A strategy to minimize reactive metabolite formation: Discovery of (S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2, 5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile as a potent, orally bioavailable corticotropin-releasing factor-1 receptor antagonist

Article abstract of DOI:10.1021/jm900716v

Detailed metabolic characterization of 8, an earlier lead pyrazinone-based corticotropin-releasing factor-1 (CRF₁) receptor antagonist, revealed that this compound formed significant levels of reactive metabolites, as measured by in vivo and in vitro biotransformation studies. This was of particular concern due to the body of evidence suggesting that reactive metabolites may be involved in idiosyncratic drug reactions. Further optimization of the structure-activity relationships and in vivo properties of pyrazinone-based CRF₁ receptor antagonists and studies to assess the formation of reactive metabolites led to the discovery of 19e, a high affinity CRF₁ receptor antagonist (IC₅₀= 0.86 nM) wherein GSH adducts were estimated to be only 0.1% of the total amount of drug-related material excreted through bile and urine, indicating low levels of reactive metabolite formation in vivo. Anovel 6-(difluoromethoxy)-2,5-dimethylpyridin-3- amine group in 19e contributed to the potency and improved in vivo properties of this compound and related analogues. 19e had excellent pharmacokinetic properties in rats and dogs and showed efficacy in the defensive withdrawal model of anxiety in rats. The lowest efficacious dose was 1.8 mg/kg. The results of a two-week rat safety study with 19e indicated that this compound was well-tolerated.

Full text of DOI:10.1021/jm900716v

J. Med. Chem. 2009, 52, 7653–7668 7653
DOI: 10.1021/jm900716v
A Strategy to Minimize Reactive Metabolite Formation: Discovery of (S)-4-(1-Cyclopropyl-2-
methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-
carbonitrile as a Potent, Orally Bioavailable Corticotropin-Releasing Factor-1 Receptor Antagonist†
Richard A. Hartz,*,‡ Vijay T. Ahuja,‡ Xiaoliang Zhuo,§ Ronald J. Mattson,‡ Derek J. Denhart,‡ Jeffrey A. Deskus,‡
Vivekananda M. Vrudhula,‡ Senliang Pan,‡ Jonathan L. Ditta,‡ Yue-Zhong Shu,§ James E. Grace,§ Kimberley A. Lentz,§
Snjezana Lelas, Yu-Wen Li, Thaddeus F. Molski, Subramaniam Krishnananthan,^ Henry Wong,^ Jingfang Qian-Cutrone,§
Richard Schartman,§ Rex Denton,§ Nicholas J. Lodge, Robert Zaczek, John E. Macor,‡ and Joanne J. Bronson‡
‡Neuroscience Discovery Chemistry and §Pharmaceutical Candidate Optimization and Neuroscience Discovery Biology and ^Department of
Chemical Synthesis, Research and Development, Bristol-Myers Squibb Company, 5 Research Parkway, Wallingford, Connecticut 06492
Received May 26, 2009
Detailed metabolic characterization of 8, an earlier lead pyrazinone-based corticotropin-releasing
factor-1 (CRF1) receptor antagonist, revealed that this compound formed significant levels of reactive
metabolites, as measured by in vivo and in vitro biotransformation studies. This was of particular
concern due to the body of evidence suggesting that reactive metabolites may be involved in
idiosyncratic drug reactions. Further optimization of the structure-activity relationships and in vivo
properties of pyrazinone-based CRF1 receptor antagonists and studies to assess the formation
of reactive metabolites led to the discovery of 19e, a high affinity CRF1 receptor antagonist (IC50=
0.86 nM) wherein GSH adducts were estimated to be only 0.1% of the total amount of drug-related
material excreted through bile and urine, indicating low levels of reactive metabolite formation in vivo.
A novel 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine group in 19e contributed to the potency and
improved in vivo properties of this compound and related analogues. 19e had excellent pharmacokinetic
properties in rats and dogs and showed efficacy in the defensive withdrawal model of anxiety in rats. The
lowest efficacious dose was 1.8 mg/kg. The results of a two-week rat safety study with 19e indicated that
this compound was well-tolerated.
Introduction
the critical role of CRF in the regulation of the endocrine,
behavioral, and autonomic responses to stress.6,9,10 Compel-
ling evidence exists to support the hypothesis that excess levels
of CRF contribute to stress-related disorders such as anxiety
and depression and that antagonists of CRF1 receptors may
be able to successfully treat these conditions.9,11-14 Patients
suffering from depression have been found to have elevated
levels of CRF in cerebrospinal fluid.15-17 In addition, a
blunted ACTH release in response to CRF infusion has been
observed,18 as well as an elevated cortisol response in the
dexamethasone/CRF test.19,20 Moreover, a variety of small
molecule CRF1 receptor antagonists, such as 1 (DMP696),21
were shown to be efficacious in behavioral models for anxiety
and depression in preclinical studies.1,5
Over the past several years, a limited number of CRF1
receptor antagonists have reached clinical development.1 In a
small open-label clinical trial with 2 (R121919, also known as
NBI30775)22 for depression, it was found that depressed
patients showed reductions in depression symptoms, as rated
by both patients and clinicians.23,24 A placebo-controlled
clinical study, designed to evaluate whether subchronic treat-
ment with 3 (NBI-34041)25 would decrease the stress hormone
responsefollowing a psychological stressor, indicated thatthis
drug candidate may improve resistance to psychological stress
by reducing stress hormone secretion.24,26 In a double-blind,
placebo-controlled clinical trial to evaluate 4 (CP-316311)27
for the treatment of major depressive disorder, it was found
Stress-related diseases, such as anxiety and depression,
affect a significant number of people. Although numerous
currently marketed drugs exist to treat these conditions,
shortcomings such as delayed onset of action, lack of efficacy
in a subset of patients, and unwanted side effects highlight the
need for new pharmacological approaches. The discovery of
corticotropin releasing factor-1 (CRF1) receptor antagonists,
which may potentially be used to treat these conditions, has
been the focus of research groups for some time.1-7 CRF,a a
41 amino acid neuropeptide first isolated by Vale and co-
workers,8 is the primary regulator of the hypothalamic-
pituitary-adrenal (HPA) axis and is primarily produced in
the paraventricular region of the hypothalamus. Upon its
secretion, CRF acts as the principal agent controlling the
release of adrenocorticotropin hormone (ACTH) and other
proopiomelanocortin (POMC)-derived peptides from the
anterior pituitary gland. Numerous studies have documented
†We acknowledge the 100th anniversary of the Division of Medicinal
Chemistry.
*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, hy-
pothalamic-pituitary-adrenal; ACTH, adrenocorticotropin hormone;
POMC, proopiomelanocortin; SAR, structure-activity relationships;
BDC, bile-duct cannulated; GSH, glutathione; cAMP, cyclic adenosine
monophosphate.
r
2009 American Chemical Society
Published on Web 09/01/2009
pubs.acs.org/jmc

7654 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
that this drug candidate lacked efficacy.28 A small number of
additional compounds, including 5 (CP-376395),29 6 (BMS-
561388),30 and 7 (BMS-562086)31 (Figure 1), were reported to
haveentered clinical trials. Theresults from theseclinicaltrials
may provide further insights into the clinical utility of CRF1
receptor antagonists. Although the outcome of the limited
number of clinical trials reported to date has been mixed,
the prospect that antagonism of CRF1 receptors may offer
therapeutic potential for the treatment of diseases resulting
from elevated levels of CRF continues to provide motivation
to discover novel CRF1 receptor antagonists.
During our investigation into the structure-activity rela-
tionships (SAR) of pyrazinone-based CRF1 receptor antago-
nists,32-34 compound 8 (Figure 2) was synthesized and was
found to have good pharmacokinetic properties in rats as well
as efficacy in the defensive withdrawal model of anxiety in
rats.34 In addition, we described how incorporation of the
1-cyclopropylethyl and difluoromethoxy groups, which were
not readily metabolized, in strategic locations of 8 contributed
to the improved pharmacokinetic properties of this com-
pound compared to others within this chemotype.34 However,
subsequent in vivo metabolism and disposition studies in bile
duct-cannulated (BDC) rats revealed that 8 was extensively
metabolized in vivo. Approximately 60% of the total drug-
related materials collected in bile (0-7 h) was identified as
pyrazinone oxidation metabolites (17%, e.g. 9 and 10,
Figure 2), glutathione (GSH)-related adducts on the pyrazi-
none ring via epoxidation of the C5-C6 double bond (18%,
e.g. 11) and GSH-related adducts on the phenyl group (25%,
e.g. 12). A detailed account of these metabolism studies is
described separately.35 The formation of excessive levels of
GSH adducts, an indication of reactive metabolite formation,
was of particular concern due to the body of circumstantial
evidence supporting the hypothesis that reactive metabolites
may be involved in idiosyncratic drug reactions.36-38 As
a result, an investigation was undertaken as part of the
medicinal chemistry optimization process to minimize reac-
tive metabolite formation, to the extent possible, by making
appropriate structural modifications.
A three-pronged optimization strategy to reduce the for-
mation of reactive metabolites found with 8 was developed,
as illustrated in Figure 3. First, to reduce the bioactivation
level of the phenyl group, we directed our efforts toward
incorporation of a pyridyl group as an isosteric replacement
for the phenyl group. In an in vitro biotransformation study
comparing a small number of pyridyl and phenyl analogues, it
was found that pyridyl groups showed substantially reduced
levels of metabolic activation relative to phenyl groups.36
Figure 1. CRF1 receptor antagonists.
Figure 2. Formation of pyrazinone oxidation metabolites and related glutathione conjugates of compound 8.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7655
Scheme 1a
Figure 3. Illustration of a three-pronged approach to reduce the
level of reactive metabolite formation on the pyrazinone and phenyl
groups of 8: (1) replacement of the phenyl group with a pyridyl
group, (2) replacement of the 5-chloro substituent with a 5-cyano
group, as the SAR allowed, and (3) inclusion of a benign metabolic
“soft spot” (methoxy group) in the molecule.
A second area of focus was to reduce the level of pyrazinone
bioactivation, measured as the sum of pyrazinone oxidation
and GSH adduct formation on the pyrazinone ring, by
decreasing the propensity of the pyrazinone ring to undergo
epoxidation. A series of thrombin inhibitors containing a
6-methylpyrazinone core had been found to generate reactive
metabolites.39 It was discovered that replacement of the C6
methyl group with a chloro group rendered the pyrazinone
less susceptible to epoxidation and led to an analogue with
diminished potential for reactive metabolite formation.36
Although 5-chloropyrazinones may be sufficiently electron
deficient so as to mitigate reactive metabolite formation in
some cases, we nevertheless sought to replace the 5-chloro
substituent with the more strongly electron-withdrawing
5-cyano group, as the SAR allowed, to further reduce the
potential for oxidation of the pyrazinone ring. Encouragingly,
we had observed in an in vitro experiment that a C5-cyano-
pyrazinone showed decreased levels of reactive metabolite
formation relative to a C5-chloropyrazinone.35 Third, we
hypothesized that including a benign metabolic “soft spot”
in the molecule would aid in diverting metabolism away from
the pyrazinone core. Itwas anticipated that metabolism at this
“soft spot” would result in the formation of innocuous
metabolites and further minimize the formation of undesired
reactive metabolites. We previously observed that including a
methoxy group in R1 resulted in an increased rate of clearance
in some analogues as a result of O-demethylation, followed to
some extent by oxidation of the resultant alcohol to a car-
boxylic acid (data not presented). Incorporating a methoxy
group within R1 may involve making a trade-off between low
clearance and minimization of GSH adduct formation.
a Reagents and conditions: (a) chloroacetonitrile, KI, K2CO3,
CH3CN, 50 ꢀC (84-96%); (b) (COCl)2, toluene, 55 ꢀC (43-71%) or
(COCl)2, dioxane/CH2Cl2, 55 ꢀC (69-74%); (c) (COBr)2, CH2Cl2, 45 ꢀC
(56-68%); (d) NaHMDS, ArNH2, THF; (e) Zn(CN)2, Pd2(dba)3, dppf,
DMF, H2O.
Scheme 2a
a Reagents and conditions: (a) MeOH, 75 ꢀC; (b) KCN, MeOH; (c)
conc HCl, 95 ꢀC (52%, 3 steps); (d) BH3 SMe2, THF (44%); (e) NaH,
3
MeI, THF (85%); (f) H2, Pd(OH)2/C, EtOH (99%).
5-cyanopyrazinone analogues (19) could also be carried out
from chloropyrazinones 17 with higher catalyst loading.
The amine groups that comprise R1 were synthesized as
previously described.33 In addition, an alternate route was
developed for the asymmetric synthesis of (S)-2-methoxy-1-
cyclopropylethylamine (25) (Scheme 2). The synthesis of 22
was completed using the method of Bayston et al.,41 whereby
condensation of commercially available cyclopropane car-
boxaldehyde (20) and (S)-1-phenethyl amine (21) resulted in
the formation of a chiral imine, which was treated in situ with
potassium cyanide. Hydrolysis of the resultant R-amino
nitrile intermediate under acidic conditions provided 22.
Borane reduction of 22 afforded 23, which was then treated
with sodium hydride and methyl iodide to provide 24 in 85%
yield. Reductive cleavage of the benzyl group furnished 25 in
high yield.
Results and Discussion
Chemistry. The synthetic route to prepare 1-alkyl-3-N-
arylpyrazin-2-ones is summarized in Scheme 1, as previously
described.33 Briefly, treatment of alkylamine hydrochlorides
13 with chloroacetonitrile in the presence of potassium
iodide and potassium carbonate in acetonitrile furnished
the aminoacetonitrile intermediate 14 in high yield. The
aminoacetonitrile intermediate was then condensed with
oxalyl chloride or oxalyl bromide to form 15 or 16, respec-
tively. Coupling of 15 or 16 with a variety of aryl amines
in the presence of NaHMDS then furnished the desired
pyrazinone products 17 or 18, respectively. Synthesis of
5-cyanopyrazinone analogues (17) was generally comp-
leted by treatment of bromopyrazinones 18 with zinc cya-
nide in the presence of a palladium catalyst.40 Synthesis of

