84 (t, J ) 7.3 Hz, 3H),
0.72-0.65 (m, 1H), 0.45-0.37 (m, 2H), 0.20-0.15 (m, 1H);
HRMS (ESI) m/e 407.1423 [(M + H)+, calcd for C20H25N4OCl2
407.1405]. HPLC: (a) 96.9%, (b) 98.5% (75% hexanes-25%
ethyl acetate with 3% methanol).
8-Eth yl-7-(4-h eptyl)-1-m eth yl-3-(2,4,6-tr im eth ylph en yl)-
3,7-d ih yd r o-1H-p u r in e-2,6-d ion e (10c). Colorless solid; mp
192-193 °C; 1H NMR (400 MHz, CDCl3) δ 6.97 (s, 2H), 4.12-
4.08 (m, 1H), 3.46 (s, 3H), 2.71 (q, J ) 7.6 Hz, 2H), 2.31 (s,
3H), 2.30-2.23 (m, 2H), 2.02 (s, 6H), 1.98-1.80 (m, 2H), 1.30-
1.20 (m, 2H), 1.19 (t, J ) 7.6 Hz, 3H), 1.16-1.05 (m, 2H), 0.90
(t, J ) 7.3 Hz, 6H); HRMS (ESI) m/e 411.2756 [(M + H)+, calcd
for C24H35N4O2 411.2760]. Anal. (C24H34N4O2) C, H, N; calcd
C 69.58, H 9.01; found C 70.21, H 8.34.
7-(1-Cyclop r op ylbu tyl)-3-(2,4-d ich lor op h en yl)-8-eth yl-
1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12i). Color-
8-Eth yl-7-(4-h eptyl)-1-m eth yl-3-(2,4,6-tr im eth ylph en yl)-
1
1
less oil; H NMR (400 MHz, DMSO-d6, 120 °C) δ 7.61 (d, J )
1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12p ). Colorless oil; H
2.4 Hz, 1H), 7.37 (dd, J ) 8.5, 2.4 Hz, 1H), 7.20 (d, J ) 8.5,
1H), 4.79 (s, 2H), 4.08-4.05 (m, 1H), 2.86 (s, 3H), 2.63 (q, J )
7.5 Hz, 2H), 2.21-2.10 (m, 1H), 2.04-1.94 (m, 1H), 1.82-1.75
(m, 1H), 1.39-1.22 (m, 1H), 1.21-1.15 (m, 4H), 0.88 (t, J )
7.3 Hz, 3H), 0.67-0.62 (m, 1H), 0.41-0.35 (m, 2H), 0.20-0.14
(m, 1H); HRMS (ESI) m/e 421.1567 [(M + H)+, calcd for
NMR (500 MHz, DMSO-d6, 30 °C) δ 6.90 (s, 2H), 4.65 (s, 2H),
4.15-4.00 m, 1H), 2.83 (s, 3H), 2.59 (q, J ) 7.3 Hz, 2H), 2.24
(s, 3H), 2.06 (s, 6H), 1.72-1.68 (m, 2H), 1.30-1.18 (m, 4H),
1.12-1.05 (m, 2H), 1.08 (t, J ) 7.3 Hz, 3H), 0.84 (t, J ) 7.3
Hz, 6H); HRMS (ESI) m/e 397.2965 [(M + H)+, calcd for
C
24H37N4O 397.2967]. Anal. (C24H36N4O) C, H, N.
C
21H27N4OCl2 421.1562]. HPLC: (a) 97.2%; (b) 96.5% (60%
3-(2-Ch lor o-4-m et h oxyp h en yl)-8-et h yl-7-(4-h ep t yl)-1-
hexanes-40% ethyl acetate with 3% methanol).
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12q). Colorless
1
3-(2,4-Dich lor op h en yl)-7-d icyclop r op ylm eth yl-8-eth yl-
1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12j). Color-
oil; H NMR (300 MHz, DMSO-d6, 80 °C) δ 7.11 (d, J ) 8.8
Hz, 1H), 7.10 (d, J ) 2.6 Hz, 1H), 6.90 (dd, J ) 8.5, 2.6 Hz,
1H), 4.71 (s, 2H), 4.60-4.45 (m, 1H), 3.81 (s, 3H), 2.85 (s, 3H),
2.65 (q, J ) 7.3 Hz, 2H), 2.20-2.08 (m, 2H), 1.85-1.75 (m,
2H), 1.30-1.10 (m, 4H), 1.17 (t, J ) 7.6 Hz, 3H), 0.88 (t, J )
7.3 Hz, 6H), HRMS (ESI) m/e 419.2215 [(M + H)+, calcd for
1
less oil; H NMR (500 MHz, DMSO-d6, 90 °C) δ 7.60 (d, J )
2.2 Hz, 1H), 7.39 (dd, J ) 8.4, 2.2.Hz, 1H), 7.20 (d, J ) 8.8
Hz, 1H), 4.81 (s, 2H), 4.08-4.01 (m, 1H), 2.81 (s, 3H), 2.72-
2.63 (m, 2H), 1.22-1.06 (m, 5H), 1.01-0.97 (m, 1H), 0.78-
0.62 (m, 1H), 0.57-0.41 (m, 2H), 0.40-0.21 (m, 3H), 0.20-
0.15 (m, 1H); HRMS (ESI) m/e 419.1402 [(M + H)+, calcd for
C
22H32N4O2Cl 419.2214]. Anal. (C22H31N4O2Cl) C, H, N; C,
63.07; found C, 62.66.
