Synthesis, structure-activity relationships, and in vivo evaluation of N3-phenylpyrazinones as novel corticotropin-releasing factor-1 (CRF1) receptor antagonists

Article abstract of DOI:10.1021/jm900301y

Evidence suggests that corticotropin-releasing factor-1 (CRF₁) receptor antagonistsmay offer therapeutic potential for the treatment of diseases associated with elevated levels of CRF such as anxiety and depression. A pyrazinone-based chemotype

Full text of DOI:10.1021/jm900301y

J. Med. Chem. 2009, 52, 4173–4191 4173
DOI: 10.1021/jm900301y
Synthesis, Structure-Activity Relationships, and In Vivo Evaluation of N3-Phenylpyrazinones as Novel
Corticotropin-Releasing Factor-1 (CRF1) Receptor Antagonists
Richard A. Hartz,*,† Vijay T. Ahuja,† Argyrios G. Arvanitis,† Maria Rafalski,† Eddy W. Yue,† Derek J. Denhart,†
William D. Schmitz,† Jonathan L. Ditta,† Jeffrey A. Deskus,† Allison B. Brenner,† Frank W. Hobbs,† Joseph Payne,†
Snjezana Lelas,‡ Yu-Wen Li,‡ Thaddeus F. Molski,‡ Gail K. Mattson,‡ Yong Peng,‡ Harvey Wong,§ James E. Grace,§
Kimberley A. Lentz,§ Jingfang Qian-Cutrone,§ Xiaoliang Zhuo,§ Yue-Zhong Shu,§ Nicholas J. Lodge,‡ Robert Zaczek,‡
Andrew P. Combs,† Richard E. Olson,† Joanne J. Bronson,† Ronald J. Mattson,† and John E. Macor†
†Neuroscience Discovery Chemistry and ‡Neuroscience Discovery Biology and §Pharmaceutical Candidate Optimization, Research and
Development, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492
Received March 10, 2009
Evidence suggests that corticotropin-releasing factor-1 (CRF1) receptor antagonists may offer therapeutic
potential for the treatment of diseases associated with elevated levels of CRF such as anxiety and
depression. A pyrazinone-based chemotype of CRF1 receptor antagonists was discovered. Structure-
activity relationship studies led to the identification of numerous potent analogues including 12p, a highly
potent and selective CRF1 receptor antagonist with an IC50 value of 0.26 nM. The pharmacokinetic
properties of 12p were assessed in rats and Cynomolgus monkeys. Compound 12p was efficacious in the
defensive withdrawal test (an animal model of anxiety) in rats. The synthesis, structure-activity relation-
ships and in vivo properties of compounds within the pyrazinone chemotype are described.
Introduction
primarily in the central nervous system, whereas CRF2 re-
ceptors are found primarily in the periphery. Numerous
studies have provided evidence that hypersecretion of CRF
is associated with various affective disorders including depres-
sion and anxiety.1-5,14 Patients suffering with depression have
been found to have elevated levels of CRF in cerebrospinal
fluid.15,16 Successful treatment of depression resulted in nor-
malization of CRF levels.10,17,18 In addition, experiments with
CRF1 receptor knockout mice have implicated the involve-
ment of CRF1 receptors in the stress response mediated by the
HPA axis.19 In the presence of increasing levels of CRF, an
increase in ACTH secretion was not observed in the pituitary
cells of mice lacking the CRF1 receptor. Experiments showed
that intracerebroventricular administration of CRF to rats
promoted behavioral effects similar to those observed in
anxiety and depression.8,10,20 In contrast, administration of
the peptide-based CRF antagonists R-helical CRF(9-41) and
astressin successfully blocked the effects of CRF.21
A number of small molecule CRF1 receptor antagonists,
including compounds 1 (CP-154526),22-25 2 (DMP696),26-29
3(DMP904),27,30 and 4(R121919, also known as NBI-30775),31-34
have been extensively studied in numerous preclinical behavioral
models and were shown to be efficacious in rat models for anxiety
and/or depression (Figure 1). These results suggest that CRF1
receptor antagonists may offer therapeutic potential for the treat-
ment of diseases resulting from elevated levels of CRF such as
anxiety and depression. The clinical efficacy results of a small
number of CRF1 receptor antagonists have slowly begun to appear
in the literature. The first clinical study published was a small, open-
label clinical trial with 4wherein it was found that depressed patients
showed reductions in depression symptoms, as rated by both
patients and clinicians.35 Compound 5 (NBI-34041)36 was sub-
sequently tested in a placebo-controlled proof-of-concept study
designed to evaluate whether subchronic treatment with this
a
Corticotropin-releasing factor-1 (CRF1 ) receptor antago-
nists are of considerable interest as a potential novel treatment
of stress-related disorders such as anxiety and depression.1-6
Corticotropin-releasing factor (CRF), a 41 amino acid neuro-
peptide first isolated by Valeand co-workers,7 functions as the
primary regulator of the hypothalamic-pituitary-adrenal
(HPA) axis, coordinating endocrine, behavioral, and auto-
nomic responses to stress.8,9 CRF mediates its action through
binding to two extensively characterized, class B subtype,
G-protein coupled receptors, CRF1 and CRF2, which are widely
distributed throughout the central and peripheral nervous
systems.10 Furthermore, three splice variants of CRF2 sub-
type receptors have been identified (R, β, and γ).11,12 CRF,
upon synthesis and release from the paraventricular region of
the hypothalamus, binds to receptors in the anterior pituitary,
promoting the subsequent release of adrenocorticotropic
hormone (ACTH), β-endorphin, and other proopiomelano-
cortin (POMC)-derived peptides. Increased levels of ACTH,
in turn, induce the production and secretion of cortisol from
the adrenal cortex, resulting in a variety of metabolic changes
that facilitate the body’s response to a stressor. It should be
noted that CRF may not originate exclusively from paraven-
tricular neurons controlling ACTH secretion as these neurons
represent only one element of an extensive system of CRF
neurons and projections throughout the brain.13
CRF1 receptors have been more extensively characterized
than the CRF2 receptor subtypes. CRF1 receptors are expressed
*To whom correspondence should be addressed. Phone: 203-677-7837.
Fax: 203-677-7702. E-mail: richard.hartz@bms.com.
a Abbreviations: CRF, corticotropin-releasing factor; HPA, hypo-
thalamic-pituitary-adrenal; ACTH, adrenocorticotropic hormone;
POMC, proopiomelanocortin; SAR, structure-activity relationships;
cAMP, cyclic adenosine monophosphate; GSH, glutathione.
r
2009 American Chemical Society
Published on Web 06/24/2009
pubs.acs.org/jmc

4174 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
Scheme 1a
a Reagents and conditions: (a) chloroacetonitrile, KI, K2CO3,
CH3CN, 50 °C (84-96%); (b) (COCl)2, toluene, 55 °C (43-76%) or
(COCl)2, dioxane/CH2Cl2, 55 °C (69-74%); (c) (COBr)2, CH2Cl2, 45 °C
(64%); (d) base, ArNH2, THF or DMF; (e) ArNH2, Pd(OAc)2, BINAP,
K2CO3, toluene.
Scheme 2a
Figure 1. CRF1 receptor antagonists.
Figure 2. Substituted pyrazinone ring system.
investigational drug would decrease the stress hormone response
following a psychological stressor.37 Briefly, the results of this study
indicated that 5 may improve resistance to psychological stress by
reducing stress hormone secretion. In a double-blind, placebo-
controlled clinical trial to evaluate 6(CP-316311)38 for the treatment
of major depressive disorder, it was found that the group treated
with 6 was not significantly different in the primary efficacy end
point from the placebo-treated group, indicating a lack of efficacy.39
Several additional CRF1 antagonists, including 7 (CP-376395),40
are reported to have entered clinical trials but the results have not yet
been disclosed.1 Although the outcome of clinical trials reported to
date have been mixed, the prospect that CRF1 receptor antagonists
may offer therapeutic potential for the treatment of diseases
resulting from elevated levels of CRF continues to drive efforts to
discover and develop viable, structurally diverse CRF1 receptor
antagonists.
In our search for structurally diverse CRF1 receptor an-
tagonists, we investigated the structure-activity relationships
(SAR) of a series of N3-phenylpyrazinones (Figure 2), a
chemotype that was first disclosed in a patent publication.41
The pyrazinone ring system was an attractive scaffold based
on its structural relationship to the well-established pharma-
cophore model of known CRF1 receptor antagonists.6 The
synthesis and SAR of these pyrazinone-based compounds is
described below.
a Reagents and conditions: (a) cyclopropylmagnesium bromide, THF
(81%); (b) NaBH(OAc)3, NH4O2CCF3, THF; (c) benzyl chloroformate,
Na2CO3, CH2Cl2/H2O, (41-78%, 2 steps); (d) Enantiomers of 16 were
separated on a Chiralpak AD column and enantiomers of 20 were
separated on a Chiralpak AS column; (e) H2, Pd/C, 4 N HCl in dioxane,
EtOH (95-100%).
yield. The cyanomethylamine intermediate was then heated in
the presence of oxalyl chloride in toluene at 55 °C to form
dichloropyrazinone intermediates 10.42 For analogues with an
ether moiety in the R1 group, improved yields were obtained by
heating the reaction mixture at 55 °C in a mixture of dioxane and
dichloromethane. Preparation of dibromopyrazinone intermedi-
ate 11 was carried out in an analogous fashion whereby cyano-
methylamine 9 was treated with oxalyl bromide.43 Coupling of
the dichloro- and dibromopyrazinones 10 and 11, respectively,
with a variety of aryl amines in the presence of a base then
furnished the desired products 12 and 13, respectively.
The various alkylamines from which R1 was comprised were
either purchased, synthesized according to literature references,
or synthesized as illustrated in Schemes 2-3.44 The synthesis of
18 began with addition of cyclopropylmagnesium bromide
to (N-methyl-N-methoxy)methoxyacetamide45 (14) (Scheme 2)
to afford cyclopropylmethoxymethyl ketone 15 in 81%
yield. The ketone was transformed to 16 via a two-step, one-
pot procedure whereby 15 was treated with sodium tri-
acetoxyborohydride in the presence of ammonium trifluoro-
acejtate. Upon completion of the reaction, the solvent was
removed under reduced pressure and the remaining material
Results and Discussion
Chemistry. The general synthetic scheme employed to prepare
1-alkyl-3-anilino-5-halopyrazin-2-ones is shown in Scheme 1.
Treatment of alkylamine hydrochlorides 8 with chloroaceto-
nitrile in the presence of potassium iodide and potassium
carbonate in acetonitrile afforded cyanomethylamines 9 in high

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4175
Scheme 4a
Scheme 3a
a Reagents and conditions: (a) Ph3CCl, Et3N, CH2Cl2 (82%); (b) NaH,
MeI, THF (100%); (c) 2 N HCl in ether, CH2Cl2/MeOH (85%).
was taken up in CH2Cl2/H2O with sodium carbonate and was
treated with benzyl chloroformate to afford 16 (78% yield, two
steps). Separation of 16 into its enantiomers was achieved by
chromatography on chiral support to provide 17 in>99% ee.46
Removal of the Cbz group by hydrogenation then afforded 18
in quantitative yield.
The synthesis of 22 was completed in three steps as
depicted in Scheme 2. Cyclopropylethyl ketone 1947 was
transformed to 20 as described for the preparation of 16
(41% yield over two steps). Separation of 20 into its en-
antiomers was achieved by chromatography on chiral sup-
port resulting in material with >99% ee.48 Removal of the
Cbz group by hydrogenation then afforded 22 in high yield.
The synthesis of 26 was carried out as illustrated in
Scheme 3. Protection of the amine of commercially available
aminoalcohol 23 with a triphenylmethyl group proceeded in
good yield. Subsequent alkylation with methyl iodide fol-
lowed by deprotection with HCl in ether afforded the desired
product (26).
Methyl pyrazinone analogues (X=Me) were prepared by
two different methods. In the first method, a four-step
procedure was employed to synthesize 3-chloro-5-methyl-
pyrazinone intermediate 30 (Scheme 4). Treatment of 27 with
chloroacetone in the presence of potassium carbonate foll-
owed by subsequent treatment with ethylchlorooxoacetate
afforded 28. Cyclization was carried out by heating 28 in
acetic acid in the presence of ammonium acetate to furnish
29. Compound 29 was heated at reflux in thionyl chloride
with a catalytic amount of DMF to give methylpyrazinone
30 in good yield. Compound 30 was then coupled with an
arylamine as described previously to afford 31.
Alternatively, methylpyrazinone analogue 31 was prepared
from the corresponding bromopyrazinone 13 by palladium-
catalyzed coupling with methylboronic acid as shown in
Scheme 5. Bromopyrazinone 13 also served as an intermediate
for other variations at the 5-position of the pyrazinone. Pre-
paration of the des-halo analogue was effected by subjecting 13
to Pd/C under hydrogen, resulting in debromination to furnish
32. Synthesis of the cyanopyrazinone was completed by coup-
ling bromopyrazinone 13 with zinc cyanide in the presence of a
palladium catalyst49 to furnish the desired cyanopyrazinone
product (33) in good yield. Alkynylpyrazinone 34 was prepared
in two steps from 13 via palladium-catalyzed coupling with
(trimethyl)silylacetylene followed by basic hydrolysis of the
TMS group. Subsequent hydrogenation of the alkyne afforded
ethylpyrazinone 35. Allylpyrazinone 36 was prepared via pall-
adium-catalyzed coupling of 13 with allyltributyltin.
a Reagents and conditions: (a) chloroacetone, K2CO3, KI, MeCN (77%);
(b) ethyl chlorooxoacetate, pyridine, CH2Cl2 (44%); (c) NH4OAc, HOAc
(58%); (d) DMF, SOCl2 (78%); (e) NaHMDS, 3-amino-6-methoxy-2-
trifluoromethylpyridine, THF (14%).
screened for binding affinity at CRF2 and for their functional
activity in Y79 cells to assess antagonist properties. The
CRF2 receptor binding titration assay employed porcine
choroid plexus homogenate and [125I]-sauvagine binding
inhibition to determine binding affinities. Compounds with
appropriate potency were evaluated in pharmacokinetic
studies and in the defensive withdrawal model of anxiety in
rats.29,50 In conjunction with the behavioral studies, com-
pounds were examined in an ex vivo binding assay to
determine CRF1 receptor occupancy. In vivo plasma levels
after oral dosing were also measured.
The CRF1 receptor binding affinities for pyrazinone-
based analogues are shown in Tables 1-6. Four points of
diversity were examined, designated as R1, X, Y, and Ar in
Figure 2. The R1 substituent SAR is summarized in Table 1
wherein the phenyl group was either a 2,4,6-trimethylphenyl
or a 2,5-dimethyl-4-methoxyphenyl group. On the basis of
SAR studies in earlier chemotypes, we focused on branched
substituents at R1.
It was found that improved CRF1 receptor binding affi-
nity was achieved when the branching point was on the
carbon atom bonded directly to the pyrazinone ring; com-
pounds with a carbon atom between the pyrazinone nitrogen
and branching point were much less potent (e.g., compare
12c vs 12d and 12k vs 12l). Incorporation of a cyclopropyl
group within R1 resulted in an improvement in potency over
the corresponding straight-chain alkyl analogues (compare
12f vs 12d and 12n vs 12l). A single methoxy group within R1
was also well-tolerated. A decrease in potency of approxi-
mately 30-fold was observed when the methoxy group in
compounds 12b and 12p was replaced with a hydroxy group
(compounds 12j and 12t).
In a limited investigation of the effect of stereogenicity
within the R1 substituent, we found little difference in
activity between enantiomers (Table 2). For example, the
binding affinities of a pair of enantiomers with a cyclopropyl
and methoxymethyl group within R1 were equivalent (com-
pare 12p vs 12u), perhaps due to the cyclopropyl and
methoxymethyl groups being fairly similar in size to each
other. In some cases, a differential in potency of 2-3 fold was
observed (e.g., 12v/12w, 12x/12y, and 12r/12z), but even this
was considered a modest difference. Compounds 12p and
Biology. Compounds were tested in a CRF1 receptor
binding titration assay using rat frontal cortex homogenate
in which inhibition of specific binding of [125I]Tyr-ovine-
CRF by our test compounds was measured to determine
their receptor binding affinity. Selected compounds were

4176 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
Scheme 5a
a Reagents and conditions: (a) MeB(OH)2, Pd(Pt-Bu3)2, K2CO3, dioxane (23%); (b) H2, 10% Pd/C, EtOH (98%); (c) Zn(CN)2, Pd2(dba)3, dppf, H2O,
DMF (71%); (d) (trimethyl)silylacetylene, Pd(PPh3)4, Et3N, DMF, 120 °C, microwave (16%); (e) 10 N NaOH, MeOH (49%); (f) H2, 10% Pd/C, MeOH
(29%); (g) allyltributyltin, Pd(PPh3)4, toluene, 120 °C, microwave (51%).
Table 1. CRF1 Receptor Binding Affinity SAR for R1 Substitution
Table 2. Comparison of CRF1 Receptor Binding Affinity for Enantiomers
a All values are the average of at least n=3 ( standard deviation unless
indicated otherwise. b Value determined by two measurements.
(CRF2 IC50 >10 μM for each compound), indicating that
these compounds are selective CRF1 receptor antagonists.
SAR results of phenyl group substituent modifications are
shown in Table 3. A comparison of compounds 12aa thru
12ac shows that a 2,4,6-substitution pattern gave optimal
CRF1 receptor binding affinity compared to compounds
with a 2,6-substitution pattern or only a 4-substituent.
Nearly a 20-fold decrease in potency occurred upon
removal of the 4-chloro group (compare 12aa vs 12ab),
a All values are the average of at least n = 3 ( standard deviation. The
IC50 of o-CRF=2.9( 1.0 nM and the IC50 of 2=1.2 ( 0.2 nM in this assay.
12u were also tested in the CRF2 receptor binding assay.51 It
was found that both compounds were inactive in this assay

