Metabolism-directed optimization of 3-aminopyrazinone acetamide thrombin inhibitors. Development of an orally bioavailable series containing P1 and P3 pyridines

Article abstract of DOI:10.1021/jm020311f

Recent efforts in the field of thrombin inhibitor research have focused on the identification of compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe a metabolism-based approach to the optimization of the 3-(2-phen

Full text of DOI:10.1021/jm020311f

J . Med. Chem. 2003, 46, 461-473
461
Articles
Meta bolism -Dir ected Op tim iza tion of 3-Am in op yr a zin on e Aceta m id e Th r om bin
In h ibitor s. Develop m en t of a n Or a lly Bioa va ila ble Ser ies Con ta in in g P 1 a n d P 3
P yr id in es
,
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‡
Christopher S. Burgey,* Kyle A. Robinson, Terry A. Lyle, Philip E. J . Sanderson, S. Dale Lewis,
‡
‡
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§
Bobby J . Lucas, J ulie A. Krueger, Rominder Singh, Cynthia Miller-Stein, Rebecca B. White, Bradley Wong,
Elizabeth A. Lyle, Peter D. Williams, Craig A. Coburn, Bruce D. Dorsey, J ames C. Barrow,
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Maria T. Stranieri, Marie A. Holahan, Gary R. Sitko, J acquelynn J . Cook, Daniel R. McMasters,
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Colleen M. McDonough, William M. Sanders, Audrey A. Wallace, Franklin C. Clayton, Dennis Bohn,
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Yvonne M. Leonard, Theodore J . Detwiler, J r., J oseph J . Lynch, J r., Youwei Yan, Zhongguo Chen,
Lawrence Kuo, Stephen J . Gardell, J ules A. Shafer, and J oseph P. Vacca
X
‡
‡
†
Departments of Medicinal Chemistry, Biological Chemistry, Drug Metabolism, Molecular Systems, Structural Biology,
and Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486
Received J uly 17, 2002
Recent efforts in the field of thrombin inhibitor research have focused on the identification of
compounds with good oral bioavailability and pharmacokinetics. In this manuscript we describe
a metabolism-based approach to the optimization of the 3-(2-phenethylamino)-6-methylpyrazi-
none acetamide template (e.g., 1) which resulted in the modification of each of the three principal
components (i.e., P1, P2, P3) comprising this series. As a result of these studies, several potent
thrombin inhibitors (e.g., 20, 24, 25) were identified which exhibit high levels of oral
bioavailability and long plasma half-lives.
In tr od u ction
tively. The limitations associated with each of these
therapies have been well documented: the requirement
for parenteral administration of LMWH limits its
chronic utility, while the delayed onset of action,
potential for drug-drug interactions, and need for
continual monitoring of blood coagulation parameters
render warfarin a challenging drug to safely administer.
Disorders resulting from the formation of an occlusive
vascular thrombus due to improper maintenance of the
blood coagulation system represent a major cause of
mortality worldwide. Among the more prominent condi-
tions that can result from excessive coagulation are deep
vein thrombosis (DVT), pulmonary embolism (PE), and
1
Recent approaches to anticoagulant therapy have
been directed toward identifying direct small molecule
inhibitors of specific coagulation cascade enzymes3
which possess the pharmacokinetic properties required
for once daily oral administration. Assuming predictable
pharmacokinetics, the direct antithrombotic action of
these inhibitors should render routine monitoring of
coagulation levels unnecessary. A prominent target to
emerge from this effort is the trypsin-like serine pro-
thromboembolic stroke. The occurrence of DVT is most
commonly associated with, but not limited to, vascularly
invasive and incapacitating procedures such as knee or
hip replacement surgery. DVT can frequently lead to
other serious, life-threatening states such as PE, which
is often difficult to detect. The severity of this condition
is evidenced by the fact that 5-15% of U.S. hospital
deaths are the result of PE, of which 70% were unsus-
pected antemortem.2 Atrial fibrillation, a prevalent
condition in the elderly, imparts a high risk of cardio-
genic thromboembolism (due to blood stasis rather than
vascular damage) and, consequently, ischemic stroke in
the absence of anticoagulant therapy.1
4
tease thrombin (Factor IIa). Thrombin occupies a
central role in hemostasis, its primary procoagulant
actions consisting of the activation of platelets and
cleavage of fibrinogen to fibrin. Fibrin subsequently
polymerizes and is cross-linked through the action of
Factor XIIIa to comprise, along with platelets, the
primary components of a vascular thrombus. Corrobora-
tion for this antithrombotic strategy is provided by the
fact that argatroban, an intravenous thrombin inhibitor,
has been approved in the U.S. for patients susceptible
to heparin-induced thrombocytopenia.