7656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
Scheme 3a
Alternatively, in method B, the 6-methyl group was installed
directly by treatment of 29 with trimethylsulfoxonium iodide
in the presence of sodium hydride and DMSO. While method
B is only a single step, method A proceeded in a higher
overall yield and proved more convenient for larger scale
preparations of 32. Completion of the synthesis of 35 began
by heating 32 under acidic conditions for 1-2 h to furnish 33
in excellent yield. The difluoromethoxy intermediate (34)
was prepared by selective O-alkylation of 33. Initial prepara-
tions involved use of sodium chlorodifluoroacetate;44 how-
ever, yields were generally low and variable. The preferred
methods involved the use of either trimethylsilyl 2,2-di-
fluoro-2-(fluorosulfonyl)acetate in combination with cesium
fluoride45,46 (method A) or 2,2-difluoro-2-(fluorosulfonyl)-
acetic acid47 (method B). Both conditions provided 34 in
high yield. Conversion of 34 to 35 was accomplished by
reduction of the nitro group with palladium on carbon under
a hydrogen atmosphere.
a Reagents and conditions: (a) HNO3, H2SO4; (b) HNO3, H2O (75%,
2 steps); (c) POCl3, 120 ꢀC (92%); (d) NaOMe, MeOH (97%);
(e) CHBr3, KOt-Bu, THF, -78 ꢀC (94%); (f) H2, Pd/C, EtOH (73%).
Scheme 4a
Biology. Compounds were tested in a CRF1 receptor
binding titration assay using rat frontal cortex homogenate.
Inhibition of specific binding of [125I]Tyr-o-CRF by test
compounds was measured to determine the receptor binding
affinity.33 Potent compounds were subsequently incubated
with rat and human liver microsomes to evaluate their
metabolic stability. Selected compounds were examined for
their functional activity in Y79 cells to assess antagonist
properties and were screened for binding affinity at CRF2
receptors. The CRF2 receptor binding titration assay em-
ployed porcine choroid plexus homogenate and [125I]-sau-
vagine binding inhibition to determine binding affinities.
Compounds with appropriate profiles were assessed in
pharmacokinetic studies, followed by evaluation in the
defensive withdrawal model of anxiety in rats.21,48 Along
with the behavioral studies, the compounds were examined
in an ex vivo binding assay to determine CRF1 receptor
occupancy. In vivo plasma levels after oral dosing were also
measured. Key compounds were also assessed for the level of
reactive metabolite formation in vitro, by incubation with
NADPH-fortified rat and human liver microsomes in the
presence of GSH, and in vivo, in BDC rats.49
a Reagents and conditions: (a) t-butylchloroacetate, KOt-Bu, THF;
(b) TFA, reflux; (c) K2CO3, DMF (65%, 3 steps); (d) NaH, Me3SOþI-,
DMSO (41%); (e) conc HCl, 110 ꢀC (94%); (f) FSO2CF2CO2SiMe3,
NaH, CsF, MeCN (92%); (g) FSO2CF2CO2H, NaH, MeCN (96%);
(h) H2, Pd/C, EtOH (89%).
Schemes 3 and 4 describe the synthesis of two pyridyl
groups that were synthesized as isosteric replacements for the
phenyl groups used in previously described analogues.33,34
Synthesis of 31 began with commercially available 3-methyl-
pyridin-2-amine (26) (Scheme 3). A modification of the two-
step, one-pot procedure of Hawkins and Roe42 afforded 27
in 75% overall yield. Subsequent treatment of 27 with
phosphorus oxychloride followed by addition of sodium
methoxide afforded 29 in high yield. Completion of the
synthesis of 31 involved incorporation of a methyl group at
the 6-position of 29 using a vicarious nucleophilic substitu-
tion reaction. Several conditions were explored, and it was
found that treatment of 29 with bromoform and potassium t-
butoxide resulted in the formation of 30 in high yield.43
Simultaneous reduction of the nitro group and debromina-
tion by treatment with hydrogen in the presence of palladium
on carbon furnished 31 in 73% yield.
Synthesis of 6-(difluoromethoxy)-2,5-dimethylpyridin-3-
amine (35) (Scheme 4) commenced with compound 29,
prepared as described in Scheme 3. We chose to investigate
alternative nucleophiles for the vicarious nucleophilic sub-
stitution reaction. Two viable reaction conditions were
identified. Method A involved treatment of 29 with t-butyl
chloroacetate in the presence of potassium t-butoxide, fol-
lowed by hydrolysis of the resultant ester to give the corre-
sponding carboxylic acid. Decarboxylation was achieved by
heating under basic conditions in DMF to provide 32.
On the basis of the SAR previously developed for N3-
phenyl pyrazinones,33 we focused on a group of analogues
wherein the 4-methoxy-2,5-dimethylaniline moiety was re-
placed with a similarly substituted pyridyl substituent
(Table 1). Compounds 17a-17d were very potent (IC50
e
1.5 nM); however, replacement of the cyclopropyl group in
R1 with a methyl group resulted in a 12-fold decrease in
potency (compare 17e vs 17b). Replacement of the chloro
substituent (X) of 17a with a cyano group (19a) resulted in
an 8-fold decrease in potency. The loss in potency could
be regained by replacement of the (S)-1-cyclopropyl-2-
methoxyethyl substituent (R1=5) with the (R)-1-cyclopro-
pylpropyl group (R1=2) (19b). The metabolic stability data
(Table 1) for compounds where R2 = OMe suggested a
moderate rate of metabolism in human liver microsomes
and, in general, a higher rate of clearance in rats. An
improved metabolic stability profile would be desirable.
On the basis of our previous experience with this chemo-
type, we recognized that replacement of the methoxy
substituent on the pyridyl moiety (R2 = OMe) with a
difluoromethoxy substituent should improve the metabolic
stability of these compounds. A group of analogues contain-
ing the difluoromethoxy group at R2 was synthesized, and
indeed, incubation of 17f-17j and 19d-19f (Table 1) with

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7657
Table 1. CRF1 Receptor Binding Affinities and Metabolic Stability Data for 17a-17j and 19a-19f
metabolic stability
(HLM, % remaining)b,c
metabolic stability
(RLM, % remaining)c,d
compd
R1
R2
X
IC50 (nM)a
17a
17b
17c
17d
17e
19a
19b
19c
17f
17g
17h
17i
5
4
2
1
3
5
2
1
1
2
3
4
5
1
5
4
OMe
OMe
Cl
Cl
1.5 ( 0.6
1.1 ( 0.2
0.28 ( 0.05
1.5 ( 0.5
14 ( 4
12 ( 3
1.7 ( 0.5
10 ( 3
0.30 ( 0.03
0.21 ( 0.03
1.6 ( 0.3
0.50 ( 0.09
0.24 ( 0.10
0.82 ( 0.09
0.86 ( 0.12
1.4 ( 0.3
48
40
24
13
5
OMe
OMe
Cl
Cl
51
77
55
8
OMe
OMe
Cl
39
60
CN
CN
CN
Cl
51
38
72
86
56
66
46
69
89
70
64
OMe
OMe
81
99
OCHF2
OCHF2
OCHF2
OCHF2
OCHF2
OCHF2
OCHF2
OCHF2
100
100
87
Cl
Cl
Cl
Cl
84
93
17j
19d
19e
19f
CN
CN
CN
100
74
78
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 1 = 1.2 ( 0.2 nM in this assay.
b HLM = human liver microsomes. c Percent remaining after 10 min. d RLM = rat liver microsomes.
human and rat liver microsomes revealed that compounds
with the difluoromethoxy group at R2 had improved meta-
bolic stability in both human and rat liver microsomes
compared to the corresponding methoxypyridyl analogues.
In addition, analogues containing the difluoromethoxy
group at R2 were more potent than the corresponding
analogues containing a methoxy group at R2. In many
cases, these compounds had subnanomolar IC50’s (Table 1).
As before, it was observed that replacement of the chloro
group at X with a cyano group resulted in a slight de-
crease in potency (compare 17f vs 19d and 17j vs 19e);
nevertheless, the binding affinities of 19d and 19e were
subnanomolar.
Shown in Table 2 are the pharmacokinetic profiles of 17a,
19b, 17i, 17j, 19d, and 19e after iv (2 mg/kg) and oral dosing
(10 mg/kg) in Sprague-Dawley rats. Compounds 17a and
19b were of interest as a result of their potency and desirable
structural features. Both compounds contained a pyridyl
group in place of a phenyl group. In addition, 17a contained
a methoxy group within R1 and 19b contained a cyano group
on the pyrazinone ring. Compound 17a had a moderately
high rate of clearance in rats (38 mL/min/kg) and 19b had a
high rate of clearance (53 mL/min/kg), most likely due to
oxidation of the top-chain alkyl group (R1). Both 17a and
19b had low oral bioavailability when dosed as an aqueous
suspension (1% polysorbate 80 in 0.5% methylcellulose).
The low aqueous solubility of 17a and 19b likely contributed
to their low oral bioavailability. The aqueous solubility of
17a (LogD=3.950) was 6 μg/mL at pH 6.5 and 116 μg/mL at
pH 1, and the aqueous solubility of 19b (LogD=4.1) was
1 μg/mL at pH=6.5 and 23 μg/mL at pH=1. The improved
solubility of 17a at low pH is likely due to its higher pKa value
compared to 19b (3.1 for 17a vs <2 for 19b).51 Because of the
low aqueous solubility of these compounds, they were also
dosed orally as a solution in an oleoyl macrogolglycerides-
based vehicle52 (Table 2). An improvement in the oral
bioavailability was observed (F = 26% and 33%, respec-
tively), although it was still modest.
On the basis of the metabolic stability results shown in
Table 1, it appeared that compounds 17i, 17j, 19d, and 19e
may have improved rat pharmacokinetic profiles relative to
17a and 19b. When dosed iv, the rate of clearance of these
four compounds was comparable to or only somewhat lower
than that of 17a (Table 2) despite their substantially greater
in vitro metabolic stability in rats (Table 1). The low correla-
tion between the in vitro metabolic stability results obtained
by incubation with rat liver microsomes and the in vivo
clearance in rats may be due to the lipophilic nature of these
compounds, resulting in a low unbound fraction53 and
relatively large volume of distribution. When compounds
17i, 17j, 19d, and 19e (R2 =OCHF2) were dosed orally, a
substantial increase in AUC, Cmax, and oral bioavailability
was observed relative to 17a and 19b (R2 = OMe). In
addition, it was discovered that the S enantiomer (17j) had
a nearly 2-fold higher exposure, as indicated by the AUC
value, and a corresponding increase in oral bioavailability
(F=86%) compared to the R enantiomer (17i) (F=48%).
After oral administration, the Cmax in plasma for 17i, 17j,
19d, and 19e occurred 2-4 h after dosing. As shown in
Table 2, 17j, 19d, and 19e each had an improved oral
bioavailability when dosed as a solution compared to
dosing as an aqueous suspension, likely due to their low
aqueous solubility. For example, the aqueous solubility
of 19e (LogD=3.9) was 4 μg/mL at pH 6.5 and 3.0 μg/mL at
pH 1. Unlike compounds 17a and 19b, there was no im-
provement in solubility at low pH, likely due to the reduced
basicity of the pyridyl nitrogen (pKa<2 for 19e). It was
found in additional studies that 19e had somewhat greater