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4177
Table 3. Effect of Phenyl Group Substituents on CRF1 Receptor Binding
Affinity
Table 5. Effect of Tieback Modifications of the Phenyl Group on CRF1
Receptor Binding Affinity
compd
R1
R2
R3
R4
Cl
R5
R6
IC50 (nM)a
compd
Z
n
R3
R4
R5
R6
IC50 (nM)a
12aa
12ab
12ac
12ad
12f
1
1
1
1
1
1
1
1
2
2
2
2
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
0.53 ( 0.18
9.3 ( 1.3
180 ( 50
6.6 ( 1.4
0.63 ( 0.14
2.4 ( 0.5
0.52 ( 0.04
0.27 ( 0.04
0.26 ( 0.04
1.5 ( 0.6
910 ( 160
280 ( 70
H
Cl
H
12an
12ao
12ap
CH2
CH2
O
1
1
2
H
H
H
OMe
OMe
OMe
H
H
H
Cl
Br
Br
0.62 ( 0.23
0.94 ( 0.20
19 ( 3
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
a All values are the average of at least n=3 ( standard deviation.
H
Me
H
12ae
12af
12n
Me
Me
H
OMe
OMe
OMe
OEt
OBn
OH
Me
H
Table 6. CRF1 Receptor Binding Affinity SAR for Substitution at X
Me
Me
Me
Me
Me
12p
H
12ag
12ah
12ai
H
H
H
a All values are the average of at least n=3 ( standard deviation.
Table 4. Effect of Aniline Alkylation on CRF1 Receptor Binding Affinity
compd
X
IC50 (nM)a
32
H
150 ( 70
1.8 ( 0.4
1.2 ( 0.3
3.8 ( 2.1
6.7 ( 1.0
5.6 ( 2.4
180 ( 20
1200 ( 400
12aq
13
Cl
Br
31
33
Me
CꢀN
CꢀCH
Et
34
35
compd
R1
R2
R3
R4
R5
R6
Y
IC50 (nM)a
36
allyl
a All values are the average of at least n=3 ( standard deviation.
12ae
12aj
12f
1
1
1
1
2
2
Me
Me
Me
Me
Cl
H
H
H
H
H
H
Me
Me
Me
Me
H
H
H
2.4 ( 0.5
1.5 ( 0.3
0.63 ( 0.14
4.6 ( 1.4
1.1 ( 0.1
34 ( 7
H
Et
H
A group of compounds was prepared to determine the
effect of alkylation of the aniline nitrogen (Table 4). In the
2,4,5-substituted aryl series, alkylation was well-tolerated
(compare 12ae vs 12aj) whereas in the 2,4,6-substituted aryl
series, alkylation led to an 8-30-fold decrease in potency
(compare 12f vs 12ak and 12al vs 12am). Analogues were also
prepared to determine the effect of tying back the ethyl
substituent at Y to form a bicyclic ring system (Table 5).54
The two indoline derivatives,55 12an and 12ao, were similar
in potency to 12al and much more potent than the corres-
ponding N-ethyl analogue 12am. When the indoline was
replaced with a 3,4-dihydro-2H-benzo[b][1,4]oxazine ring
system56 (12ap), a 20-fold decrease in binding affinity was
observed.
The SAR of substituents at X was explored with a series of
compounds containing the 1-(R)-cyclopropylethyl group at
R1 (Table 6). Compound 3257 with a hydrogen at X was
approximately 100-fold less potent than the corresponding
chloro analogue 12aq. Replacement of the chloro group with
a nitrile or an alkyne resulted in a 3- to 4-fold decrease in
potency. The limited steric tolerance at this position was
evident by the 100- and 700-fold decrease in potencies when
the chloro group was replaced by an ethyl or allyl group,
respectively. Thus, the SAR results indicate the need for a
small lipophilic group at X.
Me
Me
Me
Me
Cl
Cl
12ak
12al
12am
H
Et
H
OMe
OMe
H
Cl
H
Et
a All values are the average of at least n=3 ( standard deviation.
and when both ortho substituents in 12aa were removed, a
decrease in potency greater than 300-fold was observed
(12ac). On the basis of these results, we concluded that an
ortho substituent was required to achieve good binding
affinity to the CRF1 receptor. This observation has been
reported in the literature for other chemotypes as well. A
compound with a 2,4-dimethylphenyl group (12ad) was
approximately 10-fold less potent than the correspond-
ing 2,4,6-trimethylphenyl analogue (12f). In contrast to
the 2,4-dimethyl substituted analogue (12ad), analogues
with a 2,4,5-substitution pattern were similar in potency
to comparable 2,4,6-substituted analogues (compare 12ae
vs 12f and 12n vs 12af52). Variation of the 4-methoxy group
on the 2,4,5-substituted phenyl group was examined with
compounds 12p, 12ag, and 12ah. The results suggested
limited steric tolerance at R4, with potency decreasing as
the substituent at R4 increased in size.53 In addition, polar
groups at R4 were not well tolerated, as illustrated by
compound 12ai.

4178 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
Table 7. Discrete Pharmacokinetic Properties of 12p in Rats and Cynomo-
lgus Monkeys
PK parameters
12p in rats
12p in monkeys
iv dose (2 mg/kg)a
Cl (mL/min/kg)
170
97
21
Vss (L/kg)
t1/2 (h)
7.7
9.3
18
po dose (10 mg/kg)b
AUC (nM h)
360
52
410
32
2
3
Cmax (nM)
F%
14
a Vehicle: PEG/ethanol, 90:10 (v/v); n=2 animals. b Vehicle: 1%
Tween 80 in 0.5% methylcellulose; n=3 animals.
Compound 12p was chosen for further evaluation due to
its excellent potency (IC50=0.26 nM). The measured logD
of 12p was 4.35 at pH = 7.4.58 It was determined by
equilibrium dialysis that 12p was 2.1% unbound in plasma.
In a cell-based functional assay, the antagonist properties
of 12p were assessed by measuring inhibition of CRF-
stimulated cyclic adenosine monophosphate (cAMP) pro-
duction in human Y-79 retinoblastoma cells. 12p produced
a concentration-dependent inhibition of CRF (1nM)-in-
duced cAMP production with an IC50 value of 1.9 ( 0.6 nM
and completely suppressed CRF-stimulated cAMP produc-
tion at higher concentrations, indicating that this com-
pound behaves as an antagonist. No agonist properties
were detected at concentrations up to 10 μM. In addition,
12p was not active in a CRF2 receptor binding assay (vide
supra), indicating that this compound is a selective CRF1
receptor antagonist.
Shown in Table 7 is the pharmacokinetic profile of 12p after
iv (2 mg/kg) and oral dosing (10 mg/kg) in male Sprague-
Dawley rats and Cynomolgus monkeys. The results indicate
that in rats 12p was a high clearance compound with a clearance
higher than hepatic blood flow (55 mL/min/kg). The long half-
life of this compound is likely due to its large volume of
distribution, which in turn can be ascribed to its lipophilic
nature. 12p had modest oral bioavailability in rats (F%=14).
12p was a moderate clearance compound in monkeys (47% of
hepatic blood flow), and had a much lower volume of distribu-
tion compared to that in rats. However, 12p had only 2% oral
bioavailability in monkeys.
Compound 12p was tested in rats in the defensive with-
drawal test to determine behavioral efficacy and efficacious
plasma concentrations.29,50 This experiment was carried
out by measuring the time required for a rat to emerge from
a darkened chamber placed within an open, illuminated
field. A compound was considered to be efficacious if the
latency time period to emerge from the chamber was
significantly reduced relative to that of vehicle-treated
animals. It was found in previous studies with 2 that the
in vitro IC50 was similar to the plasma-free concentration of
2 corresponding to 50% CRF1 receptor occupancy (in vivo
IC50).28 In turn, CRF1 receptor occupancy greater than
50% was associated with anxiolytic efficacy in the defensive
withdrawal test. Figure 3 and Table 8 summarize the results
of the behavioral studies for 12p following oral dosing at 1,
3, and 10 mg/kg. CRF1 receptor occupancy in the parietal
cortex was determined by ex vivo autoradiography.28 Figure 3
shows exit latencies for each dose at 60 min after oral admin-
istration of 12p or the positive control compound 2 (dosed at
10 mg/kg). The results showed that 12p was effective at
reducing exit latency at 3 and 10 mg/kg (n=8). The lowest
Figure 3. Anxiolytic-like effects of 12p in the defensive with-
drawal test in rats at 1, 3, and 10 mg/kg with 2 as a positive control,
* p<0.05 vs vehicle, ** p<0.01 vs vehicle.
Table 8. Mean Total and Plasma Free Concentrations and CRF1 Receptor
Occupancies in the Defensive Withdrawal Test in Rats Following Oral
Doses of Compound 12p
mean total mean
oral dose plasma conc plasma free
CRF1 receptor
occupancy
(%)a,d,e
% decrease in
exit latencyb
(mg/kg)
(nM)a,b
conc (nM) a,c
1
4.9 ( 4.5
14 ( 9
76 ( 36
0.10 ( 0.09
0.29 ( 0.20
1.6 ( 0.8
37 ( 8
72 ( 10
94 ( 8
<5
60f
65g
3
10
a (SEM. b n=8. c Based on an unbound fraction of 2.1% in plasma
determined by equilibrium dialysis. d n=4. e Receptor occupancy of 2 at
10 mg/kg=92 ( 5. f p<0.05 vs vehicle. g p<0.01 vs vehicle.
effective dose of 3 mg/kg decreased exit latency by 60%
relative to vehicle treated controls. 12p decreased exit latency
by 65% at the higher dose of 10 mg/kg. 12p occupied 72% and
94% of CRF1 receptors (n=4) at doses of 3 and 10 mg/kg,
respectively. The mean plasma free concentrations were simi-
lar to, or greater than, the in vitro IC50 (0.26 nM) at doses of
3 and 10 mg/kg (Table 8).
Because of the high clearance of 12p in rats, a metabolite
ID study was conducted in human and rat liver microsomes.
LC/MS analysis of metabolites formed in microsomal in-
cubations suggested that the three major metabolites were
O-demethylation of the upper methoxy group, O-demethyl-
ation of the lower methoxy group, and O-demethylation of
both. Oxidation of the pyrazinone ring was only a minor
metabolite. In a trapping experiment performed to detect
the potential for reactive metabolite formation, 12p was
incubated with NADPH fortified human and rat liver
microsomes for 30 min at a concentration of 10 μM in the
presence of glutathione (GSH) to trap potential reactive
intermediates. The resulting GSH adducts formed via pyr-
azinone oxidation accounted for <1% of drug related
materials. The results of this study provided us with a
starting point from which to further optimize this class of
compounds.
Conclusion
In conclusion, efforts to identify structurally diverse CRF1
receptor antagonists led to the discovery of pyrazinone-based
compounds. Highly potent analogues with subnanomolar

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4179
(12.78 g, 77.6 mmol), and chloroacetonitrile (4.5 mL, 70.5 mmol).
The reaction mixture was heated at 50 °C for 16 h with vigorous
stirring. The resulting mixture was then cooled to room tempera-
ture and was filtered through a pad of celite with acetonitrile
rinsing. The filtrate was concentrated, and the product was purified
by column chromatography on silica gel (3% methanol in dichloro-
methane) to give a brown oil (8.1 g). The oil was dissolved in diethyl
ether (140 mL) and then converted to the hydrochloride salt by the
addition of 2 M HCl in diethyl ether (60 mL). The mixture was
cooled to 0 °C, and the solid was collected on a Buchner funnel
with ether rinsings and was dried under vacuum to give (R)-2-
(1-cyclopropylethylamino)acetonitrile hydrochloride (9a) (9.54 g,
85% yield) as pale-yellow solid: mp 115-116.1 °C; [R]25D +25.0
Figure 4. Summary of the metabolite ID study with compound 12p.
1
(c 0.779, CHCl3). H NMR (400 MHz, DMSO-d6) δ 9.01 (s br,
binding affinity have been identified. In particular, 12p was a
high affinity CRF1 receptor antagonist (IC50=0.26 nM) and a
potent inhibiter of CRF-stimulated cAMP production in
human Y-79 retinoblastoma cells (IC50=1.9 nM). Compound
12p was a high clearance compound in rats and a moderate
clearance compound in monkeys; nevertheless, 12p showed
efficacy in the defensive withdrawal model of anxiety at
a minimally efficacious dose of 3 mg/kg. A metabolite ID
study with 12p indicated that the major metabolites were the
result of O-demethylation. Further optimization of the phar-
macokinetic properties of pyrazinone-based CRF1 receptor
antagonists will be reported.
2H), 4.14 (s, 2H), 2.41-2.36 (m, 1H), 1.25 (d, J=6.8 Hz, 3H),
0.91-0.82 (m, 1H), 0.62-0.55 (m, 1H), 0.55-0.41 (m, 2H), 0.24-
0.18 (m, 1H). LRMS (ESI) m/e 125.0 [(M+H)+, calcd for C7H13-
N2 125.1].
All alkylaminonitrile intermediates of structure 9 (corres-
ponding to the R1 groups in compounds 12a-12i, 12u, 12w,
12y, and 12z) were prepared by the above procedure using
appropriate starting materials. In some cases, the hydrochloride
salt did not precipitate from the solution. In these cases, the
mixture was concentrated and then reconcentrated from hex-
anes (2ꢁ) and dried under vacuum to give a viscous oil. The oil
was placed in the freezer overnight to give the corresponding
hydrochloride salt as a solid.
General Procedures for Dichloropyrazinone Formation (10).
Method A: (R)-3,5-Dichloro-1-(1-cyclopropylethyl)pyrazin-2(1H)-
one (10a). Oxalyl chloride (12.7 mL, 147.0 mmol) was added
via syringe to a suspension of (R)-2-(1-cyclopropylethyl-
amino)acetonitrile hydrochloride (9a) (4.71 g, 29.4 mmol)
in toluene (130 mL) at 0 °C. After the addition was complete,
the cooling bath was removed and the reaction mixture was
heated at 55 °C for 16 h. The mixture was then cooled to room
temperature and concentrated. The residue was transferred
directly onto a silica gel column in a fume hood (caution: column
chromatography of the crude product should be performed in a
fume hood because some gas evolution occurred as residual
oxalyl chloride decomposed) and was eluted (20% ethyl acetate
in hexanes) to give (R)-3,5-dichloro-1-(1-cyclopropylethyl)pyrazin-
Experimental Section
Chemistry. All procedures were carried out under a nitrogen
atmosphere unless otherwise indicated using anhydrous 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. Column chromatography 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 LC/MS. Yields refer to chromatographically and spectro-
scopically pure compounds, except as otherwise indicated. Melt-
ing 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 Bruker 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. Elec-
trospray ionization (ESI) high-resolution mass spectra were
2(1H)-one (10a) (5.08 g, 74% yield) as a colorless solid:
1
mp 104.5-105.5 °C; [R]25 -27.5 (c 0.454, CHCl3). H NMR
D
(300 MHz, CDCl3) δ 7.47 (s, 1H), 4.26-4.20 (m, 1H), 1.46
(d, J = 6.9 Hz, 3H), 1.13-1.05 (m, 1H), 0.85-0.77 (m, 1H),
0.66-0.59 (m, 1H), 0.57-0.50 (m, 1H), 0.39-0.33 (m, 1H).
LRMS (APCI) m/e 233.0 [(M + H)+, calcd for C9H11N2OCl2
233.0].
Method B: (R)-3,5-Dichloro-1-(1-cyclopropyl-2-methoxyethyl)
pyrazin-2(1H)-one (10b). To a solution of (R)-2-(1-cyclopropyl-
2-methoxyethylamino)acetonitrile hydrochloride (9b) (6.00 g,
31.7 mmol) in dioxane (75 mL) was added CH2Cl2 (50 mL).
The mixture was cooled to 0 °C, and oxalyl chloride (13.6 mL,
159 mmol) was added slowly via syringe. After the addition was
complete, the cooling bath was removed and the reaction mixture
was heated at 55 °C for 16 h. The mixture was cooled to room
temperature and concentrated. The residue was transferred di-
rectly onto a silica gel column in a fume hood (caution: column
chromatography of the crude product should be performed in a
fume hood because some gas evolution occurred as residual
oxalyl chloride decomposed) and was eluted (20% f 25% ethyl
acetate in hexanes) to give (R)-3,5-dichloro-1-(1-cyclopropyl-
2-methoxyethyl)pyrazin-2(1H)-one (10b) (6.10 g, 74% yield) as
obtained on
a Finnigan MAT95S or Thermo Scientific
MAT900 mass spectrometer. All final products had a purity of
g95%. The purity of final products was determined by either
combustion analysis or HPLC. Combustion analyses were per-
formed by Quantitative Technologies, Inc., Whitehouse, NJ.
HPLC purity was measured using two methods for each com-
pound. Method A: Phenominex analytical C18 column (4.6 mm ꢁ
50 mm, 5 μm); mobile phase: A = H2O with 0.1% TFA, B=
acetonitrilewith0.1% TFA, 0-1 min, 20% B;1-7 min, 20%Bf
95% B; 7-8 min, 95% B; flow rate=3 mL/min; λ=254 nm; run
time=8 min. Method B: Phenominex analytical Synergi polar
RP(phenoxy) column (4.6 mmꢁ50 mm, 4 μm); mobile phase:
A = 90% H2O/10% methanol with 0.1% TFA, B = 90%
methanol/10% H2O with 0.1% TFA, 0-4 min, 40% B f
100% B; 4-6 min, 100% B; flow rate = 4 mL/min; λ = 254
nm; run time=6 min.
a colorless solid: mp 108.5-109.5 °C; [R]25 +90.9 (c 0.481,
D
CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 4.13-4.08
(m, 1H), 3.73 (dd, JAB=10.3, JAX=4.4 Hz, 1H), 3.63 (dd, JBA=10.3,
General Procedure for Alkylaminonitrile Formation (9). (R)-
2-(1-Cyclopropylethylamino)acetonitrile Hydrochloride (9a). To
a suspension of (R)-1-cyclopropylethylamine (8.53 g, 70.5 mmol)
in anhydrous acetonitrile (165 mL) at room temperature was
added potassium carbonate (29.10 g, 77.6 mmol), potassium iodide
JBX=3.0 Hz, 1H), 3.33 (s, 3H), 1.43-1.35 (m, 1H), 0.84-0.77
(m, 1H), 0.67-0.60 (m, 1H), 0.55-0.49 (m, 1H), 0.33-0.27
(m, 1H). LRMS (APCI) m/e 263.2 [(M + H)+, calcd for
C10H13N2O2Cl2 263.0].