Current therapies for the treatment of venous throm-
bosis and the prevention of cardiogenic thromboembo-
lism consist of low molecular weight heparin (LMWH)
and warfarin, in an acute and chronic setting, respec-
*
To whom correspondence should be addressed. Phone: 215-652-
2
382. Fax: 215-652-3971. E-mail: christopher_burgey@merck.com.
†
Medicinal Chemistry.
Biological Chemistry.
Drug Metabolism.
Pharmacology.
Molecular Systems.
Structural Biology.
‡
Early thrombin inhibitor development focused on the
modification of peptide structures based upon the
§
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5
⊥
sequences of known native substrates. Although this
X
approach yielded potent inhibitors indispensable in
1
0.1021/jm020311f CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/08/2003

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62 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Burgey et al.
proof-of-principle experiments, a lack of selectivity
versus homologous proteases) and/or oral bioavailabil-
Ta ble 1. Effect of P3 Benzylic Substitution on Thrombin (IIa)
and Trypsin (tryp) Inhibition (Ki) and In Vitro Anticoagulant
Potency (2xaPTT)
(
ity plagued this early class of compounds. Guided by
the wealth of enzyme structural information, impressive
advances have been made in the development of pep-
6
tidomimetic and nonpeptide thrombin inhibitors; how-
ever, many of these retain a highly basic amidine that
mimics the binding of the P1 Arg of natural peptide
substrates to Asp-189 at the base of the S1 pocket.
Although this salt-bridge interaction leads to a sub-
Ki (nM)
compd
Ar
Ph
Ph
Ph
X
IIa
0.8
tryp
2xaPTT (µM)
7
stantial amount of binding energy, the incorporation
1
2
3
4
5
6
7
8
H, H
H, Me
Me, Me
F, F
H, H
H, H
H, H
F, F
1800
1600
800
0.41
0.61
0.50
0.29
0.31
0.28
0.45
0.20
1.1
1.1
of these extremely basic moieties is generally associated
with low levels of oral bioavailability and high plasma
Ph
0.10
0.27
0.54
1.6
855
8
clearance. While a prodrug approach has been success-
2-pyr
3-pyr
4-pyr
2-pyr
3600
3800
4600
800
fully employed to produce a benzamidine analogue with
9
acceptable bioavailability (ximelagatran), the majority
0.042
of recent efforts in the field of thrombin inhibitor
development have focused on establishing compounds
that incorporate less basic P1 binding elements. These
studies have begun to produce orally bioavailable throm-
bin inhibitors, and, presumably, clinical evaluations are
in progress to determine if these candidates exhibit the
pharmacokinetic and safety profile required to displace
current anticoagulant therapies.
benzylic carbon exhibited minimal effect on thrombin
inhibition (2, 3: Ki ) 1.1 nM). Introduction of a gem-
difluoro group, however, led to an unexpected potency
enhancement in both the enzyme inhibition and 2xaPTT
clotting assay (4, Ki ) 0.10 nM, 2xaPTT ) 0.29 µM).
Replacement of the P3 phenyl ring with a pyridine ring
was well tolerated for each of the pyridine isomers: the
Recent reports from our laboratories have detailed the
development of the aminopyrazinone acetamide throm-
bin-inhibitor template, which resulted in the identifica-
tion of an efficacious, orally bioavailable compound 1
2
-pyridyl substitution (5, Ki ) 0.27 nM) improved
potency while the 3- (6, Ki ) 0.54 nM) and 4-pyridyl (7,
Ki ) 1.6 nM) isomers retained similar binding activity
relative to phenyl. The tolerance of the S3 pocket to this
change would prove to be a valuable finding, as it
presents the option of integrating a phenyl or pyridyl
P3 moiety to modulate the physicochemical properties
of the inhibitors (vide infra). Incorporation of both the
1
0
containing a moderately basic 2-aminopyridine P1.
Further study of 1 revealed three principal sites of
2
-pyridyl and gem-difluoro modifications afforded 8, a
potent thrombin inhibitor with excellent in vitro anti-
coagulant activity (Ki ) 0.042 nM, 2xaPTT ) 0.20 µM).