7658 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
Table 2. Discrete Pharmacokinetic Properties of Compounds 17a, 19b,
17i, 17j, 19d, and 19e in Rats
solubility in simulated gastric fluid (15 μg/mL) and in
simulated intestinal fluid (fed) (7.4 μg/mL).
Compounds 17j, 19d, and 19e were chosen for evaluation
of potential pyrazinone bioactivation. These compounds
incorporated structural features that were meant to minimize
the formation of reactive metabolites (vide supra). Cyto-
chrome P450 (CYP)-catalyzed pyrazinone bioactivation of
17j, 19d, and 19e was assessed in vitro by incubation with rat
and human liver microsomes for 30 min at a concentration of
10 μM in the presence of NADPH and GSH to trap potential
reactive intermediates (Table 3). The bioactivation potential
was measured as the sum of pyrazinone oxidation metabolite
formation and GSH adduct formation via the proposed
pyrazinone oxidation pathway shown in Figure 2. These
three analogues showed a substantially diminished level of
pyrazinone oxidation relative to 8, while their in vitro
metabolism turnovers were comparable to or higher than
that of 8 in human liver microsomal and rat liver microsomal
incubations. Among them, 19e produced the lowest level of
pyrazinone oxidation metabolites and related GSH adducts,
with the combined amount accounting for <2% and <4%
of the total drug related materials in human and rat liver
microsomal incubations, respectively.54 The major in vitro
metabolic pathway with 19e was identified as O-demethyla-
tion.
Compound 19e was subsequently evaluated in metabolism
and disposition studies in vivo in BDC rats (20 mg/kg, oral).
Plasma, bile, and urine samples were collected and analyzed
by HPLC/UV/MS. Compound 19e was the predominant
drug-related species in plasma, and the main circulating
metabolite was the alcohol derived from O-demethylation.
Compound 19e was extensively metabolized in rat liver with
only 3% of the total drug-related materials being excreted
as the unchanged parent drug in bile samples. Metabolic
analysis indicated that the major metabolic clearance path-
ways in liver were O-demethylation, followed by oxidation of
the resultant primary alcohol to the carboxylic acid, which to
some extent underwent subsequent glucuronidation. LC/MS
analysis did not reveal any sign of acyl migration of the
glucoronide product, a potential bioactivation mechanism.
In rat urine, the major metabolites detected were formed
through either O-demethylation or oxidation of the methyl
PK parameters
17a
19b
17i
17j
19d
19e
iv dose (2 mg/kg)a
Cl (mL/min/kg)
Vss (L/kg)
38
19
19
53
11
31
36
15
36
11
29
11
42
16
t
1/2 (h)
9.0
9.2
7.2
9.3
po dose (10 mg/kg)b
AUC (nM h)
1300
180
11
240
50
3
NDd 7000
4600
560
38
4100
860
31
3
Cmax (nM)
F%
ND
ND
770
66
po dose (10 mg/kg)c
AUC (nM h)
3000 3000
4700 9000
440 1300
9900
1400
82
8800
1600
66
3
Cmax (nM)
F%
510
26
590
33
48
86
a Vehicle: PEG/ethanol, 90:10 (v/v); n = 3 rats. b Vehicle: 1% poly-
sorbate 80 in 0.5% methylcellulose; n = 3 rats. c Vehicle: oleoyl macro-
golglycerides52/DMAC/polysorbate 80, 85:10:5; n = 3 rats. d ND = not
determined.
Table 3. Percentage of Parent Compound Remaining and Pyrazinone Oxidation Metabolites and GSH Adducts Formed via Pyrazinone
Oxidation in NADPH- and GSH-Fortified Human and Rat Liver Microsomal Incubations (30 min) with 8, 17j, 19d, and 19e at 10 μM Substrate
Concentrations
human liver microsomesa
rat liver microsomesa
% parent
compd remaining
% pyrazinone
oxidation metabolites
% parent compd
remaining
% pyrazinone
oxidation metabolites
compd
% GSH adducts
% GSH adducts
8
85
75
88
56
12
1
2
5
7
1
74
42
80
82
20
1
5
3
8
3
17j
19d
19e
<1
<1
<1
<1
a The results were obtained after incubation of each compound with human and rat liver microsomes for 30 min.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7659
groups on the pyridyl substituent. In bile and urine (0-24 h),
the amount of GSH adducts, identical to those detected in
vitro, was estimated to be only 0.1% of the total drug-related
materials.55 No pyrazinone oxidation metabolites were
detected in bile, urine, or plasma. It was previously noted
in studies with 8 (vide supra) that approximately 60% of
the total drug-related materials collected in bile (0-7 h)
was identified as pyrazinone oxidation metabolites (17%,
e.g. 9 and 10, Figure 2), GSH-related adducts on the pyr-
azinone ring via epoxidation of the C5-C6 double bond
(18%, e.g. 11, Figure 2), and GSH-related adducts on the
phenyl group (25%, e.g. 12). These results indicate that the
excessive levels of reactive metabolite formation arising from
metabolism of 8 in vivo, detected as GSH adducts and
pyrazinone oxidation metabolites, were lowered through
structural modifications in key regions of the molecule to
provide 19e, in which the level of metabolic activation was
minimal.
Additional profiling of 19e was also conducted. The anta-
gonist properties of 19e were assessed by measuring its inhibi-
tion of CRF-stimulated cyclic adenosine monophosphate
(cAMP) production in human Y-79 retinoblastoma cells.
Compound 19e produced a concentration-dependent inhibi-
tion of CRF (1nM)-induced cAMP production with an IC50
value of 1.9 ( 0.1 nM and completely suppressed CRF-
stimulated cAMP production at higher concentrations, indicat-
ing that this compound behaves as an antagonist. In addition,
19e was not active in a CRF2 receptor binding assay (CRF2
IC50 >10 μM).56 Compound 19e was also profiled in a broad
array of receptor binding assays.57 Compound 19e (10 μM)
produced <50% inhibition of binding to 43 different receptors,
transporters, and ion channels.
efficacy.21,48 CRF1 receptor occupancy and plasma concen-
trations at each dose were also measured. In this experiment,
the time required for a rat to emerge from a darkened
chamber placed within an open, illuminated field was mea-
sured. A compound was considered to have an anxiolytic
effect if the latency time period for the rat to emerge from the
chamber was significantly reduced relative to vehicle-treated
animals. Figure 4 and Table 4 summarize the results of the
behavioral studies for 19e following oral dosing at 0.56, 1,
1.8, and 3 mg/kg. Figure 4a shows exit latencies for each dose
at 60 min after oral administration of either 19e or the
positive control compound 1 (dosed at 10 mg/kg).58 The
results show that 19e was effective at reducing exit latency at
doses of 1.8 and 3 mg/kg (n =8) (Table 4). At the lowest
efficacious dose of 1.8 mg/kg, 19e reduced exit latency by
60% relative to vehicle-treated controls. CRF1 receptor
occupancy in the parietal cortex was determined by ex vivo
autoradiography.58 Compound 19e occupied CRF1 recep-
tors in a dose-dependent manner (Figure 4b). CRF1 receptor
occupancy greater than 50% was necessary to achieve a
statistically significant anxiolytic effect.
Shown in Table 5 is the pharmacokinetic profile of 19e in
beagle dogs (fasted) after iv (2 mg/kg) and oral dosing. Com-
pound 19e had a low rate of clearance (Cl=7.3 mL/min/kg),
a long iv half-life (t1/2=30 h), and a volume of distribution of
13 L/kg. Compund 19e had high plasma exposures, as measured
by the AUC values, and had excellent oral bioavailability when
dosed as either an aqueous suspension (2 mg/kg) or as a solution
(3 mg/kg) (F=53% and 70%, respectively).
Compound 19e was evaluated in a two-week rat safety
study at doses of 20, 60, and 175 mg/kg. No significant
toxicity findings, gross observations, or histopathological
findings were observed at all dose levels. The results of
this study indicated that this compound was well-tolerated.
Compound 19e was tested in rats in the defensive
withdrawal model of anxiety to determine behavioral
Figure 4. (a) Anxiolytic-like effects of 19e in the defensive withdrawal test in rats at 0.56, 1, 1.8, and 3 mg/kg with 1 as a positive control
(* p < 0.05 vs vehicle) and (b) CRF1 receptor occupancy of 19e in rats at 0.56, 1, 1.8, and 3 mg/kg with 1 as a positive control.
Table 4. Mean Total and Plasma Free Concentrations and CRF1 Receptor Occupancies in the Defensive Withdrawal Test in Rats Following Oral
Doses of Compound 19e
oral dose
(mg/kg)
mean total
plasma conc (nM)a,b
mean plasma
free conc (nM)a,c
CRF1 receptor
occupancy (%)a,d,e
% decrease in
exit latencyb
0.56
1.0
1.8
3.0
24 ( 4
45 ( 5
83 ( 7
0.84 ( 0.14
1.6 ( 0.2
2.9 ( 0.3
5.0 ( 0.4
39 ( 6
43 ( 6
71 ( 4
80 ( 5
7
28
60f
56f
140 ( 10
a (SEM. b n = 8. c Based on an unbound fraction of 3.5% in plasma determined by equilibrium dialysis. d n = 4. e Receptor occupancy of 1 at
10 mg/kg = 89 ( 5. f p<0.05 vs vehicle.

7660 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
Table 5. Discrete Pharmacokinetic Properties of Compound 19e in
Beagle Dogs
and are uncorrected. Proton NMR spectra were recorded on
either a Bruker 400 or 500 MHz NMR spectrometer. Chemical
shifts are reported in δ values relative to tetramethylsilane.
Atmosphere pressure chemical ionization (APCI) low-resolu-
tion mass spectra were obtained on a Finnigan Navigator LC/
MS single quadrupole mass spectrometer. Electrospray ioniza-
tion (ESI) high-resolution mass spectra were obtained on a
Finnigan MAT95S or Thermo Scientific MAT900 mass spectro-
meter. All final products had a purity of g95%. The purity of
final products was determined by either combustion analysis or
HPLC. Combustion analyses were performed by Quantitative
Technologies, Inc., Whitehouse, NJ. HPLC purity was mea-
sured using two methods for each compound. Method A:
Phenominex analytical C18 column (4.6 mm ꢀ 50 mm, 5 μm);
mobile phase: A=H2O with 0.1% TFA, B=acetonitrile with
0.1% TFA, 0-1 min, 20% B; 1-7 min, 20% B f 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 mm ꢀ 50 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.
PK parameters
19e in dogs
iv dose (2 mg/kg)a
Cl (mL/min/kg)
7.3
13
30
Vss (L/kg)
t1/2 (h)
po dose (2 mg/kg)b
AUC (nM h)
7600
880
53
3
Cmax (nM)
F%
po dose (3 mg/kg)c
AUC (nM h)
17000
1400
70
3
Cmax (nM)
F%
a Vehicle: PEG/ethanol, 90:10 (v/v); n = 4. b Vehicle: 0.2% polysor-
bate 80 in 0.5% methylcellulose; n = 4. c Vehicle: oleoyl macrogolgly-
cerides52/DMAC/polysorbate 80, 85:10:5; n = 2.
General procedures for the preparation of compounds 14-16
In addition, 19e did not alter motor coordination and
balance in rats at doses of up to 60 mg/kg in a rotarod study.
were previously described.33
(S)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(6-methoxy-
2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-one (17a). To a so-
lution of (S)-3,5-dichloro-1-(1-cyclopropyl-2-methoxyethyl)py-
razin-2(1H)-one (15) (4.00 g, 15.2 mmol) and 31 (2.31 g, 15.2
mmol) in THF (76 mL) at 0 ꢀC was added NaHMDS (31.9 mL,
31.9 mmol, 1 M in THF) dropwise. The reaction mixture was
stirred at 0 ꢀC for 2 h. The mixture was transferred to a
separatory funnel containing saturated aqueous NaHCO3 solu-
tion (50 mL), and the aqueous layer was extracted with ethyl
acetate (3 ꢀ 100 mL). The combined organic layers were washed
with brine, dried over MgSO4, filtered, and concentrated. The
residue was purified by column chromatography on silica gel
(10% f 40% ethyl acetate in hexanes) to afford 17a (4.94 g, 86%
yield) as a light-brown amorphous solid. The product was
recrystallized from hexanes/ethyl acetate (1:1, 10 mL) to afford
Conclusion
In conclusion, studies to minimize the formation of reactive
metabolites in the pyrazinone chemotype led to the discovery
of (S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluorometh-
oxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyr-
azine-2-carbonitrile (19e),59 a high affinity CRF1 receptor
antagonist (IC50=0.86 nM) and a potent inhibitor of CRF-
stimulated cAMP production in human Y-79 retinoblastoma
cells (IC50=1.9 nM). We have shown that excessive levels of
reactive metabolite formation arising from metabolism of 8
could be lowered to minimal levels by: (1) replacement of the
phenyl group with a pyridyl group, (2) replacement of the
chloro group on the pyrazinone with the more strongly
electron withdrawing cyano group, and (3) incorporation of
a methoxy group within R1 to act as a metabolic “soft spot”
and aid in directing metabolism away from the pyrazinone
ring. In addition, a novel 6-(difluoromethoxy)-2,5-dimethyl-
pyridin-3-amine group, which contributed to the potency and
improved in vivo properties of 19e and related analogues, was
discovered. Compound 19e had excellent pharmacokinetic
properties in rats and dogs and was efficacious in the defensive
withdrawal model of anxiety in rats. The lowest efficacious
dose was 1.8 mg/kg. The results of a two-week rat safety study
with 19e indicated that this compound was well-tolerated. On
the basis of its overall profile, 19e was selected as a candidate
for further development.
17a as a light-brown crystalline solid: mp 99-100.5 ꢀC; [R]25
-
D
52.3 (c 0.636, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.05
(s, 1H), 7.88 (s, 1H), 6.90 (s, 1H), 4.19-4.14 (m, 1H), 3.92 (s, 3H),
3.74(dd, JAB=10.3, JAX=6.2 Hz, 1H), 3.67 (dd, JBA=10.3, JBX=
3.5 Hz, 1H), 3.34 (s, 3H), 2.41 (s, 3H), 2.17 (s, 3H), 1.31-1.24
(m,1H),0.80-0.73 (m, 1H), 0.62-0.56(m,1H),0.53-0.47(m, 1H),
0.36-0.30 (m, 1H). HRMS (ESI) m/e 379.1537 [(M þ H)þ, calcd
for C18H24N4O3Cl 379.1537]. Anal. (C18H23N4O3Cl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(6-methoxy-
2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-one (17b). Com-
pound 17b was prepared according to the procedure described
for the synthesis of 17a using (R)-3,5-dichloro-1-(1-cyclopropyl-
2-methoxyethyl)pyrazin-2(1H)-one (15) (300 mg, 1.14 mmol)
and 31 (174 mg, 1.14 mmol). The product was purified by column
chromatography to afford 17b (310 mg, 72% yield) as a colorless
solid. The product was subsequently recrystallized from hexanes/
ethyl acetate to afford 17b as a pale-yellow crystalline solid: mp
Experimental Section
1
99.0-99.5 ꢀC; [R]25 þ48.2 (c 0.247, CHCl3). H NMR (400
D
MHz, CDCl3) δ 8.07 (s, 1H), 7.89 (s, 1H), 6.91 (s, 1H), 4.20-4.15
(m, 1H), 3.93 (s, 3H), 3.75 (dd, JAB=10.5, JAX=6.1 Hz, 1H), 3.67
(dd, JBA=10.3, JBX=3.4 Hz, 1H), 3.35 (s, 3H), 2.43 (s, 3H), 2.18
(s, 3H), 1.32-1.24 (m, 1H), 0.81-0.74 (m, 1H), 0.63-0.57 (m,
1H), 0.54-0.48 (m, 1H), 0.37-0.31 (m, 1H). HRMS (ESI) m/e
379.1538 [(M þ H)þ, calcd for C18H24N4O3Cl 379.1537]. Anal.
(C18H23N4O3Cl) C, H, N.
Chemistry. All procedures were carried out under a nitrogen
atmosphere unless otherwise indicated using anhydrous sol-
vents purchased from commercial sources without further pur-
ification. Reactions requiring anhydrous conditions were
performed in glassware, which was flame-dried or oven-dried
and placed under a nitrogen atmosphere. Column chromatog-
raphy was performed on silica gel using the solvent systems
indicated. Solvent systems are reported as v/v percent ratios. All
reactions were monitored by TLC using EM Science, 0.25 mm,
precoated silica gel plates or by LC/MS. Yields refer to
chromatographically and spectroscopically pure compounds,
except as otherwise indicated. Melting points were obtained on a
Laboratory Devices, Inc. Mel Temp 3.0 melting point apparatus
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(6-methoxy-2,5-dime-
thylpyridin-3-ylamino)pyrazin-2(1H)-one (17c). Compound 17c
was prepared according to the procedure described for the
synthesis of 17a using (R)-3,5-dichloro-1-(1-cyclopropylpro-
pyl)pyrazin-2(1H)-one (15) (100 mg, 0.390 mmol) and 31 (60 mg,
0.39 mmol). The product was purified by column chromatography