4180 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
Remaining dichloropyrazinone intermediates of structure 10
(10c-10i and 10k-10m with R1 groups corresponding to those
in compounds 12c-12i, 12w, 12y, and 12z) were prepared by
method A using appropriate starting materials. The dichloro-
pyrazinone intermediate 10j (with an R1 group corresponding
to that in compound 12u) was prepared by method B. Experi-
mental procedures for the preparation of these dichloropy-
razinone intermediates are included in the Supporting Infor-
mation.
>99%, method B: tR=3.73 min, >99%. (Improved yields could
be obtained using 2.2 eq. of NaH.)
Method C: (R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(2,4,5-tri-
methylphenylamino)pyrazin-2(1H)-one (12ae). To a solution of
(R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-2(1H)-one (10f)
(150 mg, 0.610 mmol), prepared according to the procedure
described for 10a using appropriate starting materials, 2,4,
5-trimethylaniline (83 mg, 0.610 mmol) and K2CO3 (590 mg,
4.30 mmol) in toluene (2 mL) in an oven-dried round-bottom flask
under N2 was added Pd(OAc)2 (7 mg, 0.031 mmol) and BINAP
(20 mg, 0.031 mmol). The reaction mixture was heated at reflux for
5 h. The reaction mixture was cooled to room temperature and
was transferred to a separatory funnel containing saturated aq
NaHCO3 solution. The aqueous layer was extracted with ethyl
acetate (3 ꢁ 15 mL). The combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated. The
product was purified by column chromatography on silica gel
(30% ethyl acetate in hexanes) to give 12ae (150 mg, 71% yield)
(R)-3,5-Dibromo-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (11).
To a suspension of (R)-2-(1-cyclopropylethylamino)acetonitrile
hydrochloride (9a) (2.50 g, 15.56 mmol) in CH2Cl2 (25 mL) at
-78 °C was added oxalyl bromide (23.3 mL, 44.7 mmol, 2 M in
CH2Cl2) via syringe. The reaction mixture was allowed to warm
to room temperature and was then heated at 45 °C for 18 h.
The mixture was cooled to room temperature and concentrated.
The residue was transferred directly onto a silica gel column in a
fume hood (caution: column chromatography of the crude pro-
duct should be performed in a fume hood because some gas
evolution occurred as residual oxalyl bromide decomposed)
and was eluted (20% ethyl acetate in hexanes) to afford (R)-3,
5-dibromo-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (11) (3.18 g,
64% yield) as a colorless solid: mp 130.5-131.5 °C; [R]25D -26.9
as an orange solid: [R]25 -11.9 (c 0.371, CHCl3). 1H NMR
D
(400 MHz, CDCl3) δ 8.18 (s, 1H), 8.06 (s, 1H), 6.95 (s, 1H), 6.74
(s, 1H), 4.05-3.99 (m, 1H), 2.27 (s, 3H), 2.26 (s, 3H), 2.20 (s, 3H),
1.96-1.74 (m, 2H), 1.09-1.00 (m, 1H), 0.92 (t, J=7.5 Hz, 3H),
0.78-0.73 (m, 1H), 0.58-0.45 (m, 2H), 0.32-0.25 (m, 1H). HRMS
(ESI) m/e 346.1677 [(M+H)+, calcd for C19H25N3OCl 346.1686].
Anal. (C19H24N3OCl) C, H, N.
1
(c 0.617, CHCl3). H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H),
4.21-4.16 (m, 1H), 1.43 (d, J=6.7 Hz, 3H), 1.10-1.05 (m, 1H),
0.82-0.76 (m, 1H), 0.62-0.57 (m, 1H), 0.52-0.47 (m, 1H), 0.36-
0.32 (m, 1H). LRMS (APCI) m/e 321.1 [(M + H)+, calcd for
C9H11N2OBr2 320.9].
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(2,4,6-trimethyl-
phenylamino)pyrazin-2(1H)-one (12b). Compound 12b was prepared
according to the procedure described for the synthesis of 12a (method
A) using (R)-3,5-dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-
2(1H)-one (10b) (50 mg, 0.190 mmol) and 2,4,6-trimethylaniline
(26 mg, 0.190 mmol). The product was purified by column chromato-
graphy to afford 12b (45 mg, 65% yield) as a colorless solid:
[R]25D+37.3 (c 0.364, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 7.52 (s, 1H), 6.90 (s, 2H), 6.86 (s, 1H), 4.21-4.16 (m, 1H), 3.73
General Procedures for Coupling of the Dichloropyrazinones
with Anilines (12). Method A: (R)-5-Chloro-1-(1-cyclopropy-
lethyl)-3-(2,4,6-trimethylphenylamino)pyrazin-2(1H)-one (12a).
To a mixture of (R)-3,5-dichloro-1-(1-cyclopropylethyl)pyrazin-
2(1H)-one (10a) (200 mg, 0.858 mmol) and 2,4,6-trimethylaniline
(0.120 mL, 0.858 mmol) in THF (4 mL) at 0 °C was added
NaHMDS (1.802 mL, 1.802 mmol, 1 M in THF) slowly while
maintaining the temperature of the reaction mixture below 10 °C.
The reaction was stirred at 0 °C for 2 h. The mixture was transferred
to a separatory funnel containing saturated aq NaHCO3 solution
(5 mL) and the aqueous layer was extracted with ethyl acetate (3ꢁ
10 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 (20 f 30% ethyl
acetate in hexanes) to afford 12a (230 mg, 81% yield) as a yellow
solid: [R]25D -19.1 (c 0.254, CHCl3). 1H NMR (300 MHz, CDCl3)
δ 7.60 (s, 1H), 6.94 (s, 2H), 6.80 (s, 1H), 4.32-4.26 (m, 1H), 2.31
(s, 3H), 2.22 (s, 6H), 1.46 (d, J=6.6 Hz, 3H), 1.18-1.14 (m, 1H),
0.83-0.75 (m, 1H), 0.65-0.57 (m, 1H), 0.56-0.45 (m, 1H), 0.45-
0.38 (m, 1H). HRMS (ESI) m/e 332.1543 [(M + H)+, calcd
for C18H23N3OCl 332.1530]. HPLC method A: tR = 5.80 min,
> 99%; method B: tR = 3.47 min, 98.9%.
(dd, JAB = 10.3, JAX = 5.8 Hz, 1H), 3.68 (dd, JBA =10.3, JBX
=
3.5 Hz, 1H), 3.35 (s, 3H), 2.27 (s, 3H), 2.19 (s, 6H), 1.32-1.24 (m,
1H), 0.79-0.73 (m, 1H), 0.64-0.57 (m, 1H), 0.53-0.47 (m, 1H),
0.39-0.34 (m, 1H). HRMS (ESI) m/e362.1637 [(M + H)+,calcdfor
C19H25N3O2Cl 362.1635]. Anal. (C19H24N3O2Cl) C, H, N.
5-Chloro-1-(2-ethylbutyl)-3-(2,4,6-trimethylphenylamino)pyrazin-
2(1H)-one (12c). Compound 12c was prepared according to the
procedure described for the synthesis of 12a (method A) using 3,
5-dichloro-1-(2-ethylbutyl)pyrazin-2(1H)-one (10c) (100 mg, 0.400 mmol)
and 2,4,6-trimethylaniline (54 mg, 0.400 mmol). The product was
purified by column chromatography to afford 12c (52 mg, 37%
1
yield) as a colorless solid. H NMR (400 MHz, CDCl3) δ 7.58
(s, 1H), 6.90 (s, 2H), 6.51 (s, 1H), 3.77 (d, J=7.6 Hz, 2H), 2.27
(s, 3H), 2.17 (s, 6H), 1.82-1.77 (m, 1H), 1.41-1.34 (m, 4H), 0.93
(t, J=7.3 Hz, 6H). HRMS (ESI) m/e 348.1845 [(M+H)+, calcd for
C19H27N3OCl 348.1843]. HPLC method A: tR= 6.56 min,
>99%; method B: tR = 3.85 min, 96.4%.
Method B: (R)-3,5-Dichloro-4-[6-chloro-4-(1-cyclopropylpro-
pyl)-3-oxo-3,4-dihydropyrazin-2-ylamino]benzonitrile (12v). To a
solution of 4-amino-3,5-dichlorobenzonitrile (93 mg, 0.500 mmol)
in DMF (1 mL) at 0 °C was added NaH (30 mg, 0.75 mmol, 60% in
mineral oil). After stirring for 20 min, (R)-3,5-dichloro-1-(1-cyclo-
propylpropyl)pyrazin-2(1H)-one (10f) (123 mg, 0.500 mmol) dis-
solved in DMF (1 mL) was then added via cannula. The reaction
mixture was warmed to room temperature and was then heated at
55 °C for 16 h. The reaction mixture was cooled to room
temperature and was transferred to a separatory funnel containing
ether (25 mL). The organic layer was washed with water (4 ꢁ
5 mL), brine (5 mL), dried over MgSO4, filtered, and concentrated.
The product was purified by column chromatography on silica gel
(5% f 10% ethyl acetate in hexanes) to afford 12v (92 mg, 46%
yield) as a colorless solid: mp 237-237.5 °C; [R]25D -14.3 (c 0.279,
CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.95 (s, 1H), 7.72 (s, 2H),
6.92 (s, 1H), 4.12-4.03 (m, 1H), 1.97-1.82 (m, 2H), 1.15-1.04
(m, 1H), 0.98 (t, J=7.4 Hz, 3H), 0.87-0.80 (m, 1H), 0.60-0.53
(m, 2H), 0.38-0.31 (m, 1H). HRMS (ESI) m/e 397.0406 [(M+H)+,
calcd for C17H16N4OCl3 397.0390]. HPLC method A: tR=5.60 min,
5-Chloro-1-(1-ethylpropyl)-3-(2,4,6-trimethylphenylamino)
pyrazin-2(1H)-one (12d). Compound 12d was prepared ac-
cording to the procedure described for the synthesis of 12a
(method A) using 3,5-dichloro-1-(pentan-3-yl)pyrazin-2
(1H)-one (10d) (50 mg, 0.210 mmol) and 2,4,6-trimethyl-
aniline (28 mg, 0.210 mmol). The product was purified by
column chromatography to afford 12d (40 mg, 67% yield)
as a colorless solid. 1H NMR (400 MHz, CDCl3) δ 7.60
(s, 1H), 6.91 (s, 2H), 6.46 (s, 1H), 4.79-4.75 (m, 1H), 2.27
(s, 3H), 2.18 (s, 6H), 1.82-1.73 (m, 2H), 1.68-1.57 (m, 2H),
0.88 (t, J=7.3 Hz, 6H). HRMS (ESI) m/e 334.1678 [(M+H)+
,
calcd for C18H25N3OCl 334.1686]. HPLC method A: tR
6.03 min, >99%; method B: tR=3.51 min, >99%.
=
5-Chloro-1-(1-propylbutyl)-3-(2,4,6-trimethylphenylamino)-
pyrazin-2(1H)-one (12e). Compound 12e was prepared accord-
ing to the procedure described for the synthesis of 12a (method A)
using 3,5-dichloro-1-(heptan-4-yl)pyrazin-2(1H)-one (10e) (150 mg,
0.570 mmol) and 2,4,6-trimethylaniline (77 mg, 0.570 mmol). The
product was purified by column chromatography to afford 12e