C
21H25N4OCl2 419.1405]. HPLC: (a) 98.0%; (b) 99.5% (75%
3-(2-Ch lor o-4-m eth oxyph en yl)-7-(1-cyclopr opylpr opyl)-
8-eth yl-1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12r ).
Colorless oil; 1H NMR (300 MHz, CDCl3) δ 7.05 (d, J ) 8.8
Hz, 1H), 6.99 (d, J ) 2.9 Hz, 1H), 6.76 (dd, J ) 8.8, 3.0 Hz,
1H), 4.79 (ABq, J AB ) 11.0, ∆υ ) 24.7 Hz, 2H), 4.22-4.15 (m,
1H), 3.79 (s, 3H), 2.95 (s, 3H), 2.69-2.66 (m, 2H), 2.38-2.08
(m, 2H), 1.35-1.22 (m, 1 H, partially obs.), 1.25 (t, J ) 7.4
Hz, 3H), 0.90 (t, J ) 7.7 Hz, 3H), 0.79-0.66 (m, 1H), 0.52-
0.35 (m, 2H), 0.30-0.17 (m, 1H); HRMS (ESI) m/e 403.2015
[(M + H)+, calcd for C21H28N4O2Cl 403.1901]. HPLC: (a) 98.3%;
(b) 97.9% (75% hexanes-25% ethyl acetate with 3% methanol).
3-(2-Ch lor o-4-m et h oxyp h en yl)-8-et h yl-1-m et h yl-7-(2-
m et h ylb en zyl)-1,2,3,7-t et r a h yd r o-6H -p u r in -6-on e (12s).
Colorless oil; 1H NMR (300 MHz, CDCl3) δ 7.22-7.11 (m, 4H),
7.03 (d, J ) 2.6 Hz, 1H), 6.81 (dd, J ) 9.1, 2.9 Hz, 1H), 6.57
(d, J ) 7.0 Hz, 1H), 5.59 (s, 2H), 4.85 (s, 2H), 3.81 (s, 3H),
2.93 (s, 3H), 2.58 (q, J ) 7.3 Hz, 2H), 2.42 (s, 3H), 1.18 (t, J )
7.7 Hz, 3H); HRMS (ESI) m/e 425.1738 [(M + H)+, calcd for
hexanes-25% ethyl acetate with 3% methanol).
7-(1-Cyclobu tyleth yl)-3-(2,4-d ich lor op h en yl)-8-eth yl-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12k ). Colorless
oil; 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J ) 2.2 Hz, 1H),
7.15 (dd, J ) 8.5, 2.4 Hz, 1H), 7.00 (d, J ) 8.5 Hz, 1H), 4.81
(ABq, J AB ) 11.5, δυ ) 36.1 Hz, 2H), 3.35-3.25 (m, 1H), 2.95
(s, 3H), 2.75 (q, J ) 7.5 Hz, 2H), 2.21-2.15 (m, 1H), 1.91-
1.71 (m, 4H), 1.58-1.51 (m, 2H), 1.52 (d, J ) 6.7 Hz, 3H), 1.27
(t, J ) 7.6 Hz, 3H); HRMS (ESI) m/e 407.1406 [(M + H)+, calcd
for C20H25N4OCl2 407.1405. HPLC: (a) 100%; (b) 100% (75%
hexanes-25% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-eth yl-7-(4-m eth oxyben zyl)-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12l). Colorless
1
amorphous solid; H NMR (300 MHz, CDCl3) δ 7.46 (d, J )
2.2 Hz, 1H), 7.20-7.14 (m, 3H), 7.07 (d, J ) 8.7 Hz, 1H), 6.87
(d, J ) 8.8 Hz, 2H), 5.49 (s, 2H), 4.86 (s, 2H), 3.79 (s, 3H),
2.95 (s, 3H), 2.65 (q, J ) 7.7, 2H), 1.18 (t, J ) 7.7 Hz, 3H);
HRMS (ESI) m/e 445.1200 [(M + H)+, calcd for C22H23N4O2Cl2
445.1198]. Anal. (C22H22N4O2Cl2) C, H, N.
C
23H26N4O2Cl 425.1744]. HPLC: (a) 99.0%; (b) 100% (60%
hexanes-40% ethyl acetate with 3% methanol).
3-(2,4-Dich lor op h en yl)-8-et h yl-7-(2-m et h ylb en zyl)-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12m ). Colorless
solid; mp 142-143 °C; 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J
) 2.5 Hz, 1H), 7.21-7.09 (m, 5H), 6.52 (d, J ) 7.6 Hz, 1H),
5.56 (s, 2H), 4.87 (s, 2H), 2.90 (s, 3H), 2.54 (q, J ) 7.4 Hz,
2H), 2.39 (s, 3H), 1.16 (t, J ) 7.6 Hz, 3H); HRMS (ESI) m/e
429.1257 [(M + H)+, calcd for C22H23N4OCl2 429.1249]. Anal.