As a result of the excellent potency and lack of obvious
metabolic soft-spots the 2,2-difluoro-2-arylethylamino
group emerged as the optimal P3.
Due to the aforementioned metabolic issues, replace-
ment of the P1 aminopyridine group was next ad-
dressed. At the outset this was projected to be a difficult
task as the aminopyridine P1 conferred not only excel-
lent potency but also high levels of oral bioavailability
metabolism: oxidation of the P3 and P2 benzylic posi-
tions and Phase II conjugation of the P1 amino group.
1
1
Assuming that metabolism is the major route of clear-
ance, elimination of these structural areas of metabo-
lism could in turn lead to the generation of compounds
with improved pharmacokinetics. In this manuscript we
describe the metabolism-directed optimization process
which resulted in the production of a series of potent,
selective, and orally bioavailable compounds containing
weakly basic pyridine P1 and P3 moieties; ultimately,
metabolic considerations dictated the modification of
each of the three principal components (i.e., P1, P2, P3)
comprising this series.
10
to this series of inhibitors. As anticipated, excision of
the amino group from 1 to give the 2-methyl-3-pyridyl
analogue 9 resulted in a substantial loss (350-fold) in
potency due to elimination of the interaction with Asp-
1
89 (Ki ) 280 nM, Table 2). Furthermore, removal of
the methyl group from 9 led to an additional 4-fold
deterioration in thrombin binding (10, Ki ) 1200 nM).
Ta ble 2. Effect of P1 Pyridine Isomers on Thrombin (IIa) and
Trypsin (tryp) Inhibition (Ki)
Resu lts a n d Discu ssion
A study to examine the effect of P3 benzylic substitu-
tion on thrombin inhibitory potency was initiated with
the fundamental goal of sterically blocking metabolism
at this site (Table 1). The inhibition constants (Ki)
versus thrombin (IIa) and trypsin, and the concentration
needed to double the activated partial thromboplastin
time (2xaPTT) in human plasma were determined for
Ki
compd
P1
IIa (nM)
tryp (µM)
9
10
2-Me-3-pyr
3-pyr
2-pyr
280
1200
390
>100
898
1
1
1
2
>1000
4-pyr
17000
251
1
2
each compound. Simple mono- or dimethylation of the

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 463
Ta ble 3. Effect of P1 Pyridine Substitution on Thrombin (IIa)
and Trypsin (tryp) Inhibition (Ki), in Vitro Anticoagulant
Potency (2xaPTT) and Dog Pharmacokinetic Parameters
of this compound established that the P1 aminopyridine
could be replaced with a less basic, simple pyridine and
still deliver potent, efficacious, selective, and orally
bioavailable thrombin inhibitors.
With 16 serving as a template, efforts were directed
toward improving the t1/2 while maintaining potency and
efficacy (Table 3). In the absence of any information
regarding the mode of clearance, it was postulated that
oxidative metabolism was occurring at the P1 pyridine
ring nitrogen or 4-methyl group. Introduction of a
Ki
IIa
tryp
2xaPTT Cmax
(µM)
3
-fluoro group onto the P1 pyridine ring could, by
compd
X
R1 R2 (nM) (µM)
(µM)a t1/2 (h) b
reducing the pyridine basicity, diminish the potential
1
1
1
1
1
1
1
2
3
4
5
6
7
8
9
0
N
H
H
H
H
F
F
F
F
H
15
1.3
>1000
47
2.2
0.89
1.7
17
for N-oxidation. Additionally, this modification could
CH
CH
N
CH
N
Cl
Me
Me
Me
Me
H
have a simultaneous effect on the putative metabolism
4.8
2.8
1.2
1.0
6.8
4.2
270
431
40
118
501
252
1
8
of the benzylic methyl group. In practice this alteration
0.59
0.72
0.48
1.26
0.67
4.12
1.83
1.66c
3.73
5.64
2.4
0.7
1.0
2.0
3.5
produced an improvement in potency (17, Ki ) 1.2 nM;
1
8, Ki ) 1.0 nM); although the good Cmax was retained,
CH
N
the half-life was not increased and the in vivo efficacy
was compromised (18, 3/6 occlusions, final plasma
concentration ) 606 ( 43 nM). The increased potency
observed upon incorporation of the 3-fluoro substituent
permitted removal of the benzylic methyl group (19, Ki
H
a
Dose ) 1 mg/kg unless otherwise noted. b po half-life. c Dose
)
0.7 mg/kg.