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4181
1
(115 mg, 56% yield) as a colorless solid: mp 189.6-190.3 °C. H
NMR (300 MHz, CDCl3) δ 7.63 (s, 1H), 6.94 (s, 2H), 6.52 (s, 1H),
5.01-4.96 (m, 1H), 2.31 (s, 3H), 2.21 (s, 6H), 1.71-1.62 (m, 4H),
1.37-1.22 (m, 4H), 0.95 (t, J=7.3 Hz, 6H). HRMS (ESI) m/e
362.2021 [(M + H)+, calcd for C20H29N3OCl 362.1999]. Anal.
(C20H28N3OCl) C, H, N.
(20% f 50% ethyl acetate in hexanes) to afford 12j (80 mg,
83% yield) as a colorless solid: [R]25D +41.7 (c 0.364, CHCl3).
1H NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 6.90 (s, 2H), 6.84
(s, 1H), 4.11-3.29 (m, 2H), 3.91 (dd, JAB=11.3, JAX=6.3 Hz, 1H),
2.27 (s, 3H), 2.18 (s, 6H), 2.12 (s br, 1H), 1.28-1.19 (m, 1H), 0.88-
0.77 (m, 1H), 0.68-0.61 (m, 1H), 0.55-0.49 (m, 1H), 0.39-0.32
(m, 1H). HRMS (ESI) m/e 348.1474 [(M + H)+, calcd for
C18H23N3O2Cl 348.1479]. Anal. (C18H22N3O2Cl) C, H, N; calcd
N, 12.08; found N, 11.65.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(2,4,6-trimethylphenyla-
mino)pyrazin-2(1H)-one (12f). Compound 12f was prepared acc-
ording to the procedure described for the synthesis of 12a (method
A) using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-2(1H)-
one (10f) (120 mg, 0.486 mmol) and 2,4,6-trimethylaniline (66 mg,
0.486 mmol). The product was purified by column chromatogra-
phy to afford 12f (72 mg, 43% yield) as a colorless solid: mp 181-
5-Chloro-1-(2-ethylbutyl)-3-(4-methoxy-2,5-dimethylphenyla-
mino)pyrazin-2(1H)-one (12k). Compound 12k was prepared
according to the procedure described for the synthesis of 12a
(method A) using 3,5-dichloro-1-(2-ethylbutyl)pyrazin-2(1H)-
one (10c) (100 mg, 0.400 mmol) and 4-methoxy-2,5-dimethyla-
niline (75 mg, 0.400 mmol). The product was purified by column
chromatography to afford 12k (80 mg, 55% yield) as a brown
solid. 1H NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.86 (s, 1H),
6.66 (s, 1H), 6.55 (s, 1H), 3.80 (s, 3H), 3.77 (d, J=7.3 Hz, 2H),
2.29 (s, 3H), 2.21 (s, 3H), 1.82-1.77 (m, 1H), 1.42-1.33 (m, 4H),
0.92 (t, J=7.3 Hz, 6H). HRMS (ESI) m/e 364.1808 [(M+H)+,
calcd for C19H27N3O2Cl 364.1792]. Anal. (C19H26N3O2Cl)
C, H, N.
5-Chloro-1-(1-ethylpropyl)-3-(4-methoxy-2,5-dimethylpheny-
lamino)pyrazin-2(1H)-one (12l). Compound 12l was prepared
according to the procedure described for the synthesis of 12a
(method A) using 3,5-dichloro-1-(pentan-3-yl)pyrazin-2(1H)-
one (10d) (100 mg, 0.426 mmol) and 4-methoxy-2,5-dimethyl-
aniline (64 mg, 0.426 mmol). The product was purified by
column chromatography to afford 12l (75 mg, 50% yield) as a
light-brown solid. 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H),
7.88 (s, 1H), 6.66 (s, 1H), 6.50 (s, 1H), 4.78-4.71 (m, 1H), 3.80
(s, 3H), 2.30 (s, 3H), 2.21 (s, 3H), 1.83-1.73 (m, 2H), 1.68-1.57
(m, 2H), 0.86 (t, J=7.3 Hz, 6H). HRMS (ESI) m/e 350.1620
[(M+H)+, calcd for C18H25N3O2Cl 350.1635]. Anal. (C18H24-
N3O2Cl) C, H, N.
5-Chloro-3-(4-methoxy-2,5-dimethylphenylamino)-1-(1-propyl-
butyl)pyrazin-2(1H)-one (12m). Compound 12m was prepared
according to the procedure described for the synthesis of 12a
(method A) using 3,5-dichloro-1-(heptan-4-yl)pyrazin-2(1H)-
one (10e) (500 mg, 1.90 mmol) and 4-methoxy-2,5-dimethylani-
line (290 mg, 1.90 mmol). The product was purified by column
chromatography to afford 12m (680 mg, 95% yield) as a brown
solid. 1H NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 7.88 (s, 1H),
6.66 (s, 1H), 6.51 (s, 1H), 4.13-4.08 (m, 1H), 3.80 (s, 3H), 2.29 (s,
3H), 2.21 (s, 3H), 1.69-1.58 (m, 4H), 1.32-1.19 (m, 4H), 0.90 (t,
J=7.6 Hz, 6H). HRMS (ESI) m/e 378.1953 [(M+ H)+, calcd for
C20H29N3O2Cl 378.1948]. Anal. (C20H28N3O2Cl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(4-methoxy-2,5-dimethyl-
phenylamino)pyrazin-2(1H)-one (12n). Compound 12n was pre-
pared according to the procedure described for the synthesis of
12a (method A) using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyr-
azin-2(1H)-one (10f) (250 mg, 1.02 mmol) and 4-methoxy-2,5-
dimethylaniline (154 mg, 1.02 mmol). The product was purified
by column chromatography to afford 12n (300 mg, 82% yield) as a
colorless solid: mp 112.3 -113.3 °C; [R]25D +58.8 (c 0.353, CHCl3).
1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.90 (s, 1H), 6.79 (s,
1H), 6.65 (s, 1H), 4.03-4.01 (m, 1H), 3.80 (s, 3H), 2.29 (s, 3H), 2.21
(s, 3H), 1.94-1.76 (m, 2H), 1.08-1.01 (m, 1H), 0.92 (t, J = 7.3 Hz,
3H), 0.79-0.73 (m, 1H), 0.54-0.45 (m, 2H), 0.31-0.26 (m, 1H).
HRMS (ESI) m/e 362.1650 [(M + H)+, calcd for C19H25N3O2Cl
362.1635]. Anal. (C19H24N3O2Cl) C, H, N.
182 °C; [R]25 -12.1 (c 0.382, CHCl3). 1H NMR (300 MHz,
D
CDCl3) δ7.60 (s, 1H), 6.94 (s, 2H), 6.72 (s, 1H), 4.09-4.05 (m, 1H),
2.31 (s, 3H), 2.22 (s, 6H), 1.94-1.80 (m, 2H), 1.10-1.02 (m, 1H),
0.97 (t, J=7.7 Hz, 3H), 0.82-0.79 (m, 1H), 0.58-0.52 (m, 2H),
0.40-0.35 (m, 1H). HRMS (ESI) m/e 346.1705 [(M+H)+, calcd
for C19H25N3OCl 346.1686]. Anal. (C19H24N3OCl) C, H, N.
(R)-5-Chloro-1-[1-(methoxymethyl)propyl]-3-(2,4,6-trimethyl-
phenylamino)pyrazin-2(1H)-one (12g). Compound 12g was pre-
pared according to the procedure described for the synthesis of
12a (methodA) using(R)-3,5-dichloro-1-(1-methoxybutan-2-yl)
pyrazin-2(1H)-one (10g) (50 mg, 0.200 mmol) and 2,4,6-tri-
methylaniline (27 mg, 0.200 mmol). The product was purified
by column chromatography to afford 12g (20 mg, 30%
1
yield) as a colorless solid: [R]25 -38.7 (c 0.336, CHCl3). H
D
NMR (400 MHz, CDCl3) δ 7.56 (s, 1H), 6.90 (s, 2H), 6.71
(s, 1H), 4.97-4.93 (m, 1H), 3.64 (dd, JAB=10.5, JAX=6.0 Hz,
1H), 3.56 (dd, JBA = 10.5, JBX = 3.5 Hz, 1H), 3.35 (s, 3H),
2.27 (s, 3H), 2.18 (s, 6H), 1.88-1.82 (m, 1H), 1.78-1.72 (m, 1H),
0.93 (t, J=7.6 Hz, 3H). HRMS (ESI) m/e 350.1646 [(M+H)+,
calcd for C18H25N3O2Cl 350.1635]. Anal. (C18H24N3O2Cl)
C, H, N.
(R)-5-Chloro-1-(2-methoxy-1-methylethyl)-3-(2,4,6-trimethyl-
phenylamino)pyrazin-2(1H)-one (12h). Compound 12h was pre-
pared according to the procedure described for the synthesis of
12a (method A) using (R)-3,5-dichloro-1-(1-methoxypropan-
2-yl)pyrazin-2(1H)-one (10h) (140 mg, 0.625 mmol) and 2,4,
6-trimethylaniline (85 mg, 0.625 mmol). The product was pur-
ified by column chromatography to afford 12h (31 mg, 15%
1
yield) as a light-brown oil: [R]25D +36.2 (c 0.350, CHCl3). H
NMR (300 MHz, CDCl3) δ 7.63 (s, 1H), 6.94 (s, 2H), 6.77 (s, 1H),
5.23-5.20 (m, 1H), 3.63-3.60 (m, 2H), 3.40 (s, 3H), 2.31 (s, 3H),
2.21 (s, 6H), 1.44 (d, J=7.0 Hz, 3H). HRMS (ESI) m/e 336.0787
[(M+H)+, calcd for C17H23N3O2Cl 336.1479]. HPLC method A:
tR=5.18 min, 99.3%; method B: tR=3.10 min, > 99%.
5-Chloro-1-[2-methoxy-1-(methoxymethyl)ethyl]-3-(2,4,6-tri-
methylphenylamino)pyrazin-2(1H)-one (12i). Compound 12i
was prepared according to the procedure described for the
synthesis of 12a (methodA)using 3,5-dichloro-1-(1,3-dimethoxy-
propan-2-yl)pyrazin-2(1H)-one (10i) (267 mg, 1.00 mmol) and
2,4,6-trimethylaniline (168 mg, 1.20 mmol). The product was
purified by column chromatography to afford 12i (126 mg, 35%
1
yield) as a colorless solid. H NMR (300 MHz, CDCl3) δ 7.58
(s, 1H), 6.93 (s, 2H), 6.89 (s, 1H), 5.24-5.20 (m, 1H), 3.78 (dd,
JAB=10.5, JAX=6.2 Hz, 2H), 3.71 (dd, JBA=10.4, JBX=4.7 Hz,
2H), 3.40 (s, 6H), 2.31 (s, 3H), 2.22 (s, 6H). HRMS (ESI) m/e
366.1580 [(M + H)+, calcd for C18H25N3O3Cl 366.1584]. Anal.
(C18H24N3O3Cl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-hydroxyethyl)-3-(2,4,6-tri-
methylphenylamino)pyrazin-2(1H)-one (12j). To a solution of
12b (100 mg, 0.28 mmol) in CH2Cl2 (6 mL) at 0 °C was added BBr3
(0.56 mL, 0.56 mmol, 1 M in CH2Cl2) slowly via syringe. The
reaction mixture was stirred at 0 °C for 1 h. The reaction mixture
was then transferred to a separatory funnel containing saturated
aq NaHCO3 solution (20 mL). The aqueous layer was extracted
with ethyl acetate (3 ꢁ 15 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
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-(4-methoxy-2,5-dimethyl-
phenylamino)pyrazin-2(1H)-one (12o). Compound 12o was pre-
pared according to the procedure described for the synthesis
of 12a (method A) using (R)-3,5-dichloro-1-(1-cyclopropylethyl)-
pyrazin-2(1H)-one (10a) (150 mg, 0.640 mmol) and 4-methoxy-2,
5-dimethylaniline (100 mg, 0.640 mmol) with DMF (3 mL) as the
solvent. The product was purified by column chromatography to
afford 12o (150 mg, 68% yield) as a tan solid: [R]25D+43.7 (c 0.205,
CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.84 (s, 1H),

4182 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
6.80 (s, 1H), 6.67 (s, 1H), 4.26-4.20 (m, 1H), 3.81 (s, 3H), 2.30 (s,
3H), 2.21 (s, 3H), 1.43 (d, J=6.8 Hz, 3H), 1.12-1.06 (m, 1H),
0.78-0.71 (m, 1H), 0.59-0.52 (m, 1H), 0.49-0.43 (m, 1H), 0.37-
0.32 (m, 1H). HRMS (ESI) m/e 348.1469 [(M+H)+, calcd for
C18H23N3O2Cl 348.1479]. Anal. (C18H22N3O2Cl) C, H, N.
(ESI) m/e 382.1524 [(M+H)+, calcd for C18H25N3O4Cl 382.1534].
Anal. (C18H24N3O4Cl3) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-hydroxyethyl)-3-(4-methoxy-2,
5-dimethylphenylamino)pyrazin-2(1H)-one (12t). Compound 12t
was prepared from 12p (200 mg, 0.53 mmol) according to the
procedure described for the synthesis of 12j. The product was
purified by column chromatography on silica gel (30% ethyl
acetate in hexanes) to afford 12t (90 mg, 47% yield) as a pale-
yellow solid: mp 181.6 - 182.1 °C; [R]25D+38.4 (c 0.450, CHCl3).
1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.81 (s, 1H), 6.86 (s,
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-methoxy-
2,5-dimethylphenylamino)pyrazin-2(1H)-one (12p). Compound
12p was prepared according to the procedure described for the
synthesis of 12a (method A) using (R)-3,5-dichloro-1-(1-cyclopro-
pyl-2-methoxyethyl)pyrazin-2(1H)-one (10b) (1.50 g, 5.73 mmol)
and 4-methoxy-2,5-dimethylaniline (865 mg, 5.73 mmol) with
DMF (28 mL) as the solvent. The product was purified by column
chromatography on silica gel (25% ethyl acetate in hexanes) to
afford 12p (1.62 g, 75% yield). The product was subsequently
recrystallized from hexanes/ethyl acetate to afford 12p as a light-
brown crystalline solid: mp 112.4 - 113.9 °C; [R]25D +58.8
1H), 6.66 (s, 1H), 4.06-4.00 (m, 2H), 3.94 (dd, JAB=12.1, JAX
=
7.3 Hz, 1H), 3.80 (s, 3H), 2.28 (s, 3H), 2.20 (s, 3H), 1.72 (s br, 1H),
1.30-1.22 (m, 1H), 0.84-0.77 (m, 1H), 0.67-0.60 (m, 1H), 0.55-
0.49 (m, 1H), 0.37-0.31 (m, 1H). HRMS (ESI) m/e 364.1416
[(M+H)+, calcd for C18H23N3O3Cl 364.1428]. Anal. (C18H22-
N3O3Cl) C, H, N.
1
(S)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-methoxy-2,
5-dimethylphenylamino)pyrazin-2(1H)-one (12u). Compound 12u
was prepared according to the procedure described for the synth-
esis of 12b (method B) using (S)-3,5-dichloro-1-(1-cyclopropyl-
2-methoxyethyl)pyrazin-2(1H)-one (10j) (150 mg, 0.570 mmol)
and 4-methoxy-2,5-dimethylaniline (90 mg, 0.570 mmol) with
DMF (2 mL) as the solvent. The product was purified by column
chromatography to afford 12u (160 mg, 56% yield) as a
yellow crystalline solid: [R]25D -40.4 (c 0.436, CHCl3). 1H NMR
(400 MHz, CDCl3) δ 8.00 (s, 1H), 7.87 (s, 1H), 6.88 (s, 1H), 6.66 (s,
(c 0.353, CHCl3). H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H),
7.86 (s, 1H), 6.80 (s, 1H), 6.66 (s, 1H), 4.20-4.15 (m, 1H), 3.80
(s, 3H), 3.75 (dd, JAB = 10.5, JAX = 6.4 Hz, 1H), 3.67 (dd,
JBA=10.5, JBX=3.6 Hz, 1H), 3.34 (s, 3H), 2.29 (s, 3H), 2.21 (s,
3H), 1.31-1.25 (m, 1H), 0.79-0.73 (m, 1H), 0.61-0.56 (m, 1H),
0.53-0.48 (m, 1H), 0.37-0.32 (m, 1H). HRMS (ESI) m/e 378.1602
[(M+H)+, calcd for C19H25N3O3Cl 378.1585]. Anal. (C19H24N3-
O3Cl) C, H, N.
(R)-5-Chloro-3-(4-methoxy-2,5-dimethylphenylamino)-1-[1-
(methoxymethyl)propyl]pyrazin-2(1H)-one (12q). Compound
12q was prepared according to the procedure described for the
synthesis of 12a (method A) using (R)-3,5-dichloro-1-(1-methoxy-
butan-2-yl)pyrazin-2(1H)-one (10g) (70 mg, 0.280 mmol) and
4-methoxy-2,5-dimethylaniline (43 mg, 0.280 mmol) with DMF
(2 mL) as the solvent. The product was purified by column
chromatography to afford 12q (20 mg, 20% yield) as a brown
solid: [R]25D+39.4 (c 0.507, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 8.03 (s, 1H), 7.80 (s, 1H), 6.72 (s, 1H), 6.66 (s, 1H), 4.97-4.91 (m,
1H), 3.80 (s, 3H), 3.63 (dd, JAB=10.4, JAX=6.3 Hz, 1H), 3.55 (dd,
1H), 4.20-4.15 (m, 1H), 3.80 (s, 3H), 3.75 (dd, JAB=10.2, JAX
=
6.1 Hz, 1H), 3.68 (dd, JBA=10.2, JBX=3.4 Hz, 1H), 3.34 (s, 3H),
2.29 (s, 3H), 2.21 (s, 3H), 1.32-1.25 (m, 1H), 0.79-0.73 (m, 1H),
0.63-0.56 (m, 1H), 0.53-0.47 (m, 1H), 0.37-0.32 (m, 1H).
HRMS (ESI) m/e 378.1585 [(M+H)+, calcd for C19H25N3O3Cl
378.1584]. Anal. (C19H24N3O3Cl) C, H, N.
(S)-3,5-dichloro-4-[6-chloro-4-(1-cyclopropylpropyl)-3-oxo-3,
4-dihydropyrazin-2-ylamino]benzonitrile (12w). Compound 12w
was prepared according to the procedure described for the
synthesis of 12v (method B) by using 4-amino-3,5-dichloroben-
zonitrile (187 mg, 1 mmol) and (S)-3,5-dichloro-1-(1-cyclopro-
pylpropyl)pyrazin-2(1H)-one (10k) (246 mg, 1 mmol). The
product was purified by column chromatography to afford
12w (161 mg, 41% yield) as a yellow solid. The product was
subsequently recrystallized from hot acetonitrile to afford 12w
as a colorless crystalline solid: mp 236-237 °C; [R]25D+15.8
JBA=10.4, JBX = 3.6 Hz, 1H), 3.33 (s, 3H), 2.28 (s, 3H), 2.20 (s,
3H), 1.88-1.70 (m, 2H), 0.92 (t, J=7.3 Hz, 3H). HRMS (ESI)
m/e 366.1573 [(M + H)+, calcd for C18H25N3O3Cl 366.1585].
HPLC method A: tR =5.59 min, >99%; method B: tR =3.58
min, 98.1%.
(R)-5-Chloro-3-(4-methoxy-2,5-dimethylphenylamino)-1-(2-
methoxy-1-methylethyl)pyrazin-2(1H)-one (12r). Compound
12r was prepared according to the procedure described for
the synthesis of 12a (method A) using (R)-3,5-dichloro-1-
(1-methoxypropan-2-yl)pyrazin-2(1H)-one (10h) (200 mg,
0.850 mmol) and 4-methoxy-2,5-dimethylaniline (130 mg,
0.850 mmol) with DMF (3 mL) as the solvent. The product
was purified by column chromatography to afford 12r
(200 mg, 67% yield) as a light-brown solid. The product was
subsequently recrystallized from hexanes/ethyl acetate to
afford 12r as a light-brown crystalline solid: mp 90.4-
90.9 °C; [R]25D+47.5 (c 0.433, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 8.01 (s, 1H), 7.82 (s, 1H), 6.74 (s, 1H), 6.66 (s, 1H),
1
(c 0.278, CHCl3). H NMR (300 MHz, CDCl3) δ 7.96 (s br,
1H), 7.72 (s, 2H), 6.92 (s, 1H), 4.12-4.03 (m, 1H), 1.97-1.82
(m, 2H), 1.12-1.07 (m, 1H), 0.98 (t, J=7.3 Hz, 3H), 0.87-0.82
(m, 1H), 0.60-0.53 (m, 2H), 0.37-0.33 (m, 1H). HRMS (ESI)
m/e 397.0372 [(M+H)+, calcd for C17H16N4OCl3 397.0390].
Anal. (C17H15N4OCl3) C, H, N.
(R)-3,5-dichloro-4-[6-chloro-4-(1-cyclopropylethyl)-3-oxo-3,
4-dihydropyrazin-2-ylamino]benzonitrile (12x). Compound 12x
was prepared according to the procedure described for the synth-
esis of 12v (method B) using 4-amino-3,5-dichlorobenzonitrile
(187 mg, 1.00 mmol) and (R)-3,5-dichloro-1-(1-cyclopropylethyl)
pyrazin-2(1H)-one (10a) (233 mg, 1.00 mmol). The product was
purified by column chromatography and was subsequently recrys-
tallized from hexanes/CH2Cl2/THF to afford 12x (159 mg, 42%
5.18-5.14 (m, 1H), 3.80 (s, 3H), 3.59 (dd, JAB=10.3, JAX
=
6.1 Hz, 1H), 3.55 (dd, JBA=10.4, JBX=4.1 Hz, 1H), 3.55 (s,
3H), 2.29 (s, 3H), 2.21 (s, 3H), 1.40 (d, J=7.1 Hz, 3H). HRMS
(ESI) m/e 352.1430 [(M + H)+, calcd for C17H23N3O3Cl
352.1428]. Anal. (C17H22N3O3Cl) C, H, N.
yield) as an off-white crystalline solid: mp 243.5-244.5 °C; [R]25
-
D
29.5 (c 0.306, CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.94
(s, 1H), 7.72 (s, 2H), 7.01 (s, 1H), 4.34-4.24 (m, 1H), 1.48 (d, J=
7.0 Hz, 3H), 1.17-1.08 (m, 1H), 0.85-0.76 (m, 1H), 0.66-0.55
(m, 1H), 0.54-0.47 (m, 1H), 0.44-0.37 (m, 1H). HRMS (ESI) m/e
383.0232 [(M + H)+, calcd for C16H14N4OCl3 383.0233]. Anal.
(C16H13N4OCl3) C, H, N.
5-Chloro-3-(4-methoxy-2,5-dimethylphenylamino)-1-[2-methoxy-
1-(methoxymethyl)ethyl]pyrazin-2(1H)-one (12s). Compound 12s
was prepared according to the procedure described for the synthesis
of 12a (method A) using 3,5-dichloro-1-(1,3-dimethoxypropan-2-
yl)pyrazin-2(1H)-one (10i) (150 mg, 0.560 mmol) and 4-methoxy-
2,5-dimethylaniline (86 mg, 0.560 mmol). The product was purified
by column chromatography to afford 12s (140 mg, 65% yield) as a
light-brown solid. 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 1H), 7.82
(s, 1H), 6.87 (s, 1H), 6.65 (s, 1H), 5.19-5.14 (m, 1H), 3.79 (s, 3H),
3.74 (dd, JAB = 10.3, JAX = 6.5 Hz, 1H), 3.66 (dd, JBA = 10.4,
(S)-3,5-dichloro-4-(6-chloro-4-(1-cyclopropylethyl)-3-oxo-3,
4-dihydropyrazin-2-ylamino)benzonitrile (12y). Compound 12y
was prepared according to the procedure described for the synth-
esis of 12v (method B) using 4-amino-3,5-dichlorobenzonitrile
(187 mg, 1.00 mmol) and (S)-3,5-dichloro-1-(1-cyclopropylethyl)
pyrazin-2(1H)-one (10l) (233 mg, 1.00 mmol). The product was
purified by column chromatography and was subsequently recrys-
JBX=4.5 Hz, 2H), 3.35 (s, 6H), 2.28 (s, 3H), 2.20 (s, 3H). HRMS