(C22H22N4OCl2) C, H, N.
3-(2,4-Dich lor op h en yl)-8-et h yl-1-m et h yl-7-(4-p h en yl-
ben zyl)-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12n ). Colorless
solid; mp 216-217 °C; 1H NMR (300 MHz, CDCl3) δ 7.56 (d, J
) 8.1 Hz, 4H), 7.47-7.32 (m, 5H), 7.24 (s, 1H), 7.20 (dd, J )
8.5, 2.2 Hz, 1H), 7.09 (d, J ) 8.4 Hz, 1H), 5.62 (s, 2H), 4.89 (s,
2H), 2.96 (s, 3H), 2.68 (q, J ) 7.3 Hz, 2H), 1.21 (t, J ) 7.3 Hz,
3-[2-Ch lor o-4-isop r op ylp h en yl]-8-eth yl-7-(4-h ep tyl)-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12t). brown oil;
1H NMR (300 MHz, CDCl3) δ 7.29 (d, J ) 1.9 Hz, 1H), 7.06
(dd, J ) 8.4, 1.8 Hz, 1H), 7.00 (d, J ) 8.1 Hz, 1H), 4.84 (s,
2H), 4.15-4.00 (m, 1H), 2.98 (s, 3H), 2.87 (pent, J ) 6.9 Hz,
1H), 2.70 (q, J ) 7.3 Hz, 2H), 2.30-2.10 (m, 2H), 1.98-1.85
(m, 2H), 1.30-1.20 (m, 13H), 0.92-0.91 (t, J ) 7.3 Hz, 6H);
HRMS (ESI) m/e 431.2581 [(M + H)+
, calcd for C24H36N4OCl
431.2578]. HPLC: (a) 98.0% (b) 98.5% (80% hexanes-20%
ethyl acetate with 3% methanol).
1-Ben zyloxym eth yl-4-[(2-ch lor o-4-isop r op yl)a m in o]-2-
eth yl-N-m eth yl-1H-im id a zole-5-ca r boxa m id e (14). 7-Ben-
zyloxymethyl-3-(2-chloro-4-isopropyl)-8-ethyl-1-methyl-3,7-
dihydro-1H-purine-2,6-dione 13 (7.88 g, 16.0 mmol) was

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4751
dissolved in THF (84 mL). The reaction mixture was cooled to
0 °C and LiAlH4 (67.6 mL, 67.6 mmol, 1 M in THF) was added
dropwise. After stirring for 1.25 h at 0 °C, the reaction was
quenched by the dropwise addition of ethanol followed by
saturated aqueous NaHCO3. The mixture was filtered through
a pad of Celite and the filtrate was extracted with CH2Cl2 (3×).
The combined organic layers were washed with brine, dried
over MgSO4, filtered and concentrated to afford 1-benzyloxy-
methyl-4-[(2-chloro-4-isopropyl)amino]-2-ethyl-N-methyl-1H-
imidazole-5-carboxamide 14 (3.50 g, 50% yield) as an orange
oil: 1H NMR (300 MHz, CDCl3) δ 8.57 (s br, 1H), 7.72 (s br,
1H), 7.44-7.30 (m, 6H), 7.18 (d, J ) 1.9 Hz, 1H), 7.04 (dd, J
) 8.5, 1.9 Hz, 1H), 5.63 (s, 2H), 4.70 (s, 2H), 2.90 (d, J ) 4.8
Hz, 3H), 2.83 (septet, J ) 7.0 Hz, 1 H, partially obs.), 2.74 (q,
J ) 7.3 Hz, 2H), 1.33 (t, J ) 7.7 Hz, 3H), 1.21 (d, J ) 7.0 Hz,
6H).
3-[2-Ch lor o-4-isop r op ylp h en yl]-8-et h yl-7-(1-et h yl-2-
m et h ylp r op yl)-1-m et h yl-1,2,3,7-t et r a h yd r o-6H -p u r in -6-
on e (12u ). Colorless oil; 1H NMR (300 MHz, CDCl3) δ 7.27
(m, 1H), 7.03 (dd, J ) 8.4, 1.8 Hz, 1H), 6.94 (d, J ) 8.0 Hz,
1H), 4.89 (s, 2H), 3.58-3.48 (m, 1H), 2.93 (s, 3H), 2.92-2.80
(m, 2H), 2.71 (q, J ) 7.3 Hz, 2H), 2.50-2.40 (m, 1H), 2.00-
1.90 (m, 1H), 1.30-1.21 (m, 9H), 1.07 (d, J ) 6.6 Hz, 3H),
0.90-0.75 (m, 6H); HRMS (ESI) m/e 417.2420 [(M + H)+, calcd
for C23H34N4OCl 417.2421]. HPLC: (a) 99.9% (b) 96.9% (80%
hexanes-20% ethyl acetate with 3% methanol).