Of the simple pyridine isomers, the 2-pyridyl analogue
1 displays the highest inherent binding affinity (Ki )
90 nM) and as a result this P1 was chosen for further
)
6.8 nM; 20, Ki ) 4.2 nM). In combination with the
1
3
pyridyl P3, this maneuver not only resulted in a
restoration of efficacy (20, 1/6 occlusions, 1 reflow; final
plasma concentration ) 799 ( 72 nM), but also excellent
pharmacokinetics. Upon dosing of 20 (1 mpk) to dogs,
a Cmax of 5.64 µM and a substantial improvement of the
t1/2 to 3.5 h were observed.
optimization.
Although 11 represents a 500-fold loss in thrombin
inhibition from the parent compound 1, the potency
enhancements established in P3 could potentially com-
pensate for this deficit. Combination of the optimized
,2-difluoro-2-pyridylethylamino P3 and the 2-pyridyl
P1 structural modifications exhibited an additive benefit
13: Ki ) 15 nM, 2xaPTT ) 2.2 µM; Table 3); however,
the functional potency was still not within the targeted
range (2xaPTT < 1 µM).13 In a previous study it had
been established that simple meta substitution of P1
aryl groups can access a lipophilic recess in the S1
pocket and lead to a large improvement in thrombin
inhibitory potency.14 Indeed, incorporation of this struc-
tural arrangement into the P1 pyridine in the form of a
The performance in the aforementioned assays
prompted further in vivo characterization of 20 (Table
2
4
). This inhibitor exhibited good to moderate bioavail-
(
ability in dog (F ) 87%, n ) 2), rat (F ) 55%, n ) 4)
and rhesus monkey (F ) 12%, n ) 4), although the half-
life decreased in the latter two species.
Ta ble 4. Pharmacokinetics Parameters of 20 in Dog, Rat, and
Rhesus Monkey
species po dose (mg/kg) po Cmax (µM)
iv t1/2 (h)
3.5
F (%)
87
dog
1
10
5
5.64
4
-chloropyridine afforded a compound with improved
rat
rhesus
2.16 ( 0.32 0.57 ( 0.07 55 ( 14
1.10 ( 0.86 0.99 ( 0.34 12 ( 10
potency (14, Ki ) 1.25 nM, 2xaPTT ) 0.89 µM) and
excellent trypsin selectivity. Cognizant of the inherent
liability associated with reactive 4-halopyridines, an
isosteric 4-methylpyridine was prepared. This modifica-
tion resulted in a modest loss in potency in both the
enzyme and coagulation (2xaPTT) assays (15, Ki ) 4.8
nM, 2xaPTT ) 1.7 µM). The functional activity could
be regained by implementing the previously established
strategy of incorporating the more basic P3 pyridine (16,
Ki ) 2.8 nM, 2xaPTT ) 0.59 µM). This 3-fold improve-
ment in the 2xaPTT (i.e., in vitro clot inhibition) is likely
due to the differences in inhibitor physicochemical
All of the P1 2-pyridyl containing thrombin inhibitors
listed in Table 3 possessed no significant activity versus
trypsin (>40 µM) or any other proteases assayed: t-PA,
plasmin, factor Xa, plasma kallikrein, activated protein
C, urokinase, and chymotrypsin.
Determination of the X-ray crystal structure of 20
complexed to thrombin revealed the same general
1
0
binding motif as observed with 1: the fluoropyridine
occupies the S1 specificity pocket, the methyl pyrazinone
fills the insertion loop, and the P3 aryl group binds in
the distal hydrophobic pocket (Figure 1). The antipar-
allel â-sheet hydrogen bonding motif between the ami-
nopyrazinone and Gly-216 is maintained (d ) 2.75 Å,
2.84 Å); similarly the Ser-214 H-bond to the inhibitor
amide is conserved (d ) 3.48 Å). The edge-to-face σ-π
interaction in S3 between the P3 aryl group and the
π-rich Trp-215 is further reinforced by the incorporation
of the electron-deficient P3 pyridine, providing greater
overall binding affinity versus its phenyl counterpart.
In agreement with this analysis is the improved potency
incurred upon incorporation of the gem-difluoro group
1
5
properties of 16 vs 15 (i.e., reduced lipophilicity).