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4183
tallized from ethyl acetate to afford compound 12y (159 mg, 42%
yield) as an off-white crystalline solid: mp 248-249 °C; [R]25D+28.5
(c0.304, CHCl3). 1H NMR (300 MHz, CDCl3)δ7.95 (s, 1H), 7.72 (s,
2H), 7.01 (s, 1H), 4.34-4.24 (m, 1H), 1.48 (d, J=7.0 Hz, 3H), 1.17-
1.08 (m, 1H), 0.85-0.76 (m, 1H), 0.66-0.59 (m, 1H), 0.57-0.47 (m,
1H), 0.44-0.38 (m, 1H). HRMS (ESI) m/e 383.0231 [(M+H)+,
calcd for C16H14N4OCl3 383.0233]. Anal. (C16H13N4OCl3)
C, H, N.
(S)-5-Chloro-3-(4-methoxy-2,5-dimethylphenylamino)-
1-(2-methoxy-1-methylethyl)pyrazin-2(1H)-one (12z). Com-
pound 12z was prepared according to the procedure described
for the synthesis of 12a (method A) using (S)-3,5-dichloro-1-
(1-methoxypropan-2-yl)pyrazin-2(1H)-one (10m) (70 mg,
0.300 mmol) and 4-methoxy-2,5-dimethylaniline (45 mg,
0.300 mmol) with DMF (1.5 mL) as the solvent. The product
was purified by column chromatography to afford compound
12z (90 mg, 86% yield) as a brown solid: [R]25D-34.4 (c 0.450,
CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 1H), 7.82 (s,
1H), 6.74 (s, 1H), 6.66 (s, 1H), 5.18-5.14 (m, 1H), 3.80 (s, 3H),
3.60 (dd, JAB=10.5, JAX=6.1 Hz, 1H), 3.55 (dd, JBA=10.5,
using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-2(1H)-one
(10f) (100 mg, 0.405 mmol) and 2,4-dimethylaniline (49 mg,
0.405 mmol). The product was purified by column chromato-
graphy to afford 12ad (72 mg, 54% yield) as a colorless solid:
[R]25D -7.9 (c 0.433, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.25
(s, 1H), 8.23 (d, J=8.1 Hz, 1H), 7.07 (d, J=8.1 Hz, 1H), 7.01 (s,
1H), 6.76 (s, 1H), 4.06-3.99 (m, 1H), 2.32 (s, 3H), 2.30 (s, 3H),
1.95-1.77 (m, 2H), 1.08-1.02 (m, 1H), 0.93 (t, J=7.3 Hz, 3H),
0.80-0.74 (m, 1H), 0.54-0.46 (m, 2H), 0.32-0.27 (m, 1H). HRMS
(ESI) m/e 332.1531 [(M+H)+, calcd for C18H23N3OCl 332.1530].
Anal. (C18H22N3OCl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(4-methoxy-2,6-dimethyl-
phenylamino)pyrazin-2(1H)-one (12af). Compound 12af was pre-
pared according to the procedure described for the synthesis of 12a
(method A) using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-
2(1H)-one (10f) (150 mg, 0.610 mmol) and 4-methoxy-2,6-dimethyl-
aniline (92 mg, 0.610 mmol). The product was purified by column
chromatography to afford 12af (170 mg, 77% yield) as a colorless
solid: [R]25D -11.5 (c 0.436, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 7.50 (s, 1H), 6.68 (s, 1H), 6.63 (s, 2H), 4.07-4.01 (m, 1H), 3.77
(s, 3H), 2.19 (s, 6H), 1.94-1.76 (m, 2H), 1.06-1.01 (m, 1H), 0.93 (t,
J=7.3 Hz, 3H), 0.81-0.73 (m, 1H), 0.55-0.46 (m, 2H), 0.33-0.29
(m, 1H). HRMS (ESI) m/e 362.1645 [(M+H)+, calcd for C19H25-
N3O2Cl 362.1635]. Anal. (C19H24N3O2Cl) C, H, N.
J
BX=4.1 Hz, 1H), 3.35 (s, 3H), 2.29 (s, 3H), 2.21 (s, 3H), 1.40
(d, J=7.1 Hz, 3H). HRMS (ESI) m/e 352.1430 [(M+H)+,
calcd for C17H23N3O3Cl 352.1428]. HPLC method A: tR=5. 24
min, >99%; method B: tR=3.40 min, 98.4%.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(2,4-dichloro-6-methyl-
phenylamino)pyrazin-2(1H)-one (12aa). Compound 12aa was
prepared according to the procedure described for the synthesis
of 12v (method B) using 2,4-dichloro-6-methylaniline (88 mg,
0.500 mmol) and (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-
2(1H)-one (10f) (123 mg, 0.500 mmol). The product was purified by
column chromatography to afford 12aa (73 mg, 38% yield) as a
colorless solid: [R]25D -14.2 (c 0.193, CHCl3). 1H NMR (300 MHz,
CDCl3) δ 7.76 (s, 1H), 7.34 (d, J=2.2 Hz, 1H), 7.21 (d, J=1.9 Hz,
1H), 6.81 (s, 1H), 4.12-4.04 (m, 1H), 2.30 (s, 3H), 1.96-1.78
(m, 2H), 1.10-1.03 (m, 1H), 0.97 (t, J=7.7 Hz, 3H), 0.85-0.79
(m, 1H), 0.59-0.52 (m, 2H), 0.38-0.32 (m, 1H). HRMS (ESI) m/e
386.0619 [(M+H)+, calcd for C17H19N3OCl3 386.0594]. Anal.
(C17H18N3OCl3) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-ethoxy-2,
5-dimethylphenylamino)pyrazin-2(1H)-one (12ag). Compound 12ag
was prepared according to the procedure described for the synth-
esis of 12a (method A) using (R)-3,5-dichloro-1-(1-cyclopropyl-
2-methoxyethyl)pyrazin-2(1H)-one (10b) (100 mg, 0.380 mmol)
and 4-ethoxy-2,5-dimethylaniline (63 mg, 0.380 mmol). The pro-
duct was purified by column chromatography to afford 12ag
(85 mg, 57% yield) as a light-brown solid: [R]25D+49.4 (c 0.457,
CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.86 (s, 1H),
6.87 (s, 1H), 6.65 (s, 1H), 4.19-4.14 (m, 1H), 3.99 (q, J=6.8 Hz,
2H), 3.74 (dd, JAB=10.5, JAX=6.3 Hz, 1H), 3.66 (dd, JBA=10.4,
JBX=3.6 Hz, 1H), 3.33 (s, 3H), 2.27 (s, 3H), 2.21 (s, 3H), 1.39 (t,
J=7.1 Hz, 3H), 1.32-1.23 (m, 1H), 0.79-0.72 (m, 1H), 0.62-0.55
(m, 1H), 0.52-0.46 (m, 1H), 0.36-0.30 (m, 1H). HRMS (ESI)m/e
392.1747 [(M+H)+, calcd for C20H27N3O3Cl 392.1741]. Anal.
(C20H26N3O3Cl) C, H, N.
(R)-5-Chloro-3-(2-chloro-6-methylphenylamino)-1-(1-cyclopropyl-
propyl)pyrazin-2(1H)-one (12ab). Compound 12ab was prepared
according to the procedure described for the synthesis of 12a
(method A) using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-2
(1H)-one (10f) (40 mg, 0.160 mmol) and 2-chloro-6-methylaniline
(23 mg, 0.160 mmol) with DMF (1 mL) as the solvent. The product
(R)-3-[4-(Benzyloxy)-2,5-dimethylphenylamino]-5-chloro-1-
(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (12ah). Com-
pound 12ah was prepared according to the procedure described
for the synthesis of 12a (method A) using (R)-3,5-dichloro-1-
(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (10b) (330 mg,
1.25 mmol) and 4-(benzyloxy)-2,5-dimethylphenylaniline (330 mg,
1.25 mmol). The product was purified by column chromatography
to afford 12ah (380 mg, 67% yield) as an amber-brown solid:
[R]25D +40.1 (c 0.464, CHCl3). 1H NMR (400 MHz, acetone-d6)
δ 8.28 (s, 1H), 7.76 (s, 1H), 7.51-7.49 (m, 2H), 7.40-7.36 (m, 2H),
7.32-7.29 (m, 1H), 7.13 (s, 1H), 6.92 (s, 1H), 5.11 (s, 2H), 4.23-
4.17 (m, 1H), 3.90 (dd, JAB=10.6, JAX=7.8 Hz, 1H), 3.69 (dd,
JBA=10.6, JBX=3.8Hz, 1H), 3.28(s, 3H), 2.25(s, 3H), 2.21(s, 3H),
1.44-1.37 (m, 1H), 0.73-0.68 (m, 1H), 0.56-0.49 (m, 2H), 0.33-
0.28 (m, 1H). HRMS (ESI) m/e 454.1899 [(M+H)+, calcd for
C25H29N3O3Cl 454.1897]. Anal. (C25H28N3O3Cl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-hydroxy-2,
5-dimethylphenylamino)pyrazin-2(1H)-one (12ai). Compound 12ai
was prepared according to the procedure described for the synth-
esis of 12a (method A) by using the appropriate dichloropyrazi-
none (10) (150 mg, 0.570 mmol) and 4-amino-2,5-dimethylphenol
(78 mg, 0.570 mmol). The product was purified by column
chromatography to afford 12ai (64 mg, 32% yield) as a tan solid:
was purified by column chromatography to afford 12ab (40 mg,
1
71% yield) as a colorless solid: [R]25 -8.6 (c 0.442, CHCl3). H
D
NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.29 (dd, J=7.6, 1.6 Hz,
1H), 7.19-7.11 (m, 2H), 6.77 (s, 1H), 4.09-4.03 (m, 1H), 2.29 (s,
3H), 1.94-1.78 (m, 2H), 1.08-1.02 (m, 1H), 0.95 (t, J=7.5 Hz, 3H),
0.79-0.76 (m, 1H), 0.55-0.50 (m, 2H), 0.36-0.31 (m, 1H). HRMS
(ESI) m/e 352.0976 [(M+H)+, calcd for C17H20N3OCl2 352.0984].
Anal. (C17H19N3OCl2) C, H, N.
(R)-5-Chloro-3-(4-chlorophenylamino)-1-(1-cyclopropylpropyl)-
pyrazin-2(1H)-one (12ac). Compound 12ac was prepared accord-
ing to the procedure described for the synthesis of 12a (method A)
using (R)-3,5-dichloro-1-(1-cyclopropylpropyl)pyrazin-2(1H)-one
(10f) (40 mg, 0.160 mmol) and 4-chloroaniline (21 mg, 0.160 mmol)
with DMF (1 mL) as the solvent. The product was purified
by column chromatography to afford 12ac (40 mg, 74% yield)
1
as a light-brown solid: [R]25D -5.3 (c 0.483, CHCl3). H NMR
(400 MHz, CDCl3) δ 8.32 (s, 1H), 7.72 (dd, J=6.8, 2.0 Hz, 2H),
7.31 (dd, J=6.8, 2.0 Hz, 2H), 6.80 (s, 1H), 4.06-4.00 (m, 1H),
1.95-1.88 (m, 1H), 1.83-1.75 (m, 1H), 1.08-1.02 (m, 1H), 0.91 (t,
J=7.6 Hz, 3H), 0.81-0.75 (m, 1H), 0.53-0.46 (m, 2H), 0.29-0.24
(m, 1H). HRMS (ESI) m/e 338.0820 [(M + H)+, calcd for
C16H18N3OCl2 338.0827]. Anal. (C16H17N3OCl2) C, H, N; calcd
N, 12.42; found: N, 11.84.
1
mp 164.5-165.5 °C; [R]25D +59.2 (c 0.412, CHCl3). H NMR
(400 MHz, CDCl3) δ 7.95 (s, 1H), 7.69 (s, 1H), 6.88 (s, 1H), 6.56 (s,
1H), 5.19 (s, 1H), 4.19-4.14 (m, 1H), 3.74 (dd, JAB=10.4, JAX
=
6.3 Hz, 1H), 3.67 (dd, JBA=10.6, JBX=3.5 Hz, 1H), 3.34 (s, 3H),
2.21 (s, 3H), 2.19 (s, 3H), 1.31-1.24 (m, 1H), 0.80-0.73 (m, 1H),
0.62-0.56 (m, 1H), 0.52-0.46 (m, 1H), 0.37-0.30 (m, 1H).
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(2,4-dimethylphenylamino)-
pyrazin-2(1H)-one (12ad). Compound 12ad was prepared according
to the procedure described for the synthesis of 12a (method A)