2-E t h yl-1-(4-h ep t yl)-4-[(4-m et h oxyb en zyl)a m in o]-N-
m eth yl-1H-im id a zole-5-ca r boxa m id e (18) a n d 8-Eth yl-7-
(4-h ep tyl)-3-(p-m eth oxyben zyl)-1-m eth yl-1,2,3,7-tetr a h y-
d r o-6H -p u r in -6-on e (19). 8-Ethyl-7-(4-heptyl)-3-(4-meth-
oxybenzyl)-1-methyl-3,7-dihydro-1H-purine-2,6-dione 17 (550
mg, 1.33 mmol) prepared according to Method A, was dissolved
in THF (17 mL) and cooled to 0 °C. Lithium aluminum hydride
(5.32 mL, 5.32 mmol, 1 M in THF) was added dropwise via
syringe. The cooling bath was removed and the reaction
mixture was stirred at room temperature for 2.5 h. The
reaction mixture was cooled to 0 °C and was quenched by the
dropwise addition of H2O (0.20 mL) followed by 15% aqueous
NaOH (0.20 mL) then H2O (0.61 mL). The mixture was filtered
through a pad of Celite and was washed with EtOAc. The
filtrate was poured into a separatory funnel containing
saturated aqueous NaHCO3 (15 mL). The layers were sepa-
rated, and the aqueous layer was extracted with EtOAc (3 ×
20 mL). The combined organic layers were washed with brine,
dried over MgSO4, filtered and concentrated. The crude
material was purified by MPLC on silica gel (30% ethyl acetate
in hexanes f 65% ethyl acetate/35% hexanes) to afford the
desired products. The higher Rf product, 8-ethyl-7-(4-heptyl)-
3-(4-methoxybenzyl)-1-methyl-1,2,3,7-tetrahydro-6H-purin-6-
one 19, was isolated as a colorless oil (193 mg, 36% yield). The
lower Rf product, 2-ethyl-1-(4-heptyl)-4-[(4-methoxybenzyl)-
amino]-N-methyl-1H-imidazole-5-carboxamide 18, was isolated
as a colorless oil (270 mg, 53% yield).
2-Ethyl-1-(4-heptyl)-4-(p-methoxybenxyl)-N-methyl-1H-imi-
dazole-5-carboxamide 18 (220 mg, 0.570 mmol) was dissolved
in toluene (18 mL) and was treated with paraformaldehyde
(600 mg) and p-TsOH‚H2O (12 mg, 0.057 mmol). The mixture
was heated at 110 °C for 10 min. The mixture was cooled to
room temperature and transferred to a separatory funnel
containing saturated aqueous NaHCO3 (25 mL). The aqueous
layer was extracted with EtOAc (3 × 25 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtered and concentrated. The residue was purified by MPLC
(30% ethyl acetate in hexanes) to afford 8-ethyl-7-(4-heptyl)-
3-(4-methoxybenzyl)-1-methyl-1,2,3,7-tetrahydro-6H-purin-6-
one 19 (170 mg, 75% yield) as a colorless oil.
Da ta for 8-Eth yl-7-(4-h ep tyl)-3-(4-m eth oxyben zyl)-1-
m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e 19. Colorless oil;
1H NMR (300 MHz, DMSO-d6, 40 °C) δ 7.12 (d, J ) 8.8 Hz,
2H), 6.81 (d, J ) 8.4 Hz, 2H), 4.61 (s, 2H), 4.34-4.20 (m, 1H),
3.71 (s, 3H), 3.25 (s, 3H), 3.22 (s, 2H), 2.66-2.58 (m, 2H), 1.88-
1.77 (m, 2H), 1.71-1.59 (m, 2H), 1.24-1.12 (m, 2H), 1.18 (t, J
) 7.7 Hz, 3H), 1.05-0.91 (m, 2H), 0.81 (t, J ) 7.3 Hz, 6H);
LRMS (APCI) m/e 399.6 [(M + H)+, calcd for C23H35N4O2
399.3].
8-E t h yl-7-(4-h ep t yl)-1-m et h yl-1,2,3,7-t et r a h yd r o-6H -
p u r in -6-on e (20). 8-Ethyl-7-(4-heptyl)-3-(4-methoxybenzyl)-
1-methyl-1,2,3,7-tetrahydro-6H-purin-6-one 19 (375 mg, 0.941
mmol) was dissolved in trifluoroacetic acid (7 mL) and was
stirred at room temperature for 1 h. The mixture was
concentrated. The residue was transferred to a separatory
funnel containing saturated aqueous NaHCO3 (30 mL), and
the aqueous layer was extracted with EtOAc (3 × 30 mL). The
combined organic layers were washed with brine, dried over
MgSO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (5% methanol in CH2-
Cl2 f 10% methanol in CH2Cl2) to afford 8-ethyl-7-(4-heptyl)-
1-methyl-1,2,3,7-tetrahydro-6H-purin-6-one 20 (250 mg, 95%
1
yield) as a colorless solid: mp 129.5-130.5 °C; H NMR (300
MHz, CDCl3) δ 4.53 (s, 1H), 4.51 (s, 2H), 4.22-4.18 (m, 1H),
2.97 (s, 3H), 2.68 (q, J ) 7.3 Hz, 2H), 2.25-2.10 (m, 2H), 1.85-
1.75 (m, 2H), 1.32 (t, J ) 7.4 Hz, 3H), 1.30-1.10 (m, 4H), 0.88
(t, J ) 7.4 Hz, 6H); LRMS (APCI) m/e 279.1 [(M + H)+, calcd
for C15H27N4O 279.1].