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7661
to afford 17c (30 mg, 21% yield) as a dark-yellow solid: [R]25 -7.5 (c
described for the synthesis of 17a using (R)-3,5-dichloro-1-(1-
methoxypropan-2-yl)pyrazin-2(1H)-one (15) (217 mg, 0.917
mmol) and 35 (200 mg, 0.917 mmol). The product was purified
by column chromatography to afford 17h (214 mg, 60% yield)
as a pale-yellow solid. The product was recrystallized from
0.265, CHCl3). 1H NMR (400 MHz, CDCl3)
δ 8.11
(s, 1H), 7.93 (s, 1H), 6.75 (s, 1H), 4.06-3.99 (m, 1H), 3.95 (s,
3H), 2.45 (s, 3H), 2.19 (s, 3H), 1.95-1.77 (m, 2H), 1.09-1.01 (m,
1H), 0.93(t, J=7.6 Hz, 3H), 0.82-0.75 (m, 1H), 0.55-0.47 (m, 2H),
0.32-0.27 (m, 1H). HRMS (ESI) m/e 363.1591 [(M þ H)þ, calcd
for C18H24N4O2Cl 363.1588]. Anal. (C18H23N4O2Cl) C, H, N.
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-(6-methoxy-2,5-dimeth-
ylpyridin-3-ylamino)pyrazin-2(1H)-one (17d). Compound 17d
was prepared according to the procedure described for the
synthesis of 17a using (R)-3,5-dichloro-1-(1-cyclopropy-
lethyl)pyrazin-2(1H)-one (15) (150 mg, 0.650 mmol) and 31
(100 mg, 0.650 mmol). The product was purified by column
chromatography to afford 17d (150 mg, 66% yield) as a pale-
yellow crystalline solid. The product was subsequently recrystal-
lized from ethyl acetate/hexanes to afford 17d a pale-yellow
crystalline solid: mp 135-136 ꢀC; [R]25D -19.1 (c 0.347, CHCl3).
1H NMR (400 MHz, CDCl3) δ 8.06 (s, 1H), 7.91 (s, 1H), 6.83
(s, 1H), 4.27-4.20 (m, 1H), 3.95 (s, 3H), 2.45 (s, 3H), 2.19 (s, 3H),
1.43 (d, J=6.9 Hz, 3H), 1.12-1.05 (m, 1H), 0.79-0.72 (m, 1H),
0.59-0.53 (m, 1H), 0.50-0.44 (m, 1H), 0.38-0.31 (m, 1H).
HRMS (ESI) m/e 349.1421 [(M þ H)þ, calcd for C17H22N4O2Cl
349.1431]. Anal. (C17H21N4O2Cl) C, H, N.
hexanes/ethyl acetate to afford 17h as a pale-yellow crystalline
1
solid: mp 106.5-107.5 ꢀC; [R]25 þ47.5 (c 0.200, CHCl3). H
D
NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 8.03 (s, 1H), 7.46 (t, J=
73.8 Hz, 1H), 6.82 (s, 1H), 5.16-5.12 (m, 1H), 3.62-3.53
(m, 2H), 3.34 (s, 3H), 2.44 (s, 3H), 2.26 (s, 3H), 1.40 (d, J=7.0 Hz,
3H). HRMS (ESI) m/e 389.1210 [(M þ H)þ, calcd for
C16H20N4O3ClF2 389.1192]. Anal. (C16H19N4O3ClF2) C, H, N.
(R)-5-Chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-[6-(difluoro-
methoxy)-2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one (17i).
Compound 17i was prepared according to the procedure de-
scribed for the synthesis of 17a using (R)-3,5-dichloro-1-(1-
cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-one (15) (238 mg,
0.906 mmol) and 35 (198 mg, 0.906 mmol). The product was
purified by column chromatography to afford compound 17i
(292 mg, 78% yield) as an off-white solid. The product was
recrystallized from hexanes/ethyl acetate to afford 17i as an
off-white crystalline solid: mp 102.5-103.5 ꢀC; [R]25 þ36.4
D
(c 0.643, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.01
(s, 1H), 7.46 (t, J=74.0 Hz, 1H), 6.96 (s, 1H), 4.19-4.14 (m, 1H),
3.75 (dd, JAB=10.3, JAX=6.3 Hz, 1H), 3.67 (dd, JBA=10.3,
(R)-5-Chloro-3-(6-methoxy-2,5-dimethylpyridin-3-ylamino)-
1-(1-methoxypropan-2-yl)pyrazin-2(1H)-one (17e). Compound
17e was prepared according to the procedure described for the
synthesis of 17a using (R)-3,5-dichloro-1-(1-methoxypropan-2-
yl)pyrazin-2(1H)-one (15) (100 mg, 0.422 mmol) and 31 (64 mg,
0.422 mmol). The product was purified by column chromatog-
J
BX=3.5 Hz, 1H), 3.34 (s, 3H), 2.44 (s, 3H), 2.26 (s, 3H), 1.32-
1.25 (m, 1H), 0.81-0.74 (m, 1H), 0.63-0.56 (m, 1H), 0.54-0.47
(m, 1H), 0.36-0.31 (m, 1H). HRMS (ESI) m/e 415.1361 [(M þ
H)þ, calcd for C18H22N4O3ClF2 415.1349]. Anal. (C18H21N4O3-
ClF2) C, H, N.
raphy to afford 17e (65 mg, 44% yield) as a tan solid: [R]25
D
þ40.2 (c 0.416, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.01 (s,
1H), 7.91 (s, 1H), 6.76 (s, 1H), 5.14 (td, J=6.74, 4.15 Hz, 1H),
3.91 (s, 3H), 3.66-3.50 (m, 2H), 3.34 (s, 3H), 2.41 (s, 3H), 2.17 (s,
3H), 1.39 (d, J=7.05 Hz, 3H). HRMS (ESI) m/e 353.1381 [(M þ
H)þ, calcd for C16H22N4O3Cl 353.1380]. HPLC method A: tR=
4.74 min, >99%; method B: tR=3.30 min, >99%.
(S)-5-Chloro-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluorome-
thoxy)-2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one (17j).
(S)-3,5-Dichloro-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-2(1H)-
one (15) (15.0 g, 57.01 mmol) and 35 (10.73 g, 57.01 mmol) were
combined in a 2 L, three-neck round-bottom flask equipped
with a thermometer and an addition funnel and placed under
N2. THF (570 mL) was added, and the mixture was cooled
to 0 ꢀC. NaHMDS (119.7 mL, 119.7 mmol, 1 M in THF) was
added dropwise via the addition funnel over 20 min (the internal
temperature was maintained below 5 ꢀC). After the addition was
complete, the reaction mixture was stirred at 0 ꢀC for an
additional 15 min. The reaction was quenched by the addition
of saturated aqueous NH4Cl solution (60 mL). The mixture was
transferred to a separatory funnel containing water (400 mL),
and the aqueous layer was extracted with ether (3 ꢀ 300 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 afford 17j (22.14 g, 94% yield) as a pale-yellow
solid, which was subsequently recrystallized from heptane to
(R)-5-Chloro-1-(1-cyclopropylethyl)-3-[6-(difluoromethoxy)-
2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one (17f). Com-
pound 17f was prepared according to the procedure described
for the synthesis of 17a using (R)-3,5-dichloro-1-(1-cyclopropyl-
ethyl)pyrazin-2(1H)-one (15) (50 mg, 0.210 mmol) and 35 (48 mg,
0.210 mmol). The product was purified by column chromato-
graphy to afford 17f (32 mg, 39% yield) as a colorless solid:
1
[R]25 -8.1 (c 0.136, CHCl3). H NMR (400 MHz, CDCl3) δ
D
8.39 (s, 1H), 8.03 (s, 1H), 7.46 (t, J=73.8 Hz, 1H), 6.88 (s, 1H),
4.24-4.20 (m, 1H), 2.44 (s, 3H), 2.26 (s, 3H), 1.43 (d, J=6.8 Hz,
3H), 1.11-1.06 (m, 1H), 0.78-0.72 (m, 1H), 0.58-0.53 (m, 1H),
0.50-0.44 (m, 1H), 0.36-0.31 (m, 1H). HRMS (ESI) m/e
385.1232 [(M þ H)þ, calcd for C17H20N4O2ClF2 385.1243].
HPLC method A: tR=6.18 min, 95.7%; method B: tR=3.82 min,
96.3%.
furnish colorless needles: mp 103.4-104.4 ꢀC; [R]25 -41.9 (c
D
0.807, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 8.02
(s, 1H), 7.46 (t, J=74.0 Hz, 1H), 6.96 (s, 1H), 4.19-4.14 (m, 1H),
3.75 (dd, JAB=10.3, JAX=6.3 Hz, 1H), 3.67 (dd, JBA=10.3, JBX
=3.5 Hz, 1H), 3.34 (s, 3H), 2.44 (s, 3H), 2.26 (s, 3H), 1.32-1.26
(m, 1H), 0.81-0.74 (m, 1H), 0.63-0.54 (m, 1H), 0.52-0.47 (m,
1H), 0.36-0.29 (m, 1H). HRMS (ESI) m/e 415.1360 [(M þ H)þ,
calcd for C18H22N4O3ClF2 415.1349]. Anal. (C18H21N4O3ClF2)
C, H, N.
(R)-5-Chloro-1-(1-cyclopropylpropyl)-3-(6-(difluoromethoxy)-
2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-one (17g). Com-
pound 17g was prepared according to the procedure described
for the synthesis of 17a using (R)-3,5-dichloro-1-(1-cyclopropyl-
propyl)pyrazin-2(1H)-one (15) (30 mg, 0.12 mmol) and 35
(26 mg, 0.12 mmol). The product was purified by column
chromatography to afford 17g (23 mg, 48% yield) as a
1
colorless solid: [R]25 -6.7 (c 0.173, MeOH). H NMR (400
D
MHz, CDCl3) δ 8.43 (s, 1H), 8.06 (s, 1H), 7.46 (t, J =
73.8 Hz, 1H), 6.79 (s, 1H), 4.05-3.98 (m, 1H), 2.44 (s, 3H),
2.26 (s, 3H), 1.96-1.86 (m, 1H), 1.80 (dt, J=14.9, 7.5 Hz, 1H),
1.10-1.01 (m, 1H), 0.92 (t, J=7.4 Hz, 3H), 0.82-0.73 (m, 1H),
0.55-0.46 (m, 2H), 0.32-0.24 (m, 1H). HRMS (ESI) m/e
399.1382 [(M þ H)þ, calcd for C18H22N4O2ClF2 399.1399].
HPLC method A: tR =6.36 min, >99%; method B: tR =
4.61 min, >99%.
(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-(6-methoxy-2,5-di-
methylpyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carboni-
trile (19a). To a solution of 17a (200 mg, 0.53 mmol) in DMF
(3 mL) and H2O (0.03 mL) was added zinc cyanide (62 mg,
0.53 mmol). Nitrogen was bubbled into the reaction mixture
for 5 min. Pd2(dba)3 (146 mg, 0.16 mmol) and dppf (216 mg,
0.39 mmol) were added, and the reaction mixture was heated at
120 ꢀC for 24 h. The mixture was cooled to room temperature
and was transferred to a separatory funnel containing ethyl
acetate. The organic layer was washed with saturated aqueous
NH4Cl solution (2ꢀ) and brine, dried over MgSO4, filtered, and
(R)-5-Chloro-3-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-
ylamino]-1-(2-methoxy-1-methylethyl)pyrazin-2(1H)-one (17h).
Compound 17h was prepared according to the procedure