4184 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
HRMS (ESI) m/e 364.1414 [(M + H)+, calcd for C18H23N3O3Cl
364.1428]. Anal. (C18H22N3O3Cl) C, H, N.
1.5 h. The mixture was transferred to a separatory funnel containing
saturated aq NaHCO3 solution (5 mL) and the aqueous layer was
extracted with ethyl acetate (3ꢁ10 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 (20% f 25% ethyl acetate in hexanes) followed by purification by
reverse-phase HPLC (acetonitrile/water containing 0.1% TFA as
mobile phase, C18 column) to afford 12an (35 mg, 14% yield) as a
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-[ethyl(2,4,5-trimethyl-
phenyl)amino]pyrazin-2(1H)-one (12aj). To a solution of 12ae
(100 mg, 0.301 mmol) in DMF (2 mL) was added NaH (36 mg,
0.900 mmol, 60% in mineral oil). After stirring for 15 min at
room temperature, ethyl iodide (104 mg, 0.660 mmol) was added
and the reaction mixture was heated at 50 °C for 16 h. The
mixture was cooled to room temperature, and the reaction was
quenched by the addition of water. The mixture was transferred
to a separatory funnel containing brine, and the aqueous
layer was extracted with ethyl acetate (3 ꢁ 15 mL). The com-
bined organic layers were dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromato-
graphy on silica gel (30% ethyl acetate in hexanes) to afford
12aj (30 mg, 28% yield) as a pale-yellow oil: [R]25D +17.1
1
yellow solid: [R]25D -11.4 (c 0.421, CHCl3). H NMR (400 MHz,
CDCl3) δ 7.01 (s, 1H), 6.72 (s, 2H), 4.32 (t, J=7.8 Hz, 2H), 4.25-4.21
(m, 1H), 3.75 (s, 3H), 3.07 (t, J=7.8 Hz, 2H), 1.41 (d, J=6.8 Hz, 3H),
1.09-1.04(m,1H),0.75-0.71 (m, 1H), 0.57-0.53 (m, 1H), 0.46-0.41
(m, 1H), 0.38-0.33 (m, 1H). HRMS (ESI) m/e 380.0934 [(M+H)+,
calcd for C18H20N3O2Cl2 380.0933]. HPLC method A: tR=5.50 min,
>99%; method B: tR=3.68 min, >99%.
(R)-3-(7-Bromo-5-methoxyindolin-1-yl)-5-chloro-1-(1-cyclo-
propylethyl)pyrazin-2(1H)-one (12ao). Compound 12ao was pre-
pared according to the procedure described for the synthesis of
12an using (R)-3,5-dichloro-1-(1-cyclopropylethyl)pyrazin-2(1H)-
one (10a) (150 mg, 0.640 mmol) and 7-bromo-5-methoxyindoline
hydrochloride (169 mg, 0.640 mmol). The residue was purified by
column chromatography to afford 12ao (30 mg, 11% yield) as a
yellow solid: [R]25D -7.7 (c 0.286, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 7.03 (s, 1H), 6.90 (s, 1H), 6.76 (s, 1H), 4.33 (t, J=7.8 Hz,
2H), 4.28-4.20 (m, 1H), 3.76 (s, 3H), 3.08 (t, J=7.8 Hz, 2H), 1.41
(d, J=6.8 Hz, 3H), 1.10-1.04 (m, 1H), 0.77-0.70 (m, 1H), 0.58-
0.52 (m, 1H), 0.48-0.42 (m, 1H), 0.38-0.32 (m, 1H). HRMS
(ESI) m/e 424.0408 [(M + H)+, calcd for C18H20N3O2BrCl
424.0428]. HPLC method A: tR=5.54 min, >99%; method B:
tR = 3.75 min, > 99%.
1
(c 0.257, CHCl3). H NMR (400 MHz, CDCl3) δ 6.93 (s, 1H),
6.74 (s, 2H), 3.91 (q, J= 8.1 Hz, 2H), 3.73-3.62 (m br, 1H),
2.18 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H), 1.79-1.62 (m, 2H), 1.21
(t, J=7.1 Hz, 3H), 0.95-0.87 (m, 1H), 0.82 (t, J=7.4 Hz, 3H),
0.71-0.64 (m, 1H), 0.45-0.33 (m, 2H), 0.18-0.13 (m, 1H).
HRMS (ESI) m/e 374.2008 [(M+H)+, calcd for C21H29N3OCl
374.1999]. HPLC method A: tR=7.34 min, 97.5%; method B:
tR=4.22 min, 96.2%.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-[ethyl(2,4,6-trimethyl-
phenyl)amino]pyrazin-2(1H)-one (12ak). Compound 12ak was
prepared according to the procedure described for the synthesis
of 12aj by using 12f (100 mg, 0.301 mmol). The residue was
purified by column chromatography to afford 12ak (35 mg, 32%
yield) as a pale-yellow oil: [R]25D+66.6 (c 0.186, CHCl3). 1H NMR
(400 MHz, CDCl3) δ NMR 6.81 (s, 2H), 6.66 (s, 1H), 3.95-3.88
(m, 1H), 3.74 (q, J=6.9 Hz, 2H), 2.24 (s, 3H), 2.09 (s, 3H), 2.07 (s,
3H), 1.77-1.58 (m, 2H), 1.21 (t, J=7.1 Hz, 3H), 0.92-0.85 (m,
1H), 0.80 (t, J=7.6 Hz, 3H), 0.66-0.61 (m, 1H), 0.40-0.30 (m,
2H), 0.15-0.11 (m, 1H). HRMS (ESI) m/e 374.2006 [(M+H)+,
calcd for C21H29N3OCl 374.1999]. Method A: tR = 7.38 min,
94.8%; method B: tR = 4.17 min, 95.6%.
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-(2,6-dichloro-4-methoxy-
phenylamino)pyrazin-2(1H)-one (12al). Compound 12al was pre-
pared according to the procedure described for the synthesis of
12v (method B) by using (R)-3,5-dichloro-1-(1-cyclopropylethyl)-
pyrazin-2(1H)-one (10a) (233 mg, 1.00 mmol) and 2,4-dichloro-
6-methoxyaniline (190 mg, 1.00 mmol). The product was purified
by column chromatography to afford 12al (170 mg, 44% yield) as a
colorless solid: mp 161.5-162 °C; [R]25D -19.8 (c 0.270, CHCl3).
1H NMR (300 MHz, CDCl3) δ 7.64 (s, 1H), 6.97 (s, 2H), 6.89 (s,
1H), 4.32-4.27 (m, 1H), 3.83 (s, 3H), 1.46 (d, J=6.6 Hz, 3H),
1.17-1.05 (m, 1H), 0.81-0.75 (m, 1H), 0.64-0.58 (m, 1H), 0.53-
0.45 (m, 1H), 0.43-0.39 (m, 1H). HRMS (ESI) m/e 388.0361
[(M+H)+, calcd for C16H17N3O2Cl3 388.0386]. Anal. (C16H16-
N3O2Cl3) C, H, N.
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-[(2,6-dichloro-4-methoxy-
phenyl)(ethyl)amino]pyrazin-2(1H)-one (12am). Compound 12am
was prepared according to the procedure described for the
synthesis of 12aj using 12al (100 mg, 0.301 mmol). The residue
was purified by column chromatography to give 12am (110 mg,
69% yield) as an amber-brown oil: [R]25D+23.1 (c 0.750, CHCl3).
1H NMR (400 MHz, CDCl3) δ 6.845 (d, J=3.0 Hz, 2H), 6.84
(s, 1H), 4.09-4.03 (m, 1H), 3.88-3.77 (m, 2H), 3.76 (s, 3H), 1.27
(d, J=6.5 Hz, 3H), 1.22 (t, J=7.0 Hz, 3H), 0.99-0.93 (m, 1H),
0.68-0.62 (m, 1H), 0.49-0.42 (m, 1H), 0.35-0.29 (m, 1H),
0.24-0.19 (m, 1H). HRMS (ESI) m/e 416.0698 [(M+H)+, calcd
for C18H21N3O2Cl3 416.0699]. HPLC method A: tR=6.91 min,
96.4%; method B: tR=4.16 min, 97.5%.
(R)-3-[5-Bromo-7-methoxy-2H-benzo[b][1,4]oxazin-4(3H)-yl]-
5-chloro-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (12ap). Com-
pound 12ap was prepared according to the procedure described
for the synthesis of 12an using (R)-3,5-dichloro-1-(1-cyclopropyl-
ethyl)pyrazin-2(1H)-one (10a) (200 mg, 0.860 mmol) and 5-bromo-
7-methoxy-3,4-dihydro-2H-benzo[b][1,4]oxazine hydrochloride
(240 mg, 0.860 mmol). The residue was purified by column
chromatography to afford 12ap (210 mg, 56% yield) as a brown
solid: [R]25 -14.1 (c 0.693, CHCl3). 1H NMR (300 MHz,
D
DMSO-d6, 100 °C) δ 7.52 (s, 1H), 6.74 (s, 1H), 6.51 (s, 1H),
4.28-3.94 (m, 5H), 3.76 (s, 3H), 1.41 (d, J=7.0 Hz, 3H), 1.38-
1.26 (m, 1H), 0.74-0.60 (m, 1H), 0.54-0.39 (m, 2H), 0.30-0.16
(m, 1H). HRMS (ESI) m/e 440.0374 [(M + H)+, calcd for
C18H20N3O3BrCl 440.0377]. Anal. (C18H19N3O3BrCl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-[6-methoxy-2-(trifluoro-
methyl)pyridin-3-ylamino]pyrazin-2(1H)-one (12aq). Compound
12aq was prepared according to the procedure described for the
synthesis of 12a (method A) using (R)-3,5-dichloro-1-(1-cyclopropyl-
ethyl)pyrazin-2(1H)-one (10a) (150 mg, 0.640 mmol) and 6-methoxy-
2-(trifluoromethyl)pyridin-3-amine (125 mg, 0.640 mmol). The pro-
duct was purified by column chromatography to afford 12aq
(120 mg, 48% yield) as a pale-yellow solid: [R]25D -19.6 (c 0.433,
CHCl3). 1H NMR (400 MHz, CDCl3)δ8.78 (d, J=9.0 Hz, 1H), 8.64
(s, 1H), 6.98 (d, J=9.0 Hz, 1H), 6.92 (s, 1H), 4.27-4.19 (m, 1H), 3.95
(s, 3H), 1.44 (d, J=6.6 Hz, 3H), 1.12-1.05 (m, 1H), 0.79-0.73 (m,
1H), 0.60-0.53 (m, 1H), 0.50-0.44 (m, 1H), 0.38-0.33 (m, 1H).
HRMS (ESI) m/e 389.0998 [(M+H)+, calcd for C16H17N4O2ClF3
389.0992]. Anal. (C16H16N4O2ClF3) C, H, N.
(R)-5-Bromo-1-(1-cyclopropylethyl)-3-[6-methoxy-2-(trifluoro-
methyl)pyridin-3-ylamino]pyrazin-2(1H)-one (13). To a solution of
(R)-3,5-dibromo-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (11) (150 mg,
0.470 mmol) and 6-methoxy-2-(trifluoromethyl)pyridin-3-amine (90 mg,
0.470 mmol) in THF (2 mL) at 0 °C was added NaHMDS (0.94 mL,
0.94 mmol, 1 M in THF). The cooling bath was removed, and the
reaction mixture was stirred at room temperature for 12 h. The mixture
was transferred to a separatory funnel containing saturated aq NaHCO3
solution (5 mL), and the aqueous layer was extracted with ethyl acetate
(3ꢁ10 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% ethyl acetate in hexanes) to
(R)-5-Chloro-3-(7-chloro-5-methoxyindolin-1-yl)-1-(1-cyclo-
propylethyl)pyrazin-2(1H)-one (12an). To a solution of (R)-3,
5-dichloro-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (10a) (150 mg,
0.640 mmol) and 7-chloro-5-methoxyindoline hydrochloride (140 mg,
0.640 mmol) in THF (6 mL) at 0 °C was added NaHMDS (2.00 mL,
2.00 mmol, 1 M in THF). The reaction mixture was stirred at 0 °Cfor

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4185
afford 13 (120 mg, 59% yield) as a tan solid: [R]25 -15.6 (c 0.450,
column (10 cmꢁ50 cm); mobile phase=94% heptane/6% ethanol;
flow rate = 300 mL/min; λ = 210 nm; 1 g per injection, 30 min
method, peak 1 (S enantiomer), peak 2 (R enantiomer). Each
enantiomer had an optical purity >99% ee as determined by ana-
lytical HPLC: Chiralpak AD column (4.6 mmꢁ250 mm, 10 μm);
mobile phase = 95% hexane/5% ethanol; flow rate = 0.8 mL/min;
λ = 210 nm; tR (S enantiomer) = 14.77 min, tR (R enantiomer) =
17.05 min. The R enantiomer (17) was used in the next step. The
D
benzene). 1H NMR (400 MHz, CDCl3)δ8.78 (d, J= 9.3 Hz, 1H), 8.63
(s,1H), 7.00 (s, 1H), 6.98 (d, J = 9.0 Hz, 1H), 4.24-4.20 (m, 1H), 3.95
(s,3H),1.44(d,J= 6.9 Hz, 3H), 1.11-1.05 (m, 1H), 0.78-0.72 (m, 1H),
0.58-0.53 (m, 1H), 0.48-0.43 (m, 1H), 0.38-0.33 (m, 1H). HRMS
(ESI) m/e 433.0494 [(M + H)+, calcd for C16H17N4O2BrF3 433.0487].
Anal. (C16H16N4O2BrF3) C, H, N.
1-Cyclopropyl-2-methoxyethanone (15). Magnesium turnings
(15.2 g, 632 mmol) were added to a 5 L round-bottom flask
equipped with an addition funnel, after which the flask and
funnel were flame-dried. A reflux condenser was then placed on
the flask. Diethyl ether (100 mL) was added to the flask,
followed by cyclopropyl bromide (5 mL, 7.55 g, 62.4 mmol)
and several crystals of iodine. After the reaction was initiated,
additional diethyl ether (400 mL) was added to the reaction
mixture followed by cyclopropyl bromide (87.28 g, 721 mmol)
slowly over 30 min with intermittent cooling of the reaction
mixture with an ice-water bath. After the addition was com-
plete and the magnesium had reacted, additional diethyl ether
(700 mL) was added and the reaction mixture was cooled to 0 °C.
The magnetic stirrer was replaced with a mechanical stirrer, and
a solution of N2-dimethoxy-N-methylacetamide (14) (42.07 g,
316 mmol) dissolved in diethyl ether (500 mL) was added slowly
over 30 min via the addition funnel. A white solid formed during
this time. After the addition was complete, the cooling bath was
removed and the mixture was stirred at room temperature for
1 h. The mixture was then cooled to 0 °C and was quenched by
the addition of 1 N HCl (700 mL, added slowly at first). After
stirring for an additional 15 min, the mixture was transferred to
a separatory funnel and the aqueous layer was extracted with
ether (3 ꢁ 500 mL). The combined organic layers were washed
with saturated aq NaHCO3 solution (400 mL), brine (400 mL),
dried over MgSO4, filtered, and concentrated with minimal
vacuum (500 mbar). The product was purified by distillation
under reduced pressure while cooling the collection flask in a dry
ice/isopropyl alcohol bath to afford 15 (29.23 g, 81% yield) as a
colorless oil: bp 35-38 °C, 5 mmHg. 1H NMR (400 MHz, CDCl3)
δ 4.13 (s, 2H), 3.43 (s, 3H), 2.11-2.07 (m, 1H), 1.10-1.06 (m, 2H),
0.94-0.89 (m, 2H). GC/MS (CI) m/e 115.1 [(M + H)+, calcd for
C6H11O2 115.1].
characterization data for the R enantiomer is as follows: mp 59.6-
D
1
60.8 °C; [R]25 +18.9 (c 0.596, CHCl3). H NMR (400 MHz,
DMSO-d6) δ 7.39-7.29 (m, 5H), 7.17 (d, J = 8.5 Hz, 1H), 5.00 (s,
2H), 3.36-3.34 (m, 2H), 3.23 (s, 3H), 3.19-3.14 (m, 1H), 0.85-0.79
(m, 1H), 0.43-0.37 (m, 1H), 0.35-0.22 (m, 2H), 0.20-0.16 (m, 1H).
13C NMR (100 MHz, CDCl3) δ 155.6, 136.9, 128.0, 127.38, 127.34,
74.0, 64.8, 57.8, 53.5, 12.6, 2.2, 1.5. LRMS (ES+) m/e 272.3 [(M +
Na)+, calcd for C14H19NO3Na 272.1].
(R)-1-Cyclopropyl-2-methoxyethanamine Hydrochloride (18).
To a solution of 17 (4.24 g, 17.0 mmol) in ethanol (80 mL) and
chloroform (3 mL) in a Parr bottle was added 4 N HCl in
dioxane (5 mL) and 10% Pd/C (476 mg, wet, Degussa type). The
reaction mixture was placed on the Parr shaker under an H2 atm
at 45 psi for 16 h. The mixture was filtered through a pad of
celite, and the filtrate was concentrated. The residue was re-
concentrated from hexanes (2ꢁ) to give 18 (2.65 g, 100% yield)
as a colorless solid: mp 190-191.1 °C; R]25 -19.5 (c 0.482,
D
1
MeOH). H NMR (400 MHz, CDCl3) δ 8.41 (s br, 3H), 3.68
(d, J = 5.6 Hz, 2H), 3.39 (s, 3H), 2.64-2.60 (m, 1H), 1.20-1.13
(m, 1H), 0.71-0.58 (m, 3H), 0.32-0.28 (m, 1H). 13C NMR
(100 MHz, CDCl3) δ 72.1, 59.2, 57.5, 10.7, 4.2, 4.1. LRMS (ESI)
m/e 231.2 [(2M + H)+, calcd for C12H27N2O2 231.2].
Benzyl 1-Cyclopropylpropylcarbamate (20). To a solution of
19 (39.6 g, 0.403 mmol) in THF (600 mL) in a three-necked
round-bottom flask equipped with a mechanical stirrer was
added ammonium trifluoroacetate (315 g, 2.42 mol). The mix-
ture was cooled to 0 °C, and sodium triacetoxyborohydride
(128 g, 0.604 mol) was added. The cooling bath was removed,
and the reaction mixture was stirred at room temperature for
12 h. The mixture was concentrated to give 1-cyclopropyl-
propan-1-amine, which was used directly in the next step.
To a solution of crude 1-cyclopropylpropan-1-amine from
the previous step in CH2Cl2 (500 mL) and water (200 mL) in a
round-bottom flask equipped with a mechanical stirrer was
added Na2CO3 (256 g, 2.42 mol). The reaction mixture was
cooled to 0 °C, and benzyl chloroformate (75.6 g, 0.443 mol)
was added via syringe. The reaction mixture was allowed to
warm up to room temperature and was stirred at room tem-
perature overnight. The mixture was poured into a separatory
funnel, diluted with ethyl acetate (400 mL), and the organic layer
was washed with water, brine, dried over MgSO4, filtered, and
concentrated. The product was purified by column chromato-
graphy on silica gel (0% f 30% ethyl acetate in hexanes) to
furnish 20 (38.64 g, 41% yield, 2 steps) as a pale-yellow solid: mp
Benzyl 1-Cyclopropyl-2-methoxyethylcarbamate (16). Com-
pound 15 (10.03 g, 87.98 mmol) in THF (1000 mL) was treated
with ammonium trifluoroacetate (115.25 g, 880 mmol), and the
mixture was cooled to 0 °C. Sodium triacetoxyborohydride (27.85 g,
133 mmol) was added, the cooling bath was removed, and the
reaction mixture was gently heated at 40 °C with a warm water bath
for 2 h. The mixture was cooled to room temperature and concen-
trated to give 1-cyclopropyl-2-methoxyethanamine, which was used
directly in the next step.
Crude 1-cyclopropyl-2-methoxyethanamine from the previous
step was dissolved in CH2Cl2/H2O (300 mL/300 mL), and
Na2CO3 (111.9 g, 1.06 mol) was added. The reaction mixture
was placed in an ice bath and benzyl chloroformate (16.46 g,
96.78 mmol) was added via syringe. During the addition, the
internal reaction mixture temperature was maintained at 15-
20 °C. After the addition was complete, the reaction mixture was
stirred at room temperature for 2 h. The mixture was poured into
a separatory funnel, diluted with H2O, (300 mL), and extracted
with CH2Cl2 (3 ꢁ 300 mL). The combined organic layers were
washed with brine (300 mL), dried over MgSO4, filtered, and
concentrated. The product was purified by column chromato-
graphy on silica gel (30% ethyl acetate in hexanes) to furnish 16
(17.17 g, 78% yield, 2 steps) as an oil, which crystallized upon
1
77.7-78.5 °C. H NMR (400 MHz, CDCl3) δ 7.35-7.28 (m,
5H), 5.08 (s, 2H), 4.64 (s br, 1H), 2.96-2.92 (m, 1H), 1.67-1.51
(m, 2H), 0.94 (t, J = 7.3 Hz, 3H), 0.78-0.72 (m, 1H), 0.53-0.47
(m, 1H), 0.43-0.36 (m, 1H), 0.35-0.30 (m, 1H), 0.24-0.22
(m, 1H). 13C NMR (100 MHz, CDCl3) δ 156.2, 136.8, 128.5,
128.0, 66.5, 57.1, 28.6, 16.0, 10.3, 3.7, 2.4. LRMS (ESI) m/e 234.3
[(M + H)+, calcd for C14H20NO2 234.1].
(R)-1-Cyclopropylpropylcarbamate (21). Racemic 20 was se-
parated into its enantiomers by super critical fluid chromatogra-
phy (SFC): Chiralpak AS column (3 cm ꢁ 25 cm); mobile phase
= 10% isopropyl alcohol in CO2; flow rate = 120 mL/min @ 15
°C and 100 bar; λ = 220 nm; 150 mg per injection per 4.2 min,
Peak 1 (S enantiomer), peak 2 (R enantiomer). Each enantiomer
had an optical purity >99% ee as determined by analytical SFC:
Chiralcel OD-H column (4.6 mm ꢁ 250 mm, 5 μm); mobile phase =
5% heptane/isopropyl alcohol (1:1) in CO2; flow rate = 2 mL/min
@ 35 °C and 150 bar; λ = 210 nm; tR (R enantiomer) = 6.95 min,
tR (S enantiomer) = 7.87 min. The R enantiomer (21) was used in
1
standing: mp 190.5-192 °C. H NMR (400 MHz, DMSO-d6)
δ 7.39-7.29 (m, 5H), 7.17 (d, J = 8.5 Hz, 1H), 5.00 (s, 2H), 3.36-
3.34 (m, 2H), 3.23 (s, 3H), 3.19-3.14 (m, 1H), 0.85-0.79 (m, 1H),
0.43-0.37 (m, 1H), 0.35-0.22 (m, 2H), 0.20-0.16 (m, 1H).
LRMS (ESI) m/e 250.3 [(M + H)+, calcd for C14H20NO3 250.1].
(R)-Benzyl 1-Cyclopropyl-2-methoxyethylcarbamate (17). Race-
mic 16 was separated into its enantiomers by HPLC: Chiralpak AD