8-E t h yl-7-(4-h ep t yl)-1-m et h yl-3,7-d ih yd r o-1H -p u r in e-
2,6-d ion e (21). 8-Ethyl-7-(4-heptyl)-3-(4-methoxybenzyl)-1-
methyl-3,7-dihydro-1H-purine-2,6-dione 17 (4.30 g, 10.4 mmol),
prepared according to Method A, was dissolved in trifluoro-
acetic acid (20 mL) and was heated at 105 °C in a sealed tube
for 14 h. The mixture was cooled to room temperature and
concentrated. The residue was transferred to a separatory
funnel containing saturated aqueous NaHCO3 (50 mL), and
the aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layers were washed with brine, dried over
MgSO4, filtered and concentrated. The residue was purified
by column chromatography on silica gel (5% methanol in CH2-
Cl2) to afford 8-ethyl-7-(4-heptyl)-1-methyl-3,7-dihydro-1H-
purine-2,6-dione 21 (2.96 g, 97% yield) as a colorless solid: mp
152.5-153 °C; 1H NMR (300 MHz, CDCl3) δ 12.00 (s, 1H),
4.20-4.10 (m, 1H), 3.41 (s, 3H), 2.94 (q, J ) 7.3 Hz, 2H), 2.33-
2.19 (m, 2H), 1.94-1.83 (m, 2H), 1.42 (t, J ) 7.4 Hz, 3H), 1.26-
1.06 (m, 4H), 0.90 (t, J ) 7.3 Hz, 6H); LRMS (APCI) m/e 293.1
[(M + H)+, calcd for C15H25N4O2 293.2].
8-E t h yl-7-(4-h ep t yl)-3-(4-m et h oxy-2-m et h ylp yr id in -3-
yl)-1-m eth yl-3,7-dih ydr o-1H-pu r in e-2,6-dion e (10d). 8-Eth-
yl-7-(4-heptyl)-1-methyl-3,7-dihydro-1H-purine-2,6-dione 21
(3.30 g, 11.3 mmol), 6-methoxy-2-methyl-3-pyridineboronic acid
(2.84 g, 17.0 mmol), copper(II) acetate (3.07 g, 17 mmol), and
4 Å powdered molecular sieves (3.0 g) were combined in a 200
mL round-bottom flask. CH2Cl2 (25 mL) and pyridine (1.81
mL, 22.6 mmol) were added, and the reaction mixture was
stirred at room temperature for 2 days. Additional 6-methoxy-
2-methyl-3-pyridineboronic acid (1.50 g, 8.98 mmol) and cop-
per(II) acetate (2.0 g, 11.0 mmol) were added, and the reaction
mixture was stirred at room temperature for an additional 2
days. The mixture was poured into a separatory funnel
containing saturated aqueous NH4Cl (100 mL) and concd NH4-
OH (150 mL). The aqueous layer was extracted with EtOAc
(4 × 100 mL). The combined organic layers were washed with
brine, dried over MgSO4, filtered and concentrated. The
residue was purified by column chromatography on silica gel
(30% ethyl acetate in hexanes then 75% ethyl acetate/25%
hexanes to recover starting material) to furnish 8-ethyl-7-(4-
heptyl)-3-(4-methoxy-2-methylpyrid-3-yl)-1-methyl-3,7-dihydro-
1H-purine-2,6-dione 10d (1.92 g, 41% yield) and recovered
starting material (1.72 g, 52% recovery). The product was
Da t a for 2-E t h yl-1-(4-h ep t yl)-4-[(4-m et h oxyb en zyl)-
a m in o]-N-m eth yl-1H-im id a zole-5-ca r boxa m id e 18. Color-
less oil;1H NMR (300 MHz, DMSO-d6) δ 7.37 (s br, 1H), 7.29
(d, J ) 8.5 Hz, 2H), 6.85 (d, J ) 8.5 Hz, 2H), 5.13 (s br, 1H),
4.25 (d, J ) 6.3 Hz, 2H), 3.72 (s, 3H), 2.66 (d, J ) 4.3 Hz, 3H),
2.59 (q, J ) 7.3 Hz, 2H), 2.00-1.60 (m, 4H), 1.25-0.96 (m,
4H), 1.18 (t, J ) 7.3 Hz, 3H), 0.81 (t, J ) 7.3 Hz, 6H); LRMS
(APCI) m/e 387.6 [(M + H)+, calcd for C22H35N4O2 387.3].
1
isolated as a colorless solid: mp 124-126 °C; H NMR (300
MHz, CDCl3) δ 7.45 (d, J ) 8.5 Hz, 1H), 6.70 (d, J ) 8.8 Hz,
1H), 4.18-4.05 (m, 1H), 3.97 (s, 3H), 3.46 (s, 3H), 2.72 (q, J )
7.7 Hz, 2H), 2.35-2.20 (m, 1H), 2.25 (s, 3H), 1.95-1.85 (m,

4752 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Hartz et al.