7662 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
concentrated. The product was purified by column chromato-
graphy on silica gel (30% ethyl acetate in hexanes) to afford 19a
(120 mg, 62% yield) as a tan solid: mp 120-121 ꢀC; [R]25 -77.8
(c 0.573, CHCl3). 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H),
7.86 (s, 1H), 7.48 (s, 1H), 4.14 (ddd, J=10.1, 5.1, 3.1 Hz, 1H),
3.92 (s, 3H), 3.73 (dd, J=10.3, 5.3 Hz, 1H), 3.65 (dd, J=10.3, 3.0
Hz, 1H), 3.34 (s, 3H), 2.41 (s, 3H), 2.18 (s, 3H), 1.41-1.29
(m, 1H), 0.86-0.76 (m, 1H), 0.68-0.58 (m, 1H), 0.52 (dq, J=
9.6, 4.8 Hz, 1H), 0.35-0.26 (m, 1H). HRMS (ESI) m/e 370.1885
[(MþH)þ, calcd for C19H24N5O3 370.1879]. Anal. (C19H23-
N5O3) C, H, N.
(R)-4-(1-Cyclopropylpropyl)-6-(6-methoxy-2,5-dimethylpyrid-
in-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile (19b).
Part 1. To a solution of (R)-3,5-dibromo-1-(1-cyclopropylpro-
pyl)pyrazin-2(1H)-one (16) (18.0 g, 54.0 mmol) and 31 (8.22 g,
54.0 mmol) in THF (200 mL) at 0 ꢀC was added NaHMDS (113
mL, 113 mmol, 1 M in THF). The reaction mixture was stirred at
0 ꢀC for 1 h. The mixture was transferred to a separatory funnel
containing saturated aqueous NaHCO3 solution (200 mL), and
the aqueous layer was extracted with ethyl acetate (3 ꢀ 250 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 (10% f 30% ethyl acetate
in hexanes) to afford (R)-5-bromo-1-(1-cyclopropylpropyl)-
3-(6-methoxy-2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-
one (18) (20.12 g, 94% yield) as a light-brown solid. An analy-
tical sample was recrystallized from cyclohexane then tritrated
with MeOH to furnish a colorless crystalline solid: mp 114.5-
115.7 ꢀC; [R]25 -9.5 (c 0.443, EtOH). 1H NMR (400 MHz,
CDCl3) δ 8.10 (s, 1H), 7.92 (s, 1H), 6.82 (s, 1H), 4.00 (q, J=8.8
Hz, 1H), 3.93 (s, 3H), 2.43 (s, 3H), 2.18 (s, 3H), 1.94-1.76
(m, 2H), 1.08-1.00 (m, 1H), 0.92 (t, J=7.3 Hz, 3H), 0.81-0.73
(m, 1H), 0.54-0.45 (m, 2H), 0.31-0.24 (m, 1H). HRMS (ESI)
m/e 407.1083 [(M þ H)þ, calcd for C18H24N4O2Br 407.1083].
Anal. (C18H23N4O2Br) C, H, N.
mixture was heated under N2 at 100 ꢀC for 1.5 h. The mixture was
then cooled to room temperature and transferred to a separatory
funnel containing saturated aqueous NaHCO3 solution (15 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 (20% ethyl acetate in
hexanes) to furnish 19c (105 mg, 80% yield) as a colorless solid:
mp 138-140 ꢀC; [R]25D -36.6 (c 0.477, CHCl3). 1H NMR (400
MHz, CDCl3) δ 8.00 (s, 1H), 7.88 (s, 1H), 7.37 (s, 1H), 4.24-4.20
(m, 1H), 3.92 (s, 3H), 2.41 (s, 3H), 2.18 (s, 3H), 1.44 (d, J=6.8 Hz,
3H), 1.10-1.06 (m, 1H), 0.82-0.78 (m, 1H), 0.61-0.57 (m, 1H),
0.51-0.48 (m, 1H), 0.36-0.31 (m, 1H). HRMS (ESI) m/e
340.1786 [(M þ H)þ, calcd for C18H22N5O2 340.1774]. HPLC
method A: tR = 5.28 min, >99%; method B: tR = 3.30 min,
>99%.
(R)-4-(1-Cyclopropylethyl)-6-(6-(difluoromethoxy)-2,5-di-
methyl-pyridin-3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbo-
nitrile (19d). Compound 19d was prepared according to the two-
step procedure described for the synthesis of 19b using (R)-3,
5-dibromo-1-(1-cyclopropylethyl)pyrazin-2(1H)-one (16) (15.0 g,
46.9 mmol) and 35 (8.8 g, 46.9 mmol). Purification by column
chromatography on silica gel (0 f 30% ethyl acetate in hexanes)
afforded 19d (12.4 g, 93% yield, 2 steps) as a pale-yellow solid.
Subsequent recrystallization from heptane and a minimal amount
of ethyl acetate afforded 19d as white needles: mp 149-150 ꢀC;
[R]25D -24.47 (c 0.509, MeOH). 1H NMR (400 MHz, CDCl3) δ
8.37 (s, 1H), 8.01 (s, 1H), 7.46 (t, J=73.5 Hz, 1H), 7.43 (s, 1H),
4.26-4.18 (m, 1H), 2.44 (s, 3H), 2.27 (s, 3H), 1.45 (d, J=6.8 Hz,
3H), 1.13-1.06 (m, 1H), 0.85-0.78 (m, 1H), 0.64-0.57 (m, 1H),
0.54-0.47 (m, 1H), 0.36-0.29 (m, 1H). HRMS (ESI) m/e376.1595
[(M
þ
H)þ, calcd for C18H20N5O2F2 376.1585]. Anal.
(C18H19N5O2F2) C, H, N.
(S)-4-(1-Cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-
2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-car-
bonitrile (19e). (S)-2-(1-Cyclopropyl-2-methoxyethylamino)ace-
tonitrile hydrochloride (14) (15.0 g, 78.7 mmol) was suspended
in anhydrous dichloromethane (300 mL) in a 1 L, three-neck
round-bottom flask equipped with an addition funnel. The
mixture was cooled to -60 ꢀC and oxalyl bromide (41.3 mL,
440 mmol) was added dropwise over 15 min. After addition was
complete, the cooling bath was removed and the reaction
mixture was allowed to warm to room temperature and was
then heated at 40 ꢀC for 3 h. The mixture was cooled to room
temperature and concentrated under vacuum. The residue was
transferred directly onto a silica gel column in a fume hood
(caution: this purification should be performed in a fume hood
as some gas evolution occurred as residual oxalyl bromide
decomposed) and was eluted (5% f 20% ethyl acetate in
hexanes) to give (S)-3,5-dibromo-1-(1-cyclopropyl-2-methox-
yethyl)pyrazin-2(1H)-one (16) (15.5 g, 56% yield) as an off-
white solid: mp 98.5-100.5 ꢀC; [R]25D -71.8 (c 1.19, CHCl3). 1H
NMR (400 MHz, CDCl3) δ 7.61 (s, 1H), 4.08-4.04 (m, 1H), 3.72
(dd, JAB=10.5, JAX=4.5 Hz, 1H), 3.61 (dd, JBA=10.3, JBX=3.0
Hz, 1H), 3.32 (s, 3H), 1.41-1.36 (m, 1H), 0.82-0.76 (m, 1H),
0.65-0.59 (m, 1H), 0.54-0.48 (m, 1H), 0.32-0.27 (m, 1H).
LRMS (APCI) m/e 351.1 [(M þ H)þ, calcd for C10H13N2O2Br2
350.9].
(S)-3,5-Dibromo-1-(1-cyclopropyl-2-methoxyethyl)pyrazin-
2(1H)-one (16) from the above procedure (18.1 g, 51.7 mmol)
and 35 (9.70 mg, 51.7 mmol) were combined in a 2 L, three-
necked round-bottom flask equipped with a thermometer and
an addition funnel and placed under N2. THF (360 mL) was
added and the mixture was cooled to 0 ꢀC. NaHMDS (109 mL,
109 mmol, 1 M in THF) was added dropwise via the addition
funnel over 15 min (the internal temperature was maintained
below 5 ꢀC). After the addition was complete, the reaction
mixture was stirred at 0 ꢀC for an additional 30 min. The
reaction was quenched by the addition of saturated aqueous
NH4Cl solution (120 mL). The mixture was transferred to a
Part 2. To a solution of (R)-5-bromo-1-(1-cyclopropylpro-
pyl)-3-(6-methoxy-2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-
one (18) (16.0 g, 39.3 mmol) from above in DMF (200 mL) and
H2O (2.0 mL) was added zinc cyanide (4.60 mg, 39.3 mmol).
Nitrogen was bubbled into the reaction mixture for 10 min.
Pd2(dba)3 (0.83 g, 2.00 mmol) and dppf (2.60 g, 4.70 mmol) were
added, and the reaction mixture was heated at 120 ꢀC for 7 h.
The mixture was cooled to room temperature, filtered through
celite, and the filtrate was transferred to a separatory funnel
containing ethyl acetate. The organic layer was washed with
saturated aqueous NH4Cl solution (2ꢀ) and brine, dried over
MgSO4, filtered, and concentrated. The product was purified by
column chromatography on silica gel (10% f 30% ethyl acetate
in hexanes) to afford a pale-green solid, which was subsequently
recrystallized from cyclohexane to furnish 19b (5.50 g). Mixed
fractions from the column chromatography, and the filtrate
from the recrystallization were combined, repurified by column
chromatography, and recrystallized from cyclohexane to give an
additional 6.65 g of product. A total of 12.15 g (88% yield) of
19b was isolated as a pale-yellow crystalline solid: mp 150.2-
1
151.2 ꢀC; [R]25 -23.0 (c 0.364, EtOH). H NMR (400 MHz,
CDCl3) δ 8.05 (s, 1H), 7.91 (s, 1H), 7.28 (s, 1H), 4.04-3.98 (m,
1H), 3.93 (s, 3H), 2.42 (s, 3H), 2.18 (s, 3H), 2.02-1.77 (m, 2H),
1.09-1.01 (m, 1H), 0.93 (t, J=7.5 Hz, 3H), 0.88-0.79 (m, 1H),
0.58-0.49 (m, 2H), 0.29-0.24 (m, 1H). HRMS (ESI) m/e
354.1925 [(M þ H)þ, calcd for C19H24N5O2 354.1930]. Anal.
(C19H23N5O2) C, H, N.
(R)-4-(1-Cyclopropylethyl)-6-(6-methoxy-2,5-dimethylpyridin-
3-ylamino)-5-oxo-4,5-dihydropyrazine-2-carbonitrile (19c). To a
solution of (R)-5-bromo-1-(1-cyclopropylethyl)-3-(6-methoxy-
2,5-dimethylpyridin-3-ylamino)pyrazin-2(1H)-one (18) (153 mg,
0.389 mmol), prepared according to the procedure described in
Part 1 for the preparation of 19b using appropriate starting
materials in DMF (1 mL) was added zinc cyanide (46 mg, 0.389
mmol) and Pd(PPh3)4 (135 mg, 0.117 mmol) and the reaction