4186 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
the next step. The characterization data for the R enantiomer is as
(27) (5.00 g, 41.1 mmol) and potassium carbonate (17.1 g, 124 mmol)
in acetonitrile (200 mL) at 0 °C was added chloroacetone (3.3 mL,
41.1 mmol) via syringe followed by potassium iodide (7.55 g,
345.5 mmol). The cooling bath was removed, and the reaction mixture
was stirred at room temperature for 4 h. The mixture was filtered
through a pad of celite, and the filtrate was concentrated. The product
was purified by column chromatography on silica gel (10% f 15%
MeOH in CH2Cl2)togive(R)-1-(1-cyclopropylethylamino)propan-2-
one (4.46 g, 77% yield), which was used immediately in the next step.
The product readily decomposes if stored at room temperature. 1H
NMR (400 MHz, CDCl3) δ 4.71 (s, 1H), 3.60 (s, 2H), 2.12 (s, 3H),
1.83-1.76 (m, 1H), 1.14 (d, J = 6.5 Hz, 3H), 0.72-0.64 (m, 1H),
0.55-0.48 (m, 1H), 0.44-0.39 (m, 1H), 0.23-0.17 (m, 1H), 0.06-
0.00 (m, 1 H).
To a solution of (R)-1-(1-cyclopropylethylamino)propan-2-
one (4.46 g, 31.6 mmol) in CH2Cl2 (300 mL) at -78 °C was
added pyridine (9.0 mL, 111 mmol) followed by ethyl chloro-
oxoacetate (4.5 mL, 40.40 mmol). The reaction mixture was
stirred at -78 °C for 5 min and then warmed to room tempera-
ture and stirred at room temperature for 30 min. The reaction
mixture was transferred to a separatory funnel and diluted with
saturated aq NaHCO3 solution, and the aqueous layer was
extracted with ethyl acetate (3 ꢁ 200 mL). The combined
organic layers were washed with brine, dried over MgSO4,
filtered, and concentrated. The product was purified by column
chromatography on silica gel (45% f 55% ethyl acetate in
follows: [R]25 +2.6 (c 0.393, EtOH). 1H NMR (400 MHz,
D
CDCl3) δ 7.35-7.28 (m, 5H), 5.08 (s, 2H), 4.65 (s br, 1H),
2.96-2.92 (m, 1H), 1.67-1.51 (m, 2H), 0.94 (t, J = 7.3 Hz,
3H), 0.78-0.72 (m, 1H), 0.53-0.47 (m, 1H), 0.43-0.36 (m, 1H),
0.35-0.30 (m, 1H), 0.24-0.22 (m, 1H). 13C NMR (100 MHz,
CDCl3) δ 156.2, 136.8, 128.5, 128.0, 66.5, 57.1, 28.6, 16.0, 10.3,
3.7, 2.4. LRMS (ESI) m/e 234.3 [(M + H)+, calcd for
C14H20NO2 234.1].
(R)-1-Cyclopropylpropan-1-amine Hydrochloride (22). To a
solution of 21 (39.0 g, 167.2 mmol) in ethanol (250 mL) in a Parr
bottle was added 4 N HCl in dioxane (46.3 mL) and 10% Pd/C
(1.2 g). The reaction mixture was placed on the Parr shaker
under an H2 atm at 40 psi for 2 h. The mixture was filtered
through a pad of celite with methanol rinsing, and the filtrate
was concentrated. The residue was reconcentrated from hexanes
(2ꢁ) to give 22 (21.5 g, 95% yield) as a colorless solid: mp 254.5-
1
255.2 °C; [R]25 -13.5 (c 0.443, EtOH). H NMR (400 MHz,
D
CDCl3) δ 8.47 (s br, 3H), 2.36-2.33 (m, 1H), 1.97-1.85 (m, 2H),
1.10 (t, J = 7.3 Hz, 3H), 1.11-1.03 (m, 1H), 0.69-0.56 (m, 3H),
0.35-0.30 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 59.6, 27.0,
13.7, 10.3, 4.8, 3.4. GC/MS (CI) m/e 100 [(M + H)+, calcd for
C6H14N 100].
(R)-2-(Tritylamino)butan-1-ol (24). Triethylamine (80 mL,
0.57 mol) was added dropwise at 0 °C to a solution of triphe-
nylmethyl chloride (156 g, 560 mmol) in methylene chloride
(400 mL). (R)-(-)-2-Amino-1-butanol (23) (50 g, 0.56 mol) in
methylene chloride (100 mL) was then added dropwise at 0 °C.
The mixture was stirred at 0 °C under nitrogen for 4 h and then
slowly warmed to room temperature overnight. The mixture
was diluted with ether (1 L) and then washed with water and
brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. The residue was purified by column chromatography
on silica gel (10% f 35% ethyl acetate in hexanes) to afford 24
hexanes) to furnish 28 (3.32 g, 44% yield) as a brown oil:
1
[R]25 -26.8 (c 0.900, CHCl3). H NMR (400 MHz, CDCl3)
D
δ 4.35-4.26 (m, 2H), 4.09 (d, J = 2.6 Hz, 2H), 3.07-3.03 (m,
1H), 2.21 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H), 1.24 (d, J = 6.6 Hz,
3H), 0.89-0.81 (m, 1H), 0.65-0.60 (m, 1H), 0.52-0.45 (m, 1H),
0.41-0.31 (m, 1H), 0.28-0.23 (m, 1H). LRMS (APCI) m/e
242.3 [(M + H)+, calcd for C12H20NO4 242.1].
(R)-1-(1-Cyclopropylethyl)-5-methylpyrazine-2,3(1H,4H)-
dione (29). A solution of 28 (3.24 g, 13.43 mmol) in acetic acid
(185 mL) was treated with ammonium acetate (10.36 g, 134 mmol)
and the reaction mixture was heated at 100 °C for 2.5 h. The
mixture was cooled to room temperature and concentrated. The
residue was transferred to a separatory funnel containing saturated
aq NaHCO3 solution (500 mL, some bubbling occurred). The
aqueous layer was extracted with ethyl acetate (7 ꢁ 250 mL). The
combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated. The product was purified
by column chromatography on silica gel (5% f 10% MeOH in
CH2Cl2) to afford 29 (1.51 g, 58% yield) as a light-brown
solid: mp 196-197 °C; [R]25D +18.0 (c 0.338, CHCl3). 1H NMR
(400 MHz, CDCl3)δ11.22 (s, 1H), 6.15 (s, 1H), 4.22-4.15 (m, 1H),
2.13 (s, 3H), 1.35 (d, J = 6.8 Hz, 3H), 1.03-0.97 (m, 1H), 0.71-
0.64 (m, 1H), 0.52-0.39 (m, 2H), 0.36-0.30 (m, 1H). LRMS
(APCI) m/e 195.5 [(M + H)+, calcd for C10H15N2O2 195.1].
(R)-3-Chloro-1-(1-cyclopropylethyl)-5-methylpyrazin-2(1H)-
one (30). A solution of 29 (1.50 g, 7.72 mmol) and DMF (120 μL,
1.55 mmol) in thionyl chloride (75 mL) was heated at 75 °C for 1 h.
The mixture was cooled to room temperature and was concen-
trated then reconcentrated from hexanes. The product was purified
by column chromatography on silica gel (15% f 30% ethyl
1
(152 g, 82% yield) as a light-yellow oil. H NMR (300 MHz,
CDCl3) δ 7.56-7.53 (m, 6H), 7.31-7.19 (m, 9H), 3.21 (dd, J =
10.0, 2.0 Hz, 1H), 3.05 (dd, J = 10.0, 4.0 Hz, 1H), 2.55-2.48 (m,
1H), 2.10-1.95 (m, 1H), 1.95-1.75 (m, 1H), 1.30-1.18 (m, 1H),
1.02-0.86 (m, 1H), 0.63 (t, J = 7.4 Hz, 3H).
(R)-1-Methoxy-N-tritylbutan-2-amine (25). To a suspension
of sodium hydride (20.2 g, 505 mmol, 60% in mineral oil) in
THF (1 L) was added 24 (152 g, 459 mmol) in THF (200 mL)
dropwise at 0 °C. The mixture was stirred at 0 °C under nitrogen
for 1 h. Iodomethane (31.8 mL, 510 mmol) was added dropwise
at 0 °C. The mixture was slowly warmed to room temperature
and stirred under nitrogen overnight. The mixture was cooled to
0 °C and quenched carefully with water. The mixture was
transferred to a separatory funnel and was extracted with ethyl
acetate (3ꢁ), and the combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo to
1
provide 25 (158 g, quantitative yield) as a light-yellow oil. H
NMR (300 MHz, CDCl3) δ 7.59-7.56 (m, 6H), 7.28-7.14 (m,
9H), 3.06 (s, 3H), 2.91 (dd, J = 10.0, 4.0 Hz, 1H), 2.50-2.35 (m,
2H), 2.15 (br s, 1H), 1.40-1.25 (m, 2H), 0.70 (t, J = 7.4 Hz, 3H).
(R)-1-Methoxybutan-2-amine hydrochloride (26). To a solu-
tion of 25 (158 g, 459 mmol) in methylene chloride (500 mL) and
methanol (500 mL) was added HCl (700 mL, 700 mmol, 1.0 M in
ether) at 0 °C over a period of 10 min. The mixture was stirred at
0 °C for 1 h and then at room temperature overnight. The
solvents were removed in vacuo, and the residue was washed
with ether and hexanes (1:9) to provide crude product (56 g) as a
light-yellow solid, which was purified by recrystallization from
ethyl acetate to give 26 (54.8 g, 85% yield) as a white solid: mp
134-136 °C; [R]25D -24.3° (c 1.03, H2O); [R]25D -16.6° (c 1.03,
CH3OH). 1H NMR (300 MHz, DMSO-d6) δ 8.26 (br s, 3H), 3.46
(ddd, J = 16.0, 8.0, 4.0 Hz, 2H), 3.30 (s, 3H), 3.21-3.07 (m, 1H),
1.70-1.48 (m, 2H), 0.91 (t, J = 7.5 Hz, 3H). LRMS (APCI) m/e
104.0 [(M + H)+, calcd for C5H14NO 104.1].
acetate in hexanes) to afford 30 (1.28 g, 78% yield) as a pale-yellow
1
solid: mp 78.5-80 °C; [R]25 +4.7 (c 0.469, CHCl3). H NMR
D
(400 MHz, CDCl3) δ 7.12 (s, 1H), 4.47-4.21 (m, 1H), 2.28 (s, 3H),
1.41 (d, J = 6.8 Hz, 3H), 1.09-1.05 (m, 1H), 0.79-0.72 (m, 1H),
0.57-0.50 (m, 2H), 0.49-0.31 (m, 1H). LRMS (APCI) m/e 213.3
[(M + H)+, calcd for C10H14N2OCl 213.1].
(R)-1-(1-Cyclopropylethyl)-3-[6-methoxy-2-(trifluoromethyl)pyri-
din-3-ylamino]-5-methylpyrazin-2(1H)-one (31). Method A (from
30). To a solution of 30 (114 mg, 0.536 mmol) and 6-methoxy-2-
(trifluoromethyl)pyridin-3-amine (103 mg, 0.536 mmol) in THF
(4 mL) at 0 °C was added NaHMDS (1.1 mL, 1.12 mmol) dropwise
via syringe. The cooling bath was removed, and the reaction
mixture was stirred at room temperature for 8 h. The mixture
was transferred to a separatory funnel containing saturated aq
(R)-Ethyl 2-[(1-Cyclopropylethyl)(2-oxopropyl)amino]-2-oxoacetate
(28). To a suspension of (R)-1-cyclopropylethanamine hydrochloride