3H), 1.23 (t, J ) 7.4 Hz, 3H), 1.20-1.05 (m, 4H), 0.94-0.87
(m, 6H); HRMS (ESI) m/e 414.2506 [(M + H)+, calcd for
6H-purin-6-one 12x (57 mg, 86% yield) as a colorless solid:
mp 144-145.5 °C; H NMR (300 MHz, CDCl3) δ 7.44 (d, J )
1
C
22H32N5O3 414.2505]. Anal. (C22H31N5O3) C, H, N.
2.2 Hz, 1H), 7.14 (dd, J ) 8.4, 2.2 Hz, 1H), 6.88 (d, J ) 8.8 Hz,
1H), 4.85 (q, J ) 6.3 Hz, 1H), 4.10-4.02 (m, 1H), 2.94 (s, 3H),
2.71 (q, J ) 7.7 Hz, 2H), 2.50-1.75 (m, 4H), 1.54 (d, J ) 6.2
Hz, 3H), 1.40-1.00 (m, 4H), 1.28 (t, J ) 7.3 Hz, 3H), 1.06-
0.85 (m, 6H); HRMS (ESI) m/e 437.1871 [(M + H)+, calcd for
8-E t h yl-7-(4-h ep t yl)-3-(4-m et h oxy-2-m et h ylp yr id in -3-
yl)-1-m eth yl-1,2,3,7-tetr ah ydr o-6H-pu r in -6-on e (12v). 8-Eth-
yl-7-(4-heptyl)-3-(4-methoxy-2-methylpyrid-3-yl)-1-methyl-3,7-
dihydro-1H-purine-2,6-dione 10d (1.84 g, 4.46 mmol) was
dissolved in THF (50 mL) and was cooled to 0 °C. Lithium
aluminum hydride (8.92 mL, 8.92 mmol, 1 M in THF) was
added dropwise via syringe. The cooling bath was removed,
and the reaction mixture was stirred at room temperature for
30 min. The reaction mixture was cooled to 0 °C and was
quenched by the dropwise addition of H2O (0.34 mL) followed
by 15% aqueous NaOH (0.34 mL) then H2O (1.02 mL). The
mixture was filtered through a pad of Celite and was washed
with EtOAc. The filtrate was poured into a separatory funnel
containing saturated aqueous NaHCO3 (50 mL). The layers
were separated, and the aqueous layer was extracted with
EtOAc (3 × 50 mL). The combined organic layers were washed
with brine, dried over MgSO4, filtered and concentrated. Two
major products were formed in the reaction. The crude
material was purified by column chromatography on silica gel
(30% ethyl acetate in hexanes f 50% ethyl acetate in hexanes)
to afford the higher Rf product (306 mg) and the lower rf
product, 2-ethyl-1-(4-heptyl)-4-[(4-methoxy-2-methylpyrid-3-
yl)amino]-N-methyl-1H-imidazole-5-carboxamide 22, 800 mg,
46% yield) as a green oil. The higher Rf product was purified
further by column chromatography on silica gel (2% MeOH in
CH2Cl2) to afford 8-ethyl-7-(4-heptyl)-3-(4-methoxy-2-meth-
ylpyrid-3-yl)-1-methyl-1,2,3,7-tetrahydro-6H-purin-6-one 12v
(240 mg, 14% yield) as a pale green solid.
C
22H31N4OCl2 437.1875]. Anal. (C22H30N4OCl2) C, H, N.
3-(2,4-Dich lor op h en yl)-2,8-d ieth yl-7-(4-h ep tyl)-1-m eth -
yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12y). A solution of
4-[(2,4-dichlorophenyl)amino]-2-ethyl-N-methyl-1-(3-pentyl)-
1H-imidazole-5-carboxamide 11 (68 mg, 0.178 mmol) in toluene
(8 mL) was treated with propionaldehyde (1.0 mL, 0.81 g, 13.9
mmol) and p-TsOH‚H2O (8.0 mg, 0.042 mmol). The reaction
mixture was heated at reflux for 1 h in a flask equipped with
a Dean-Stark trap. The mixture was cooled to room temper-
ature and was transferred to a separatory funnel containing
saturated aqueous NaHCO3 (10 mL). The aqueous layer was
extracted with EtOAc (3 × 15 mL). The combined organic
layers were washed with brine, dried over MgSO4, filtered and
concentrated. The residue was purified by preparative TLC
(two 1000 µM silica plates, 60% hexanes/35% CH2Cl2/5% ethyl
acetate/0.2% MeOH) to furnish 3-(2,4-dichlorophenyl)-2,8-
diethyl-7-(4-heptyl)-1-methyl-1,2,3,7-tetrahydro-6H-purin-6-
one 12y (59 mg, 74% yield) as a colorless solid: mp 116.5-
1
117.5 °C; H NMR (300 MHz, CDCl3) δ 7.43 (d, J ) 2.6 Hz,
1H), 7.15 (dd, J ) 8.7, 2.5 Hz, 1H), 6.95 (d, J ) 8.8 Hz, 1H),
4.70 (t, J ) 7.4 Hz, 1H), 4.10-4.00 (m, 1H), 3.02 (s, 3H), 2.70
(q, J ) 7.3 Hz, 2H), 2.40-1.75 (m, 6H), 1.40-1.00 (m, 4H),
1.27 (t, J ) 7.7 Hz, 3H), 1.01 (t, J ) 7.6 Hz, 3H), 0.96-0.85
(m, 6H); HRMS (ESI) m/e 451.2040 [(M + H)+, calcd for
C
23H33N4OCl2 451.2031]. Anal. (C23H32N4OCl2) C, H, N.