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7663
separatory funnel containing water (100 mL), and the aqueous
layer was extracted with ethyl acetate (3ꢀ250 mL). The com-
bined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated to afford (S)-5-bromo-
1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluoromethoxy)-2,5-di-
methylpyridin-3-ylamino]pyrazin-2(1H)-one (18) (22.40 g, 95%
yield) as a brown solid, which was used in the next step without
further purification. 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H),
7.98 (s, 1H), 7.53 (s, 1H), 7.46 (t, J=73.7 Hz, 1H), 4.16-4.12
from heptane to furnish a colorless solid: mp 131-132 ꢀC;
[R]25D þ56.8 (c 0.264, CHCl3). 1H NMR (400 MHz, CDCl3) δ
8.40 (s, 1H), 7.99 (s, 1H), 7.54 (s, 1H), 7.46 (t, J=73.7 Hz, 1H),
4.14 (ddd, J=10.3, 5.2, 3.1 Hz, 1H), 3.73 (dd, J=10.3, 5.3 Hz,
1H), 3.66 (dd, J=10.3, 3.0 Hz, 1H), 3.34 (s, 3H), 2.43 (s, 3H),
2.27 (s, 3H), 1.41-1.31 (m, 1H), 0.87-0.78 (m, 1H), 0.68-0.59
(m, 1H), 0.57-0.49 (m, 1H), 0.35-0.27 (m, 1H). HRMS (ESI)
m/e 406.1692 [(M þ H)þ, calcd for C19H22N5O3F2 406.1691].
Anal. (C19H21N5O3F2) C, H, N.
(m, 1H), 3.74 (dd, JAB=10.5, JAX=5.2 Hz, 1H), 3.66 (dd, JBA
=
(S)-2-Cyclopropyl-2-[(S)-1-phenylethylamino]ethanol (23). To
a solution of 2241 (400 g, 1.82 mol) in dry THF (5.2 L) at 0 ꢀC was
added borane dimethylsulfide (neat) (485 g, 6.39 mol) with
vigorous stirring. The reaction mixture was stirred overnight
at room temperature. Reaction progress was monitored by
HPLC. Stirring was continued until the acid was consumed
completely (18-20 h). Upon completion, the reaction mixture
was cooled to 0 ꢀC and methanol (6 L) was added dropwise. The
mixture was concentrated under vacuum, and the residue was
dissolved in chloroform (5 L). The organic layer was washed
with 10% aqueous NaHCO3 solution (2 ꢀ 1 L) followed by
brine, dried over Na2SO4, filtered, and concentrated to afford a
tan-yellow oil. The oil was distilled under reduced pressure to
10.3, JBX=3.3 Hz, 1H), 3.35 (s, 3H), 2.44 (s, 3H), 2.27 (s, 3H),
1.39-1.34 (m, 1H), 0.85-0.79 (m, 1H), 0.66-0.62 (m, 1H),
0.56-0.51 (m, 1H), 0.34-0.29 (m, 1H). HRMS (ESI) m/e
459.0864 [(M þ H)þ, calcd for C18H22N4O3BrF2 459.0843].
(S)-5-Bromo-1-(cyclopropyl-2-methoxyethyl)-3-[6-(difluo-
romethoxy)-2,5-dimethylpyridin-3-ylamino]pyrazin-2(1H)-one
(18) from the above procedure (22.40 g, 48.8 mmol) was
dissolved in dimethylformamide (480 mL) and water (24 mL)
at room temperature. Nitrogen was bubbled through the reac-
tion mixture for 15 min. Zinc cyanide (6.00 g, 51.0 mmol),
Pd2(dba)3 (2.23 g, 2.40 mmol), and dppf (3.24 g, 5.85 mmol)
were added and the reaction mixture was heated at 120 ꢀC for 3
h. The reaction mixture was cooled and filtered through a pad of
celite. The filtrate was concentrated in vacuo, and the residue
was transferred to a separatory funnel containing saturated
aqueous NH4Cl solution (200 mL). The aqueous layer was
extracted with ethyl acetate (3 ꢀ 400 mL). The combined
organic layers were washed with brine (300 mL), dried over
MgSO4, filtered, and concentrated. Purification by column
chromatography on silica gel (70% f 100% CH2Cl2 in hexanes
then 2% f 5% ethyl acetate in CH2Cl2) afforded 19e (14.23 g,
72% yield) as a pale-yellow solid. Recrystallization from a
mixture of ethanol:2-butanol (10:1) afforded 19e as pale-yellow
needles: mp 146.8-147.7 ꢀC; [R]25D -63.7 (c 0.486, CHCl3). 1H
NMR (400 MHz, CDCl3) δ 8.40 (s, 1H), 7.98 (s, 1H), 7.53
(s, 1H), 7.46 (t, J=73.5 Hz, 1H), 4.16-4.12 (m, 1H), 3.74 (dd,
afford 23 (166 g, 44% yield) as a colorless oil: bp 175-184 ꢀC,
1
0.1 mmHg; [R]25 -52.1 (c 1.0, CHCl3). H NMR (400 MHz,
D
CDCl3) δ 7.33-7.20 (m, 5H), 3.91-3.86 (m, 1H), 3.67 (dd, JAB
=
10.6, JAX=3.8 Hz, 1H), 3.39 (dd, JBA=10.6, JBX=4.3 Hz, 1H),
2.40 (s br, 2H), 1.70-1.65 (m, 1H), 1.35 (d, J=6.6 Hz, 3H),
0.88-0.82 (m, 1H), 0.47-0.37 (m, 2H), 0.05- -0.06 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 146.8, 127.6, 126.2, 125.9,
63.0, 60.5, 54.4, 24.4, 13.3, 3.0, 1.5; LRMS (ESI) m/e 206.3 [(M þ
H)þ, calcd for C13H20NO 206.2].
(S)-1-Cyclopropyl-2-methoxy-N-[(S)-1-phenylethyl]ethana-
mine (24). To a solution of 23 (29.2 g, 0.143 mol) in THF (700
mL) at 0 ꢀC was added sodium hydride (6.29 g, 0.157 mol, 60%
dispersion in mineral oil). The cooling bath was removed and the
reaction mixture was allowed to warm to room temperature and
was stirred at room temperature for 30 min. Methyl iodide
(20.30 g, 0.143 mol) was then added dropwise via syringe. Some
warming occurred soon after the addition was complete. The
temperature of the reaction mixture was controlled at ∼25 ꢀC
with a water bath containing a small amount of ice. The reaction
mixture was stirred at room temperature for 4 h. The reaction
mixture was then slowly quenched with saturated aqueous
NaHCO3 solution and was transferred to a separatory funnel
containing saturated aqueous NaHCO3 solution (400 mL). The
aqueous layer was extracted with ethyl acetate (3 ꢀ 300 mL).
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated. The crude product was
purified by column chromatography on silica gel (5% MeOH
in 1:1 ethyl acetate/hexanes) to afford 24 (26.52 g, 85% yield) as
a light-brown oil: [R]25D -61.5 (c 0.72, CHCl3). 1H NMR (400
MHz, CDCl3) δ 7.30-7.25 (m, 4H), 7.20-7.16 (m, 1H), 3.98-
3.93 (q, J=6.8 Hz, 1H), 3.50 (dd, JAB=9.5, JAX=3.5 Hz, 1H),
3.35 (s, 3H), 3.34 (dd, JBA=9.5, JBX=5.8 Hz, 1H), 1.79-1.74
(m, 2H), 1.33 (d, J=6.5 Hz, 3H), 0.73-0.67 (m, 1H), 0.36-0.34
(m, 2H), 0.03- -0.06 (m, 2H). GC/MS (ESI) m/e 220.2 [(M þ
H)þ, calcd for C14H22NO 220.2].
(S)-1-Cyclopropyl-2-methoxyethanamine Hydrochloride (25).
A mixture of 24 (100 g, 458 mmol), Pd(OH)2/C (50 g, 20% on
carbon), and ethanol (1.2 L) in a Parr bottle was placed under a
H2 atmosphere (15 psi) and was shaken for 18 h. The reaction
mixture was then filtered through a pad of celite into a flask
containing 2 N HCl in Et2O (360 mL) with stirring. The resulting
filtrate was concentrated to a yellow solid, which was then
coevaporated with Et2O (500 mL). The resulting solid was dried
overnight in vacuo to give 25 (69 g, 99% yield) as a white solid:
mp 190-192 ꢀC; [R]25D þ16.3 (c 0.446, MeOH). 1H 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)
JAB=10.3, JAX=5.0 Hz, 1H), 3.67 (dd, JBA=10.4, JBX=3.0 Hz,
1H), 3.35 (s, 3H), 2.44 (s, 3H), 2.27 (s, 3H), 1.39-1.33
(m, 1H), 0.85-0.79 (m, 1H), 0.67-0.61 (m, 1H), 0.56-0.50
(m, 1H), 0.34-0.29 (m, 1H). HRMS (ESI) m/e 406.1704
[(MþH)þ, calcd for C19H22N5O3F2 406.1691]. Anal. (C19H21-
N5O3F2) C, H, N.
When the chiral amine intermediate (compound 25) was
prepared via Scheme 2, residual amounts of the undesired
enantiomer of 19e (∼97% ee) were removed by super critical
fluid chromatography (SFC) on chiral support: Chiralpak OD-
H column (5 cm ꢀ 25 cm), mobile phase=15% isopropanol/
acetonitrile (1:1) in CO2; flow rate=200 mL/min, pressure=100
bar, temperature=35 ꢀC, λ=254 nm, 5 mL of 91 mg/mL in
isopropanol/acetonitrile (1:1) per injection per 6 min. The
purified material had an optical purity >99% ee as determined
by analytical SFC: Chiralcel OD-H column (4.6 mm ꢀ 250 mm,
5 μm); mobile phase=8% ethanol in CO2; flow rate=2 mL/min
@ 150 bar; λ=215 nm; tR=6.5 min. Compound 19e was then
recrystallized from anhydrous 2-butanol.
(R)-4-(1-Cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-
2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-car-
bonitrile (19f). Nitrogen was bubbled into a mixture of 17i (500
mg, 1.21 mmol), zinc cyanide (143 mg, 1.21 mmol), and zinc dust
(39 mg, 0.604 mmol) in DMF (25 mL) and H2O (0.3 mL) for
5 min. Pd2(dba)3 (332 mg, 0.362 mmol) and dppf (335 mg,
0.604 mmol) were added, and the reaction mixture was heated at
120 ꢀC for 5 h. The mixture was cooled to room temperature and
was transferred to a separatory funnel containing saturated
aqueous NH4Cl solution. The aqueous layer was extracted with
ethyl acetate (3 ꢀ 25 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 afford 19f (400 mg, 82% yield)
as an off-white solid, which was subsequently recrystallized