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4187
NaHCO3 solution (15 mL). The aqueous layer was extracted with
ethyl acetate (3 ꢁ 20 mL). The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated.
The product was purified via column chromatography on silica gel
(10% f 30% ethyl acetate in hexanes) to afford 31 (27 mg, 14%
yield) as a pale-yellow solid. See method B for 31 (below) for
characterization data.
13 (300 mg, 0.69 mmol), Et3N (190 μL, 1.38 mmol), (trimethylsilyl)
acetylene (136 mg, 1.38 mmol), and Pd(PPh3)4 (160 mg,
0.14 mmol) were combined in DMF (2 mL), and the reaction mix-
ture was heated at 120 °C in a microwave for 2 h. The mixture was
cooled to room temperature and was transferred to a separatory
funnel containing brine. The aqueous layer was extracted with ethyl
acetate (3 ꢁ 15 mL). The combined organic layers were dried
over MgSO4, filtered, and concentrated. The residue was purified
by column chromatography on silica gel (15% ethyl acetate
in hexanes) to afford (R)-1-(1-cyclopropylethyl)-3-(6-methoxy-2-
(trifluoromethyl)pyridin-3-ylamino)-5-((trimethylsilyl)ethynyl)pyrazin-
2(1H)-one as a yellow solid: [R]25D -29.1 (c 0.350, CHCl3). 1H NMR
(400 MHz, CDCl3)δ8.88 (d, J= 9.1 Hz, 1H), 8.55 (s, 1H), 7.15 (s 1H),
6.97 (d, J = 9.0 Hz, 1H), 4.23-4.17 (m, 1H), 3.93 (s, 3H), 1.44 (d, J =
6.8 Hz, 3H), 1.15-1.09 (m, 1H), 0.78-0.72 (m, 1H), 0.59-0.52 (m,
1H), 0.48-0.42 (m, 1H), 0.39-0.31 (m, 1H), 0.24 (s, 9H). 19F NMR
(375 MHz, CDCl3) δ -64.8.
A solution of (R)-1-(1-cyclopropylethyl)-3-[6-methoxy-2-(triflu-
oromethyl)pyridin-3-ylamino]-5-[(trimethylsilyl)ethynyl]pyrazin-2
(1H)-one (50 mg, 0.118 mmol) from above, 10 N NaOH (1 mL),
and MeOH (6 mL) was stirred at room temperature for 1 h. The
mixture was transferred to a separatory funnel containing brine.
The aqueous layer was extracted with ethyl acetate (3 ꢁ 10 mL).
The combined organic layers were dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromato-
graphy on silica gel (20% ethyl acetate in hexanes) and was then
taken up in acetonitrile/water and was frozen and placed on the
(R)-1-(1-Cyclopropylethyl)-3-[6-methoxy-2-(trifluoromethyl)
pyridin-3-ylamino]-5-methylpyrazin-2(1H)-one (31). Method B
(from 13). Bromopyrazinone 13 (50 mg, 0.120 mmol), potassium
carbonate (50 mg, 0.360 mmol), methylboronic acid (8 mg, 0.132
mmol), and Pd(Pt-Bu3)2 (13 mg, 0.024 mmol) were combined in
dioxane (1 mL) in a sealed vial. Nitrogen was bubbled through the
mixture for several minutes, and the reaction mixture was heated
at 120 °C for 24 h. The mixture was cooled to room temperature
and transferred to a separatory funnel containing saturated
aq NaHCO3 (5 mL). The aqueous layer was extracted with ethyl
acetate (3 ꢁ 5 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 (15% ethyl
acetate in hexanes) to afford 31 (10 mg, 23% yield) as a colorless
solid: [R]25D -4.0 (c 0.329, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 8.98 (d, J = 9.0 Hz, 1H), 8.61 (s, 1H), 6.94 (d, J = 9.0 Hz, 1H),
6.65 (s, 1H), 4.25-4.21 (m, 1H), 3.94 (s, 3H), 2.18 (s, 3H), 1.42 (d,
J= 6.8 Hz, 3H), 1.12-1.07 (m, 1H), 0.75-0.69(m, 1H), 0.53-0.41
(m, 2H), 0.37-0.32 (m, 1H). HRMS (ESI) m/e 369.1540 [(M +
H)+, calcd for C17H20N4O2F3 369.1539]. HPLC method A: tR
5.76 min, >99%; method B: tR = 3.35 min, 97.5%.
=
lyophilizer to afford 34 (20 mg, 49% yield) as a tan solid: [R]25
-
D
29.1 (c 0.350, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.87 (d, J =
9.0 Hz, 1H), 8.56 (s, 1H), 7.19 (s, 1H), 6.96 (d, J = 9.1 Hz, 1H),
4.24-4.19 (m, 1H), 3.93 (s, 1H), 3.03 (s, 1H), 1.44 (d, J = 6.8 Hz,
3H), 1.13-1.07 (m, 1H), 0.79-0.72 (m, 1H), 0.59-0.52 (m, 1H),
0.49-0.43 (m, 1H), 0.37-0.32 (m, 1H). HRMS (ESI) m/e 379.1363
[(M + H)+, calcd for C18H18N4O2F3 379.1382]. Anal. (C18H17-
N4O2F3) C, H, N.
(R)-1-(1-Cyclopropylethyl)-3-[6-methoxy-2-(trifluoromethyl)-
pyridin-3-ylamino]pyrazin-2(1H)-one (32). Bromopyrazinone 13
(30 mg, 0.069 mmol) was dissolved in EtOH (2 mL) in a test
tube placed inside a Parr bottle and was treated with 10% Pd/C
(30 mg, wet, Degussa type). The mixture was placed on a Parr
shaker under a hydrogen atmosphere at 50 psi for 1 h. The
mixture was filtered through celite with methanol rinsing, and
the filtrate was concentrated. The residue was purified by
column chromatography on silica gel (10% f 20% ethyl acetate
in hexanes) to afford 32 (24 mg, 98% yield) as a pale-yellow
(R)-1-(1-Cyclopropylethyl)-5-ethyl-3-[6-methoxy-2-(trifluoro-
methyl)pyridin-3-ylamino]pyrazin-2(1H)-one (35). Compound 34
(200 mg, 0.53 mmol) was dissolved in MeOH (5 mL) and was
treated with 10% Pd/C (50 mg, wet, Degussa type). The mixture
was stirred under hydrogen (1 atm) for 6 h. The mixture was
filtered through celite with methanol rinsing, and the filtrate was
concentrated. The residue was purified by column chromato-
graphy on silica gel (15% ethyl acetate in hexanes) to afford 35
(59 mg, 29% yield) as a pale-yellow amorphous solid: [R]25D -3.9
solid: [R]25 +27.7 (c 0.294, CHCl3). 1H NMR (400 MHz,
D
CDCl3) δ 8.72 (d, J = 8.8 Hz, 1H), 8.41 (s, 1H), 6.85 (d, J =
4.7 Hz, 1H), 6.84 (d, J = 10.0 Hz, 1H), 6.79 (d, J = 4.9 Hz, 1H),
4.18-4.11 (m, 1H), 3.83 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.05-
0.98 (m, 1H), 0.67-0.60 (m, 1H), 0.46-0.41 (m, 1H), 0.40-
0.32 (m, 1H), 0.27-0.20 (m, 1H). HRMS (ESI) m/e 355.1379
[(M + H)+, calcd for C16H18N4O2F3 355.1382]. HPLC method
A: tR = 5.61 min, 95.3%; method B: tR = 3.08 min, 98.0%.
(R)-4-(1-Cyclopropylethyl)-6-[6-methoxy-2-(trifluoromethyl)pyridin-
3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile (33). To a solution
of bromopyrazinone 13 (40 mg, 0.092 mmol) in DMF (5 mL) and
water (0.075 mL) was added zinc cyanide (11 mg, 0.092 mmol).
Nitrogen gas was bubbled through the suspension for 1 min. Pd2(dba)3
(4.2 mg, 0.005 mmol) and 1,1-bis(diphenylphosphino)ferrocene (dppf)
(6.1 mg, 0.011 mmol) were added and the reaction mixture was heated
under N2 at 120 °C for 5 h. The mixture was then cooled to room
temperature and transferred to a separatory funnel containing satu-
rated aq NH4Cl solution (20 mL). The aqueous layer was extracted
with ethyl acetate (2 ꢁ 10 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% f 40% ethyl acetate in hexanes) to afford a yellow film. The
product was taken up in acetonitrile/water and was frozen and placed
on the lyophilizer to afford 33 (25 mg, 71% yield) as a yellow solid:
[R]25D -33.5 (c0.442, CHCl3). 1H NMR (400 MHz, CDCl3)δ8.76 (d,
J = 9.0 Hz, 1H), 8.60 (s, 1H), 7.47 (s, 1H), 6.99 (d, J = 9.1 Hz, 1H),
4.25-4.21 (m, 1H), 3.96 (s, 3H), 1.46 (d, J = 6.8 Hz, 3H), 1.13-1.06
(m, 1H), 0.85-0.78 (m, 1H), 0.64-0.57 (m, 1H), 0.54-0.48 (m, 1H),
0.37-0.32 (m, 1H). HRMS (ESI) m/e 380.1329 [(M + H)+, calcd for
C17H17N5O2F3 380.1334]. Anal. (C17H16N5O2F3) C, H, N.
1
(c 0.207, CHCl3). H NMR (400 MHz, CDCl3) δ 9.02 (d, J =
9.0 Hz, 1H), 8.62 (s, 1H), 6.93 (d, J = 9.1 Hz, 1H), 6.65 (s, 1H),
4.27-4.14 (m, 1H), 3.93 (s, 3H), 2.46 (q, J = 7.3 Hz, 2H), 1.41 (d,
J = 6.8 Hz, 3H), 1.21 (t, J = 7.6 Hz, 3H), 1.14-1.06 (m, 1H),
0.75-0.68 (m, 1H), 0.54-0.40 (m, 2H), 0.37-0.31 (m, 1H). HRMS
(ESI) m/e 383.1679 [(M + H)+, calcd for C18H22N4O2F3
383.1695]. HPLC method A: tR = 6.37 min, >99%; method B:
tR = 3.72 min, >99%.
(R)-5-Allyl-1-(1-cyclopropylethyl)-3-[6-methoxy-2-(trifluoro-
methyl)pyridin-3-ylamino]pyrazin-2(1H)-one (36). Bromopyrazi-
none 13 (150 mg, 0.35 mmol), allyltributyltin (172 mg, 0.52 mmol),
and Pd(PPh3)4 (81 mg, 0.07 mmol) were combined in toluene (1 mL),
and the reaction mixture was heated at 120 °C in a microwave for
2 h. The mixture was cooled to room temperature and was
transferred to a separatory funnel containing brine. The aqueous
layer was extracted with ethyl acetate (3 ꢁ 15 mL). The combined
organic layers were dried over MgSO4, filtered, and concentrated.
The residue was purified by column chromatography on silica gel
(20% ethyl acetate in hexanes) to afford 36 (70 mg, 51% yield) as a
colorless solid: [R]25D -5.8 (c 0.257, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 9.00 (d, J = 9.1 Hz, 1H), 8.62 (s, 1H), 6.92 (d, J = 9.0 Hz,
1H), 6.66 (s, 1H), 6.03-5.93 (m, 1H), 5.18-5.12 (m, 2H), 4.26-4.19
(m, 1H), 3.93 (s, 3H), 3.21 (d, J = 6.8 Hz, 2H), 1.41 (d, J = 6.8 Hz,
3H), 1.14-1.05 (m, 1H), 0.75-0.68 (m, 1H), 0.54-0.40 (m, 2H),
0.36-0.30(m,1H).HRMS(ESI)m/e395.1695 [(M+H)+,calcdfor
(R)-1-(1-Cyclopropylethyl)-5-ethynyl-3-[6-methoxy-2-(trifluoro-
methyl)pyridin-3-ylamino]pyrazin-2(1H)-one (34). Bromopyrazinone

4188 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14
Hartz et al.
C19H22N4O2F3 395.1695]. HPLC method A: tR=6.61 min, >99%;
method B: tR = 3.82 min, >99%.
via a jugular vein catheter at 0, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h
postdose for the intravenous experiment and at 0, 0.25, 0.5, 1, 2, 4,
6, 8, and 24 h postdose for the oral experiment. Plasma was
separated by centrifugation and stored frozen at -20 °C until
analysis. Concentrations were determined by LC/MS/MS.
Cynomolgus Monkey Pharmacokinetic Studies. Pharmacoki-
netic properties were estimated in Cynomolgus monkeys (n = 3)
following a 2 mg/kg intravenous dose. Intravenous doses were
prepared in a vehicle consisting of PEG:ethanol, 90:10 (v/v) at a
volume of 1 mL/kg. Blood samples were collected via a jugular
vein at 0, 0.08, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose.
Plasma was separated by centrifugation and stored frozen at
-20 °C until analysis. Plasma concentrations were determined
by LC/MS/MS. Pharmacokinetic properties were estimated in
Cynomolgus monkeys (n = 3) following a 10 mg/kg oral dose.
Oral doses were prepared as a suspension in a vehicle consisting
of 1% Tween in 0.5% methylcellulose at a volume 3 mL/kg.
Blood samples were collected via a femoral vein at 0, 0.25, 0.5,
0.75, 1, 2, 4, 6, 8, and 24 h postdose. Plasma was separated by
centrifugation and stored frozen at -20 °C until analysis.
Plasma concentrations were determined by LC/MS/MS.
LC/MS/MS Conditions. Sample preparation was conducted
as follows. Aliquots (typically, 50 μL) of the biological matrix
from in vivo study and standard/QC samples were treated with
acetonitrile (200 μL) containing an appropriate internal stan-
dard, followed by vortex mixing for 2 min. The supernatant was
then separated from the precipitated proteins after a 20 min
centrifugation at 3000 rpm, and 200 μL was transferred to a
96-well plate. The supernatant was evaporated under nitrogen
using a TurboVap, with the plate heater set at 37 °C, and then
reconstituted using 75 μL of 0.1% formic acid.
An aliquot (5 μL) was injected onto a Synergi Fusion-RP column
(2 mm ꢁ 50 mm, 4 μm) (Phenomenex, Torrance, CA) at room
temperature for LC/MS/MS-based analysis (mobile phase = 0.1%
formic acid in water (A) and 0.1% formic acid in acetonitrile (B);
flow rate = 0.4 mL/min). A combination of isocratic and linear
gradients were used for peak separation. The HPLC was interfaced
to a Micromass Quattro Ultima LC/MS/MS tandem mass spectro-
meter (Micromass, Manchester, UK) equipped with an electro-
spray interface operating in the positive ionization mode. Detection
of each analyte was achieved through selected reaction monitoring.
Behavioral Studies: Subjects. Male Sprague-Dawley rats
weighing 180-300 g were purchased from Charles River Labora-
tories (Wilmington, MA.). The rats were housed individually in
suspended wire cages in a colony room maintained at constant
temperature (21 ( 2 °C) and humidity (50 ( 10%). The room was
illuminated 12 h per day (lights on at 0600 h). The rats had ad
libitum access to food and water throughout the study. Behavior-
al studies wereconducted between0600 and 1300 h. Animals were
maintained in accordance with the guidelines of the Committee
on Animals of the Bristol-Myers Squibb Company, the “Guide
for Care and Use of Laboratory Animals” (Institute of Animal
Laboratory Resources, 1996), and the guidelines published in the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Biology. Binding Assays. 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 homo-
genate 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 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 3 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 γ counter. IC50 values
were determined in a five-point (five drug concentrations) or
ten-point (ten drug concentrations) competition assay using
nonlinear regression by Microsoft Excel-fit.
Materials. Rat frontal cortex and porcine choroid plexus
were obtained from Analytical Biological Services, Inc.
(Wilmington, DE). 125I-ovine-CRF (2200 Ci/mmol) and 125I-
sauvagine (2200 Ci/mmol) were obtained from PerkinElmer
Life Sciences, Inc. (Boston, MA).
Functional Assay (Y-79 Cells). Human Y-79 retinoblastoma
cells were suspended in assay buffer (Hank’s Balanced Salt Solu-
tion containing 2 mM CaCl2, 5mMMgCl2, 20mMHEPES, 1mM
IBMX) and plated at 20000 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 homo-
geneous time-resolved florescence (HTRF) cAMP XL665 conju-
gate followed by HTRF anti-cAMP cryptate conjugate (CIS bio
International). Plates were subsequently incubated at room tem-
perature 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.
Research protocols were approved by the Bristol-Myers
SquibbCompany Institutional Animal Care andUse Committee.
Defensive Withdrawal. The defensive withdrawal procedure
was used as previously described.29 Briefly, the testing apparatus
consisted of an opaque plexiglass open field (106 cm length ꢁ 92 cm
width ꢁ 50 cm height) containing a cylindrical galvanized chamber
(14 cm length, 10 cm diameter) that was positioned lengthwise
against one wall, with the open end 40 cm from the corner. The
open field was illuminated by a 60 W incandescent bulb, and
illumination was titrated by a powerstat transformer to a 23 lx
reading at the entrance to the cylinder. Rats were habituated to
handling by gently stroking their dorsal surface for approximately
one minute the day before testing. To initiate testing, each rat was
placed within the cylinder that was then secured to the floor.
Behavior was assessed for 15 min by a trained observer (unaware of
Protein Binding Studies. Unbound fraction of test compounds
in rats was determined in vitro by equilibrium dialysis using the
Dianorm dialysis system. Rat plasma was spiked with the test
compound and equilibrated against isotonic phosphate buffer for
3 h at 37 °C. Following the incubation period, plasma and buffer
samples were analyzed for compound concentrations using LC/
MS/MS. Unbound fraction was calculated based on the ratio
between buffer concentration and the plasma concentration.
Rat Pharmacokinetic Studies. Pharmacokinetic parameters
were estimated in Spague-Dawley rats following intravenous
(2 mg/kg; n = 3) and oral (10 mg/kg; n = 3) dosing. Intravenous
doses were prepared in a vehicle consisting of PEG:ethanol, 90:10
(v/v) at a volume of 1 mL/kg. The oral doses were prepared in a
vehicle consisting of 1% Tween 80 in 0.5% methylcellulose
suspension at a volume of 3 mL/kg. Blood samples were collected

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4189
treatment assignment). The latency to exit the chamber, defined by
the placement of all four paws into the open field was recorded
(in seconds). The plexiglass chamber and the cylinder were cleaned
with 1.0% glacial acetic acid between animals to prevent olfactory
cues from influencing the behavior of subsequently tested animals.
All compounds were prepared in 0.25% methylcellulose suspen-
sion and beadmilled overnight. They were administered po, 1 h
before testing in a volume of 2 mL/kg body weight. Data were
analyzed using an analysis of variance, followed by individual
mean comparisons using Fisher’s least significant difference test.
The significance level was set at p < 0.05.
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Estimation of In Vivo CRF1 Receptor Occupancy: Ex Vivo
Binding Autoradiography Method. CRF1 receptor occupancy
studies were undertaken using the brain from rats tested in the
situational anxiety model. In brief, following oral dosing of the
test compound and evaluation in the situational anxiety beha-
vioral test, the rats were sacrificed and the brains and pituitaries
were collected, frozen, and sectioned in a Cryostat (20 μM) and
the slide-mounted sections were stored at -80 °C. For the ex
vivo ligand binding studies,28 the sections from drug- and
vehicle-treated rats were brought to 22-24 °C, air-dried for
20 min, and preincubated for 1 min in an assay solution contain-
ing 50 mM HEPES (pH 7.2), 10 mM MgCl2, 2 mM EGTA,
100 KIU/mL aprotinin, 0.1 M bacitracin, and 0.1% ovalbumin.
The sections were then incubated for 20 min at 22-24 °C in the
same assay solution also containing 0.15-0.20 nM [125I-Tyro]
sauvagine. For CRF1 occupancy studies, nonspecific binding
was defined in adjacent brain sections from vehicle-treated rats
by including 1 μM of 2 in the assay solution. After incubation,
the sections were rinsed in cold phosphate buffer saline for 40 s
and subsequently dried under a stream of cold air. The slides of
sections were then placed in cassettes against iodine-sensitive
storage phosphor-imaging screens (PerkinElmer Life Sciences)
for 12-16 h, and the screens were then scanned with a Cyclone
storage phosphor-imaging system (PerkinElmer Life Sciences).
Captured storage phosphor images were analyzed with
OptiQuant Acquisition and Analysis software (PerkinElmer
Life Sciences). CRF1 receptor occupancy was calculated as
100% minus % (total [125I-Tyro]sauvagine binding in drug-
treated minus nonspecific [125I-Tyro]sauvagine binding in vehi-
cle-treated)/(total [125I-Tyro]sauvagine binding in vehicle-trea-
ted minus nonspecific [125I-Tyro]sauvagine binding in vehicle-
treated).
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L. M.; Gold, L. H.; Chen, R.; Marchuk, Y.; Hauser, C.; Bentley, C.
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Acknowledgment. We thank Jennifer Kuehne for perform-
ing large scale separations on chiral support, Baoqing Ma
and Qi Gao for structure confirmation of key compounds by
X-ray crystallography, and the Discovery Analytical Sciences
Department for obtaining high-resolution MS analysis and
optical rotations.
(20) Koob, G. F.; Heinrichs, S. C.; Pich, E. M.; Menzaghi, F.; Baldwin,
H.; Miczek, K.; Britton, K. T.; Chadwick, D. J.; Marsh, J.; Ackrill,
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Supporting Information Available: Tables of elemental analysis
data, high-resolution mass spectral data with HPLC purity data
for compounds lacking elemental analysis data, and experimental
procedures for the preparation of the dichloropyrazinone inter-
mediates 10c-10m (corresponding to those used for compounds
12c-12i, 12u, 12w, and 12y-12z). This material is available free of
charge via the Internet at http://pubs.acs.org.
(22) Schulz, D. W.; Mansbach, R. S.; Sprouse, J.; Braselton, J. P.;
Collins, J.; Corman, M.; Dunaiskis, A.; Faraci, S.; Schmidt, A. W.;
Seeger, T.; Seymour, P.; Tingley, F. D.; Winston, E. N.III;
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Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4191
(57) 6-Methoxy-3-nitro-2-(trifluoromethyl)pyridine was prepared from
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W. E.; Olson, R. E.; Gilligan, P. J.; Robertson, D. W. CRF Ligands
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Chem. Lett. 2003, 13, 289-291. Subsequent reduction of the nitro
group by hydrogenation provided the desired product in 97% yield.
(58) LogD measurements were determined by partitioning of the com-
pound between octanol and water at pH=7.4.