Da t a for 2-E t h yl-1-(4-h ep t yl)-4-[(4-m et h oxy-2-m et h -
ylp yr id -3-yl)a m in o]-N-m et h yl-1H -im id a zole-5-ca r b ox-
a m id e 22. Green oil; 1H NMR (300 MHz, CDCl3) δ 7.14 (s br,
1H), 7.01 (d br, J ) 7.3 H, 1H), 6.47 (d, J ) 8.5 Hz, 1H), 5.42
(s br, 1H), 4.15-4.05 (m, 1H), 3.87 (s, 3H), 2.77 (d, J ) 5.1
Hz, 3H), 2.73 (q, J ) 7.3 Hz, 2H), 2.45 (s, 3H), 1.90-1.75 (m,
4H), 1.31 (t, J ) 7.3 Hz, 3H), 1.40-1.23 (m, 2H), 1.22-1.10
(m, 2H), 0.91 (t, J ) 7.4 Hz, 6H); LRMS (APCI) m/e 388.3 [(M
+ H)+, calcd for C21H34N5O2 388.3].
3-(2,4-Dich lor op h en yl)-7-(4-h ep t yl)-1-m et h yl-1,2,3,7-
tetr a h yd r o-6H-p u r in -6-on e (12z). Colorless oil; 1H NMR
(300 MHz, CDCl3) δ 7.47 (d, J ) 2.2 Hz, 1H), 7.39 (s, 1H),
7.20 (dd, J ) 8.4, 2.2 Hz, 1H), 7.09 (d, J ) 8.4 Hz, 1H), 4.94-
4.85 (m, 1H), 4.87 (s, 2H), 2.98 (s, 3H), 1.92-1.80 (m, 4H),
1.38-1.22 (m, 4H), 0.92 (t, J ) 7.3 Hz, 6H); HRMS (ESI) m/e
395.1410 [(M + H)+, calcd for C19H25N4OCl2 395.1405]. Anal.
(C19H24N4OCl2) C, H, N.
Da ta for 8-Eth yl-7-(4-h ep tyl)-3-(4-m eth oxy-2-m eth ylp y-
r id -3-yl)-1-m eth yl-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e 12v.
Pale green solid; mp 120-122 °C; 1H NMR (300 MHz, DMSO-
d6, 80 °C) δ 7.39 (d, J ) 8.7 Hz, 1H), 6.63 (d, J ) 8.8 Hz, 1H),
4.69 (s, 2H), 4.55-4.44 (m, 1H), 3.87 (s, 3H), 2.86 (s, 3H), 2.63
(q, J ) 7.7 Hz, 2H), 2.37 (s, 3H), 2.19-2.07 (m, 2H), 1.84-
1.73 (m, 2H), 1.32-1.07 (m, 4H), 1.15 (t, J ) 7.3 Hz, 3H), 0.87
(t, J ) 7.3 Hz, 6H); HRMS (ESI) m/e 400.2715 [(M + H)+, calcd
for C22H34N5O2 400.2713]. Anal. (C23H33N5O2) C, H, N.
Biology. Bin d in g Assa ys. Frozen rat frontal cortex (source
of CRF1 receptor) or frozen porcine choroid plexus (source of
CRF2 receptor) were thawed rapidly in assay buffer containing
50 mM HEPES (pH 7.0 at 23 °C), 10 mM MgCl2, 2 mM EGTA,
1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A,
0.005% Triton X-100, 10U/mL bacitracin and 0.1% ovalbumin
and homogenized. The suspension was centrifuged at 32000g
for 30 min. The resulting supernatant was discarded and the
pellet resuspended by homogenization in assay buffer and
centrifuged again. The supernatant was discarded and the
pellet resuspended by homogenization in assay buffer and
frozen at -70 °C. On the day of the experiment aliquots of
the homogenate were thawed quickly and homogenate (25 µg/
well rat frontal cortex or 10 µg/well porcine choroid plexus)
added to ligand (150 pM 125I-ovine-CRF for CRF1 binding or
100 pM 125I-sauvagine for CRF2 binding) and drugs in a total
volume of 100 µL of assay buffer. The assay mixture was
incubated for 2 h at 21 °C. Bound and free radioligand were
then separated by rapid filtration, using glass fiber filters
(Whatman GF/B, pretreated with 0.3% PEI) on a Brandel Cell
Harvester. Filters were then washed multiple times with ice
cold wash buffer (PBS w/o Ca2+ and Mg2+, 0.01% Triton X-100
(pH 7.0 at 23 °C)). Nonspecific binding was defined using 1
µM DMP696 in the CRF1 binding assay and 1 µM R-helical
CRF (9-41) in the CRF2 binding assay. Filters were then
counted in a Wallac Wizard gamma counter.