7664 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
δ 72.1, 59.2, 57.5, 10.7, 4.2, 4.1. LRMS (ESI) m/e 231.2 [(2 Mþ
H)þ, calcd for C12H27N2O2 231.2].
recrystallized from 10% ethyl acetate in hexanes (300 mL) and
air-dried to give 28 (139 g, 92% yield) as a white solid, which was
used in the next step without further purification. 1H NMR (300
MHz, CDCl3) δ 9.04 (d, J=2.5 Hz, 1H), 8.33 (d, J=2.2 Hz, 1H),
2.50 (s, 3H).
3-Methyl-5-nitropyridin-2-ol (27). (Caution: Careful tempera-
ture control must be maintained while performing the following
nitration procedure to avoid the risk of an uncontrolled exother-
mic reaction. A detailed procedure follows.) A three-necked, 2
L, round-bottomed flask equipped with a mechanical stirrer, an
addition funnel, and a thermometer was placed in an ice-water
bath. Conc H2SO4 (150 mL) was added to the flask. 2-Amino-3-
methylpyridine (26) (50.0 g, 0.463 mol, Lancaster, CAS 1603-40-
3, mp 29 ꢀC, prewarmed in a warm water bath to melt it) was
weighed out in a 125 mL Erlenmeyer flask and was subsequently
added in small portions. (The Erlenmeyer flask was kept in a
warm water bath during the addition to prevent the starting
material from solidifying.) The temperature rose to ca. 45 ꢀC
during the addition, and white smoke/fog formed within the
flask. The Erlenmeyer flask was rinsed with conc H2SO4 (100 mL),
which was then added to the reaction flask. The resulting
mixture was a milky-white suspension. A solution of conc
H2SO4 (35 mL) and 70% nitric acid (35 mL) was premixed with
ice-water bath cooling and transferred into the addition funnel.
After the internal temperature of the reaction mixture had
cooled to 10-15 ꢀC (but not below 10 ꢀC), the premixed
H2SO4/HNO3 acid mixture was added dropwise at a rate such
that the internal reaction temperature rose to 20-25 ꢀC (5-10
min addition time). After the addition was complete, the ice-
water bath was replaced with a tap water bath. The reaction
temperature slowly increased to ca. 30 ꢀC and then cooled down
to room temperature. The reaction should be monitored during
this time to ensure that the temperature does not rise too high.
The reaction mixture was then stirred overnight at room tem-
perature. 70% Nitric acid (35 mL) was then added dropwise via
the addition funnel to the dark red-brown mixture at a rate of
addition such that the temperature did not exceed 35 ꢀC. During
this time, the reaction flask was in a room temperature water
bath. Water (500 mL) was then added to the reaction flask in
portions via the addition funnel. The first ∼150 mL of water was
added dropwise while allowing the internal temperature to climb
slowly to 50-60 ꢀC. The rate of stirring was increased in order to
break up any foaming that occurred. Brown gas evolved during
the addition of this initial ∼150 mL of water. The remaining
∼350 mL of water was added at a faster rate as the gas evolution
had stopped and a temperature increase was no longer observed.
The reaction turned from a dark cloudy brown to a clear-orange
solution. Some yellow precipitate may form as the reaction cools
to below 50 ꢀC. The water bath was then removed and replaced
with a heating mantle, and the addition funnel was replaced with
a condenser. The reaction mixture (a light-orange or bright-
yellow solution) was then heated at 115-118 ꢀC for 1.75-2 h.
Additional gas evolution occurred at ∼115 ꢀC during this time.
The reaction mixture was then cooled to room temperature with
the aid of an ice-water bath and was then cooled further to 0 ꢀC
by adding ice directly into the reaction mixture. The solid that
formed was collected on a Buchner funnel and was washed with
cold water followed by a minimal amount of cold ethanol
followed by a minimal amount of cold ether. The solid was then
dried under vacuum to afford 27 (53.5 g, 75% yield) as a pale
yellow solid: mp 228-229 ꢀC. 1H NMR (400 MHz, DMSO-d6) δ
12.55 (s, br, 1H), 8.54 (d, J=3.0 Hz, 1H), 8.04 (d, J=2.0 Hz, 1H),
2.04 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 162.2, 135.4, 130.0,
129.5, 128.1, 15.8. LRMS (ESI) m/e 152.96 [(M - H)-, calcd for
C6H5N2O3, 153.03]. Anal. (C6H6N2O3) C, H, N.
2-Methoxy-3-methyl-5-nitropyridine (29). Compound 28 (139
g, 0.806 mol) from the above procedure was divided into two
portions and placed in two 2 L round-bottom flasks with
methanol (500 mL). The solutions were cooled in dry ice/
isopropyl alcohol baths as solid sodium methoxide (26.5 g,
0.467 mol) was added portionwise to each flask such that the
temperature remained below 20 ꢀC. When the additions were
complete, the resulting mixtures were heated to reflux for 1 h.
The mixtures were cooled and diluted with ice-water (500 mL)
to give white precipitates, which were collected by filtration. The
combined filtrates were washed with water and air-dried to give
1
29 (127 g, 97% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 8.91 (d, J=2.0 Hz, 1H), 8.16 (s, 1H), 4.06 (s, 3H), 2.25
(s, 3H). 13C NMR (100 MHz, CDCl3) δ 165.8, 141.9, 139.4,
132.9, 121.8, 54.8, 15.8. An analytical sample was recrystallized
from hexane to give white needles, mp 95-96.5 ꢀC. Anal.
(C7H8N2O3) C, H, N.
2-(Dibromomethyl)-6-methoxy-5-methyl-3-nitropyridine (30).
To a three-neck, 22 L round-bottom flask equipped with a
mechanical stirrer and an addition funnel was added potassium
t-butoxide (450 g, 4.00 mol) and THF (4 L). The mixture was
stirred vigorously, and a solution of 29 (100 g, 0.595 mol) and
bromoform (195.4 g, 0.773 mol) in 500 mL of dry THF was
added dropwise at such a rate that the temperature did not rise
above -74 ꢀC (3 h addition time). The reaction mixture was
stirred at -78 ꢀC for an additional 15 min. The reaction mixture
was then quenched by the dropwise addition of a mixture of
methanol (400 mL) and conc HCl (600 mL) while maintaining
the temperature below -68 ꢀC. Water (1 L) was then added, and
the aqueous layer was extracted with ethyl acetate (3 ꢀ 4 L). The
combined organic layers were washed with brine, dried over
MgSO4, filtered, and concentrated. The product was purified by
column chromatography on silica gel (0% f 7% ethyl acetate in
hexanes) to give 30 (190.0 g, 94% yield) as a red-brown solid:
mp 58.7-61.2 ꢀC. 1H NMR (300 MHz, CDCl3) δ 8.04 (s, 1H),
7.60 (s, 1H), 4.15 (s, 3H), 2.26 (s, 1H). LRMS (ESI) m/e 339.0
[(M þ H)þ, calcd for C8H9N2O3Br2, 338.9].
6-Methoxy-2,5-dimethylpyridin-3-amine (31). To a solution of
30 (110 g, 0.323 mol) in EtOH (1.1 L) in a Parr bottle was added
triethylamine (135 mL, 0.970 mol) followed by 10% Pd/C (11.0
g). The mixture placed on a Parr shaker under a hydrogen
atmosphere at 45 psi for 18 h. The reaction mixture was filtered
through celite, and the filtrate was concentrated. The residue
was partitioned between water and ethyl acetate, and the
organic layer was washed with water, brine, dried over MgSO4,
filtered, and concentrated. The residue was purified by column
chromatography on silica gel (10% f 50% ethyl acetate in
hexanes) to give 31 (35.7 g, 73% yield) as a light-brown solid:
mp 39.8-41 ꢀC. 1H NMR (400 mHz, DMSO-d6) δ 6.82 (s, 1H),
4.37 (s, 2H), 3.73 (s, 3H), 2.16 (s, 3H), 2.00 (s, 3H). 13C NMR
(100 MHz, DMSO-d6) δ 152.7, 135.8, 134.8, 126.3, 116.1, 52.1,
19.2, 14.7. LRMS (ESI) m/e 153.2 [(M þ H)þ, calcd for
C8H13N2O, 153.1].
2-Methoxy-3,6-dimethyl-5-nitropyridine (32). Method
A
(three steps): A solution of 29 (68.8 g, 409 mmol) and tert-butyl
2-chloroacetate (77.0 g, 511 mmol) in THF (1 L) was stirred and
cooled to -20 ꢀC in a dry ice/isopropyl alcohol bath. Potassium
tert-butoxide (115 g, 1.02 mol) was added at a rate such that the
reaction temperature was less than -10 ꢀC. The reaction
mixture turned dark purple. When the addition was complete,
the cooling bath was removed and the reaction was stirred for
30 min. The stirred reaction mixture was quenched with HCl
(500 mL, 2.4 N). The purple solution turned pale yellow, and the
mixture separated into two layers. The organic layer was
separated, washed three times with brine, and concentrated in
2-Chloro-3-methyl-5-nitropyridine (28). Compound 27 (134 g,
0.872 mol) was divided into three portions and placed in three 1
L round-bottom flasks. Phosphorus oxychloride (200 mL) was
added to each flask, and the mixtures were heated at reflux for
2 h. The solutions were cooled, and the excess phosphorus oxy-
chloride was removed in vacuo. The residues were poured into
ice water (1 L) with stirring and the precipitate was collected by
filtration and air-dried for 20 min. The combined products were

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7665
vacuo. Hexane was added to the amber residue. The mixture was
concentrated in vacuo and then dried under high vacuum to give
tert-butyl 2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)acetate
(83.4 g, 72% yield) as a tan solid. An analytical sample was re-
crystallized from hexane to give white needles: mp 71-72.5 ꢀC.
1H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H), 4.09 (s, 2H), 4.02
(s, 2H), 2.21 (s, 3H), 1.44 (s, 9H). 13C NMR (100 MHz, CDCl3)
δ 168.8, 163.6, 147.9, 139.4, 135.2, 120.8, 81.7, 54.7, 44.5, 28.0,
15.3. Anal. (C13H18N2O5) C, H, N.
A solution of tert-butyl 2-(6-methoxy-5-methyl-3-nitropyri-
din-2-yl)acetate from the previous step (83.0 g, 294 mmol) in
trifluoroacetic acid (200 mL) was heated in a hot water bath for
1 h. The solution was concentrated in vacuo to give a brown oil.
The oil was diluted with hexane and stirred. The resulting solid
was collected by filtration and air-dried to give 2-(6-methoxy-5-
methyl-3-nitropyridin-2-yl)acetic acid (62.8 g, 94% yield) as a
tan solid. An analytical sample was recrystallized from hexane:
mp 135-137 ꢀC. 1H NMR (400 MHz, CDCl3) δ 8.70 (s br, 1H),
8.20 (s, 1H), 4.25 (s, 2H), 4.03 (s, 3H), 2.24 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ 175.6, 163.8, 146.5, 139.2, 135.3, 121.5,
54.86, 42.9, 15.3. Anal. (C9H10N2O5) C, H, N.
A mixture of 2-(6-methoxy-5-methyl-3-nitropyridin-2-yl)-
acetic acid from the previous step (62.5 g, 276 mmol), potassium
carbonate (20.0 g, 145 mmol), and DMF (100 mL) was heated to
90 ꢀC with stirring in a hot water bath for 1 h. Gas evolution
was noted during the heating period and had ceased after 1 h.
The mixture was poured into stirred ice water (600 mL) with
washing of the reaction flask with a small volume of acetone.
The resulting precipitate was collected by filtration and air-dried
to give 32 (48.5 g, 96% yield) as a tan solid. An analytical sample
was recrystallized from hexanes to give tan needles: mp 85.9-
90.5 ꢀC. 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 4.02 (s, 3H),
2.76 (d, 3H), 2.19 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 163.4,
151.6, 139.4, 135.0, 119.4, 54.5, 24.2, 15.2. Anal. (C8H10N2O3)
C, H, N.
[(M þ H)þ, calcd for C7H9N2O3 169.1]. Anal. (C7H8N2O3)
C, H, N.
2-(Difluoromethoxy)-3,6-dimethyl-5-nitropyridine (34). Meth-
od A: Sodium hydride (6.63 g, 166 mmol, 60% in mineral oil)
was washed with hexanes (100 mL) to remove the mineral oil
and was then suspended in dry acetonitrile (1500 mL) at room
temperature. Compound 33 (27.9 g, 166 mmol) was added in
portions over 30 min to give a yellow suspension. During the
addition, there was some bubbling but negligible temperature
change. Cesium fluoride (2.50 g, 16.6 mmol) was then added
followed by the slow addition of trimethylsilyl 2,2-difluoro-
2-(fluorosulfonyl)acetate (36.0 mL, 182 mmol) over 30 min.
(Caution: trimethylsilyl 2,2-difluoro-2-(fluorosulfonyl)acetate
can cause skin damage and should always be handled with
gloves in a fume hood.) During the addition there was some
bubbling, the temperature rose from 23 to 30 ꢀC, and the
suspension became noticeably less turbid. After stirring for
15 min, TLC indicated that starting material still remained, so
additional trimethylsilyl 2,2-difluoro-2-(fluorosulfonyl)acetate
(6.5 mL, 33 mmol) was added over 10 min. After an additional
15 min, TLC indicated consumption of starting material. The
reaction was quenched by the addition of water (20 mL) drop-
wise at such a rate that the bubbling did not become too
vigorous. After bubbling ceased, additional water (200 mL)
was added. Most of the solvent was removed in vacuo, and
the aqueous residue was extracted with ethyl acetate (3 ꢀ 200
mL). The combined organic layers were dried over Na2SO4 and
evaporated to a brown syrup which solidified upon standing.
This residue was dissolved in ethanol (400 mL), and decolorizing
charcoal (15 g) was added. The suspension was heated at 70 ꢀC
for 20 min and then filtered through a pad of celite and sand. The
filtrate was collected, and the solvent was evaporated. The
residue was dissolved in methylene chloride, and the solution
was evaporated to give 34 (33.4 g, 92% yield) as a pale-yellow
solid identical to that prepared by method B: mp 51-52 ꢀC. 1H
NMR (400 MHz, CDCl3) δ 8.21 (s, 1 H), 7.55 (t, J=72.0 Hz, 1
H), 2.76 (s, 3 H), 2.30 (s, 3 H). Anal. (C8H8N2O3F2) C, H, N.
2-(Difluoromethoxy)-3,6-dimethyl-5-nitropyridine (34). Meth-
od B: To a suspension of 33 (700 mg, 4.17 mmol) in acetonitrile
(70 mL) was added sodium hydride (450 mg, 11.3 mmol, 60% in
mineral oil). After stirring at room temperature for 15 min, 2,2-
difluoro-2-(fluorosulfonyl)acetic acid (0.73 mL, 7.09 mmol) was
added dropwise over several minutes. (Caution: 2,2-difluoro-
2-(fluorosulfonyl)acetic acid can cause skin damage and should
always be handled with gloves in a fume hood.) Some bubbling
occurred during the addition. After stirring the reaction mixture
at room temperature for 15 min, the reaction was quenched by
the slow addition of water (10 mL). The acetonitrile was
removed in vacuo, and the residue was transferred to a separa-
tory funnel containing water (50 mL). The aqueous layer was
extracted with ethyl acetate (3 ꢀ 50 mL). The combined organic
layers were washed with brine, dried over MgSO4, filtered, and
concentrated. The residue was purified by column chromatog-
raphy on silica gel (10% ethyl acetate in hexanes) to afford 34
(870 mg, 96% yield) as a colorless solid: mp 48-49 ꢀC. 1H NMR
(400 MHz, CDCl3) δ 8.21 (s, 1 H), 7.54 (t, J=72.4 Hz, 1 H), 2.76
(s, 3 H), 2.30 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ 158.1,
151.0, 142.0, 137.0, 120.0, 113.9 (t, J=255.8 Hz), 23.5, 14.7.
6-(Difluoromethoxy)-2,5-dimethylpyridin-3-amine (35). To a
solution of 34 (33.4 g, 153 mmol) in methylene chloride (100 mL)
and ethanol (600 mL) was added 10% palladium on carbon (3.3 g).
The resulting suspension was hydrogenated on a Parr shaker
at 40 psi H2 for 1 h. TLC was used to monitor the reaction. An
additional 3.3 g of palladium on carbon was added hourly until
no starting material remained. A total of 13.2 g of Pd/C was
added. The reaction mixture was kept under an H2 atmosphere
for 2 h after the last addition of catalyst. The reaction mixture
was filtered through celite and sand and the collected solids were
washed with ethyl acetate (2ꢀ100 mL). The filtrate was con-
centrated in vacuo to give a gray oil, which was purified by
2-Methoxy-3,6-dimethyl-5-nitropyridine (32). Method B: Di-
methyl sulfoxide (35 mL) was added to a dry round-bottomed
flask containing sodium hydride (1.82 g, 45.5 mmol, 60% in
mineral oil). The resulting suspension was heated at 70 ꢀC for
35 min, during which time the suspension became a solution. The
reaction mixture was cooled to room temperature, trimethyl-
sulfoxonium iodide (10.0 g, 45.5 mmol) was added, and the
mixture was stirred at room temperature for 30 min. Compound
29 (4.50 g, 26.80 mmol) was added, and the resulting dark-red
solution was stirred at room temperature for 30 min, at which
time TLC showed complete consumption of starting material.
The reaction mixture was transferred to a separatory funnel
containing water (30 mL), and the aqueous layer was extracted
with ethyl acetate (3 ꢀ 100 mL). The combined organic layers
were washed with brine, dried over MgSO4, filtered, and con-
centrated. The residue was purified by column chromatography
on silica gel (20% ethyl acetate in hexanes) to afford 32 (2.00 g,
41% yield) as a colorless solid identical to that prepared by
1
Method A: mp 85.5-86.2 ꢀC. H NMR (400 MHz, CDCl3) δ
8.08 (s, 1H), 4.02 (s, 3H), 2.77 (s, 3H), 2.20 (s, 3H).
3,6-Dimethyl-5-nitropyridin-2-ol (33). A solution of 32 (32.3 g,
182 mmol) in conc hydrochloric acid (300 mL) was heated at
100 ꢀC for 1 h. Analysis by TLC indicated that some starting
material remained. The reaction was heated at 110 ꢀC for an
additional 45 min. The reaction mixture was cooled to room
temperature and poured onto ice (400 g). When the ice had
melted and the temperature of the resulting thick brown sus-
pension was still less than 0 ꢀC, the mixture was filtered.
The solid cake was washed with water (100 mL) and allowed
to dry on the filter for 30 min. The solid was then resuspended
in cold (-10 ꢀC) ethanol (150 mL), filtered, washed with
cold ethanol (50 mL), and air-dried on the filter for 1 h to
afford 33 (28.0 g, 94% yield) as a tan powder: mp 263 ꢀC.
1H NMR (400 MHz, DMSO-d6) δ 12.42 (s br, 1 H), 8.03
(s, 1 H), 2.61 (s, 3 H), 2.01 (s, 3 H). LRMS (ESI) m/e 169.3