3-(4-Ch lor oph en yl)-8-eth yl-7-(4-h eptyl)-1-m eth yl-1,2,3,7-
tetr a h yd r o-6H-p u r in -6-on e (12w ). Colorless solid, mp 99-
1
100 °C; H NMR (500 MHz, DMSO-d6, 100 °C) δ 7.45 (d, J )
2.2 Hz, 2H), 7.32 (d, J ) 2.2 Hz, 2H), 4.98 (s, 2H), 4.65-4.55
(m, 1H), 2.93 (s, 3H), 2.72 (q, J ) 7.6 Hz, 2H), 2.14-2.08 (m,
2H), 1.85-1.78 (m, 2H), 1.31-1.27 (m, 2H), 1.27 (t, J ) 7.6
Hz, 3H), 1.18-1.00 (m, 2H), 0.87 (t, J ) 7.3 Hz, 6H); HRMS
(ESI) m/e 389.2103 [(M + H)+, calcd for C21H30N4OCl 389.2108].
Anal. (C21H29N4OCl) C, H, N.
3-(2,4-Dich lor op h en yl)-1,2-d im et h yl-8-et h yl-7-(4-h ep -
tyl)-1,2,3,7-tetr a h yd r o-6H-p u r in -6-on e (12x). A solution of
4-[(2,4-dichlorophenyl)amino]-2-ethyl-N-methyl-1-(3-pentyl)-
1H-imidazole-5-carboxamide 11 (58 mg, 0.151 mmol) in toluene
(8 mL) was treated with acetaldehyde dimethylacetal (0.40 mL,
3.79 mmol) and p-TsOH‚H2O (8.0 mg, 0.042 mmol). The
reaction mixture was heated at reflux for 1.25 h in a flask
equipped with a Dean-Stark trap. The mixture was cooled to
room temperature and was transferred to a separatory funnel
containing saturated aqueous NaHCO3 (10 mL). The aqueous
layer was extracted with EtOAc (3 × 15 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtered and concentrated. The residue was purified by pre-
parative TLC (two 1000 µM silica plates, 60% hexanes/35%
CH2Cl2/5% ethyl acetate/0.2% MeOH) to furnish 3-(2,4-dichlo-
rophenyl)-1,2-dimethyl-8-ethyl-7-(4-heptyl)-1,2,3,7-tetrahydro-
Ma ter ia ls. Rat frontal cortex and porcine choroid plexus
were obtained from Analytical Biological Services, Inc. (Wil-
mington, DE). 125I-ovine-CRF (2200 Ci/mmol) and 125I-sau-
vagine (2200 Ci/mmol) were obtained from PerkinElmer Life
Sciences, Inc. (Boston, MA).
F u n ction a l Assa y (Y-79 cells). Human Y-79 retinoblas-
toma cells were suspended in assay buffer (Hank’s Balanced
Salt Solution containing 2 mM CaCl2, 5 mM Mg Cl2, 20 mM

Corticotropin Releasing Factor1 Receptor Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4753
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W.; Chalmers, D. T.; DeSouza, E. B.; Oltersdorf, T. Cloning and
characterization of a functionally distinct corticotropin-releasing
factor receptor subtype from rat brain. Proc. Natl. Acad. Sci.
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HEPES, 1 mM IBMX) and plated at 20 000 cells/well in a 96-
well black plate. CRF antagonists (typically 0.01 to 10 000 nM)
were then added to wells as needed and allowed to equilibrate
with the cells for 30 min at 37 °C. CRF (1 nM; CRF EC50
)
1.11 ( 0.14 nM, n ) 6), dissolved in assay buffer + 0.1% BSA,
was then added to the wells (30 min at 37 °C) to stimulate
the production of cAMP. The reaction was terminated by the
addition of a lysis solution containing homogeneous time-
resolved florescence (HTRF) cAMP XL665 conjugate followed
by HTRF anti-cAMP cryptate conjugate (CIS bio Interna-
tional). Plates were subsequently incubated at room temper-
ature for 1 h prior to reading the time-resolved fluorescence
signal. The amount of cAMP produced was estimated from a
standard curve prepared using known concentrations of cAMP.
The percentage inhibition of CRF-induced cAMP production
was determined for each compound (triplicate determinations).
The effect of CRF antagonists on basal cAMP production (i.e.
in the absence of CRF) was also determined.
Ra t Ca ssette P h a r m a k ok in etic Stu d y. Compound 12d
was coadministered intravenously with 10 other compounds
to Sprague-Dawley rats (n ) 3) at a dose of 0.5 mg/kg for
each compound. In addition, the same set of compounds was
administered to rats (n ) 3) orally at a dose of 2 mg/kg. Plasma
samples were collected at 0, 0.1, 0.25, 0.5, 1, 2, 4, 6, 8, and 24
h postdose for the intravenous experiment, and at 0, 0.5, 1, 2,
4, 6, 8, and 24 h postdose for the oral experiment. Drug
concentrations were determined in the plasma samples using
LC/MS/MS.
Ack n ow led gm en t. The authors gratefully thank
Gail Mattson and Anne Marshall for performing the in
vitro binding studies.
Su p p or tin g In for m a tion Ava ila ble: Tables of elemental
analysis data and high-resolution mass spectral data with
HPLC purity data for compounds lacking elemental analysis
data. This information is available free of charge via the
Internet at http://pubs.acs.org.
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