7666 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Hartz et al.
column chromatography on silica gel (35% f 50% ethyl acetate
in hexanes) to furnish 35 (25.7 g, 89% yield) as a pale-yellow oil,
which solidified upon cooling in a refrigerator. The product was
recrystallized from hexanes below 0 ꢀC to afford white needles:
mp 40-42 ꢀC. 1H NMR (400 MHz, CDCl3) δ 7.35 (t, J =
74.0 Hz, 1 H), 6.84 (s, 1 H), 2.27 (s, 3 H), 2.15 (s, 3 H). LRMS
(ESI) m/e 189.2 [(MþH)þ, calcd for C8H11N2OF2 189.1]. Anal.
(C8H10N2OF2) C, H, N.
Biology. Metabolic Stability Assay. Estimates of rates of
compound turnover (pmol/min/mg protein) were generated
using pooled human and rat liver microsomes (BD Biosciences,
Woburn, MA). The stability in liver microsomes was deter-
mined by a high throughput in-house assay in which 3 μM of
compound was incubated with 1.3 mM NADPH and 1 mg/mL
of microsomal protein at 37 ꢀC. Incubations were performed in
sodium phosphate buffer (100 mM), pH 7.4 and were termi-
nated at 0 and 10 min following the start of the incubations. The
final organic concentration of solvent was 0.015% DMSO and
0.985% acetonitrile. Compound concentrations were deter-
mined by LC/MS/MS.
Protein Binding Studies. Unbound fraction in human serum
was determined in vitro by equilibrium dialysis using the
Dianorm dialysis system or HTDialysis LLC HTD 96B system.
Human serum was spiked with compound and equilibrated
against isotonic phosphate buffer for 3 or 7 h at 37 ꢀC for the
different systems, respectively. Following the incubation period,
plasma and buffer samples were analyzed for compound con-
centrations using LC/MS/MS. Unbound fraction was calcu-
lated based on the ratio between buffer concentration and the
serum 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% polysorbate 80 in 0.5% methylcel-
lulose suspension or Labrafil M 1944CS/DMAC/polysorbate
80, 85:10:5 (v/v) at a volume of 3 mL/kg. Blood samples were
collected 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.
Beagle Dog Discrete Studies. Pharmacokinetic properties
were estimated in male beagle dogs (n=4) following a 2 mg/kg
intravenous dose. Intravenous doses were prepared in a vehicle
consisting of propylene glycol:ethanol, 90:10 (v/v) at a volume
of 1 mL/kg over 5 min at a constant rate of 0.2 mL/kg/min.
Pharmacokinetic properties were estimated in male beagle dogs
(fasted) following a 2 mg/kg oral dose in 0.2% polysorbate 80 in
0.5% methylcellulose suspension (n=4) or following a 3 mg/kg
oral dose in Labrafil M 1944CS/DMAC/polysorbate 80, 85:10:5
(v/v) (n=2). The dose volume was 1 mL/kg. Blood samples were
collected via a femoral vein at 0, 0.17, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8,
and 24 h postdose for the intravenous experiment and at 0, 0.25,
0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose for the oral experiments.
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 spectrometer (Micromass, Manchester, UK)
equipped with an electrospray interface operating in the positive
ionization mode. Detection of each analyte was achieved
through selected reaction monitoring.
In Vitro Metabolism Using Liver Microsomes. In vitro meta-
bolic reaction mixtures (1 mL) contained 100 mM of potassium
phosphate buffer (pH 7.4), 10 μM of a substrate, and pooled rat
or human liver microsomes (final concentration as 1 mg/mL;
BD Biosciences, Bedford, MA). The reactions were initiated by
adding NADPH-regenerating reagents (final concentrations as
1.3 mM NADPþ, 3.3 mM β-D-glucose-6-phosphate, 3.3 mM
magnesium chloride, and 0.5 unit/mL of glucose-6-phosphate
dehydrogenase) and carried out at 37 ꢀC. At the time of 0 and
30 min, an aliquot (300 μL) of reaction mixture was collected and
reactions were terminated by adding an equal volume of acet-
onitrile. Following centrifugation at 10000 rpm for 5 min using
an Eppendorf 5415C centrifuge (Hamburg, Germany), 30 μL of
the supernatant was injected into HPLC/UV/MS for metabolite
profiling. In separate glutathione trapping experiments, 5 mM
of GSH was added to the reaction mixtures while the other
reaction components and sample preparation procedures re-
mained the same mentioned above. The relative abundance of
each drug-related component detected in liver microsomal in-
cubations was estimated based upon the ratio of the UV peak
area (330-340 nm) of each particular component vs the com-
bined UV peak areas (330-340 nm) of all recovered drug-
related components at 30 min.
In Vivo Metabolism Using Bile Duct Cannulated (BDC) Rats.
All animal procedures were reviewed and approved by the
Animal Care and Use Committee at Bristol-Myers Squibb Co.
BDC rats (male Sprague-Dawley) were obtained from Hilltop
Lab Animals (Scottdale, PA). The dose was formulated in 90%
poly ethylene glycol (PEG) and 10% ethanol (v/v). Three BDC
rats were orally administered with 19e at a dose of 20 mg/kg of
body weight. Bile and urine samples were collected between 0
and 24 h, and plasma samples were collected at 0, 0.25, 0.5, 1, 2,
4, 6, 8, and 24 h. Three additional BDC rats were administered
with the dosing vehicle (orally) as a control, and bile, urine, and
plasma were collected accordingly. An aliquot (150 μL) of
pooled bile or urine samples was mixed with an equivalent
volume of acetonitrile. After precipitation by centrifugation at
10000 rpm for 5 min, the supernatant (30 μL) was injected
directly onto the HPLC/UV/MS. For plasma sample prepara-
tion, three volumes of acetonitrile were added to one volume of
pooled plasma (250 μL) to precipitate proteins. Following
centrifugation at 10000 rpm for 5 min, the supernatant was
collected and dried using a N2 stream. The residues were
redisolved in 150 μL of 50% acetonitrile and 50% H2O contain-
ing 0.1% formic acid, and a 30 μL aliquot was introduced onto
the HPLC/UV/MS for analysis. The relative abundance of each
drug-related component detected in plasma, bile, or urine
samples was estimated based upon the ratio of the UV peak
area (330-340 nm) of each particular component vs the com-
bined UV peak areas (330-340 nm) of all recovered drug-
related components in the particular matrix.
HPLC/UV/MS for Metabolite Identification. The HPLC/
UV/MS system consisted of a Waters 2695 Separations Module
(Waters, Milford, MA), which was connected with a Surveyor
PDA detector (ThermoFisher Scientific, Waltham, MA) and an
LCQ-Deca XP ion trap mass spectrometer (ThermoFisher
Scientific) equipped with an electrospray ionization source (ESI).
Separation of antagonists and their metabolites was achieved
on a Synergi Hydro-RP column (4 μm, 3 mm ꢀ 150 mm;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7667
Phenomenex, Torrance, CA) using solvent A (5% acetonitrile
and 95% H2O containing 0.1% formic acid) and solvent B
(100% acetonitrile). At a flow rate of 0.3 mL/min, the initial
gradient (98% A and 2% of solvent B) was held constant for the
first 2 min followed by a linear increase to 70% of solvent B over
the next 8 min and finally ramped up to 100% of solvent B over
an additional 15 min. The gradient was maintained for 5 min
before returning to the initial condition for a 10 min equilibra-
tion period. The eluate from the column was routed to the PDA
detector scanning from 200 to 550 nm. The mass spectrometer
was set for a full scan and data- or list-dependent scan modes
and was operated in a positive ion mode. Adjusted to achieve the
best sensitivity, the electrospray voltage was 5.0 kV, heated
capillary temperature was 350 ꢀC, sheath gas was 40 (arbitrary
unit), auxiliary gas was 90 (arbitrary unit), and normalized
collision energy (NCE for MSn) was 45%.
Metabolite Identification. UV and mass spectrometry data
were acquired and analyzed using Xcalibur version 1.3
(ThermoFisher Scientific). Metabolite ID (ThermoFisher Scien-
tific) and Mass Frontier (ThermoFisher Scientific) were also
employed to help identify metabolites according to a predicted
metabolite list derived from an in-house biotransformation
database at Bristol-Myers Squibb Co. and the metabolite pre-
diction software, Meteor (Lhasa Limited, Leeds, UK).60
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Acknowledgment. We thank Gail Mattson for performing
the in vitro binding assay, Yong Peng for performing the
functional assays, Dauh-Rurng Wu and Leslie W. Leith for
performing large scale separations on chiral support, and the
Discovery Analytical Sciences Department for obtaining
high-resolution MS analysis and optical rotations.
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Supporting Information Available: Tables of elemental ana-
lysis data and high-resolution mass spectral data with HPLC
purity data for compounds lacking elemental analysis data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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compound between octanol and water at pH=7.4.
(51) The pKa values were determined in the pH range between 2 and 11,
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pKa was measured using the spectrophotometric titration method
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(55) The relative abundance of each drug-related component detected in
plasma, bile, or urine samples was estimated based upon the ratio
of the UV peak area (330-340 nm) of each particular component vs
the combined UV peak areas (330-340 nm) of all recovered drug-
related components in the particular matrix.
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