As the primary objective of the current study was the
identification of thrombin inhibitors with superior phar-
macokinetics, compounds meeting our established po-
tency criteria were directly subjected to dog pharmaco-
kinetic experiments (Table 3). Upon oral dosing of 16
1 mg/kg), a 4.12 µM maximum plasma concentration
Cmax) and a 2.4 h plasma half-life (t1/2) were achieved
F ) 68%). Additionally, 16 demonstrated complete
(
(
(
efficacy in the rat FeCl3 arterial thrombosis model upon
iv dosing (0/6 occlusions, final plasma concentration )
36 ( 31 nM). The in vitro and in vivo performance
1
6
19
7
which inductively reinforces this σ-π interaction. In

4
64 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Burgey et al.
F igu r e 1. X-ray crystal structures of 20 and 18 bound in the thrombin active site. The solvent accessible surface is depicted for
1
8 (the lipophilic surface created by the sidechains of Tyr-228 and Val-213 is highlighted in blue).
Ta ble 5. Effect of P2 Pyrazinone 6-Substitution on Thrombin
(IIa) and Trypsin (tryp) Inhibition (Ki), in Vitro Anticoagulant
Potency (2xaPTT) and Dog Pharmacokinetic Parameters
Ki
IIa
(nM)
tryp
(µM)
2xaPTT
(µM)
Cmax
(µM)
a
t1/2 (h)b
compd
R
2
2
2
2
2
0
2
3
4
5
Me
H
CF3
Cl
4.2
140
44
5.2
2.3
252
>1000
136
0.67
5.64
3.5
202
213
0.78
0.41
1.38
1.17
6.6
9.7
CN
F igu r e 2. Primary metabolism of 20 upon incubation with
human liver microsomes.
a
Dose ) 1 mg/kg unless otherwise noted. b po half-life.
S1 the fluoropyridine does not interact with Asp-189:
the ring nitrogen projects toward the enzyme backbone
and the fluorine is solvent exposed and appears not to
be involved in any specific interaction. The structure of
chloro group afforded a compound (24, Ki ) 5.2 nM) that
was nearly equipotent to the parent, whereas incorpora-
tion of a nitrile led to a more potent inhibitor (25, Ki )
2.3 nM). More importantly, and as anticipated from the
metabolism study, both the chloro (t1/2 ) 6.6 h) and
cyano (t1/2 ) 9.7 h) modifications imparted significantly
improved half-lives versus 20. While chloropyrazinone
24 maintained good in vivo efficacy (2/6 occlusions, final
plasma concentration ) 245 ( 9 nM), the cyanopyrazi-
none analogue 25 demonstrated compromised perfor-
mance in the rat thrombosis assay (5/6 occlusions, final
plasma concentration ) 460 ( 46 nM).
Having established the 6-Cl and 6-CN substitutions
as acceptable replacements for the 6-Me group, a
reinvestigation into the P1 structure-activity relation-
ship was undertaken (Table 6); particular attention was
focused on the impact of these modifications on plasma
half-life. Substitution of the 3-fluoro with a chlorine
degraded potency (26, Ki ) 7.9 nM, 2xaPTT ) 1.41 µM)
and, significantly, afforded lower plasma levels after
oral administration (Cmax ) 0.062 µM). As in the
6-methylpyrazinone series, incorporation of either a
4-methyl or 3-fluoro-4-methylpyridine P1 yielded potent
1
8 bound to thrombin revealed that incorporation of the
4
-Me group induces a 180° rotation of the P1 fluoropy-
ridine ring. This conformational change is driven by the
lipophilic binding interactions between the 4-Me and 3-F
groups and the surface created by the side chains of Tyr-
2
28 and Val-213 (Figure 1, blue surface), consistent with
the overall potency of 18 versus 20.
Although 20 represents an inhibitor which displayed
good bioavailability across three species, the plasma
half-life could be improved. Toward this end, a study to
identify the primary sites of metabolism of 20 revealed
that the 6-hydroxymethylpyrazinone 21 was the major
metabolite upon incubation with human liver mi-
crosomes (Figure 2).20 In view of these data, metaboli-
cally resistant replacements for the 6-methyl group were
investigated (Table 5). Elimination of the 6-methyl
group resulted in a significant loss of potency (22, Ki )
1
(
40 nM), as did incorporation of a trifluoromethyl group
23, Ki ) 44 nM).21 Substitution of the methyl with a

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 465
Ta ble 6. Effect of P1 Pyridine Substitution on Thrombin (IIa)
and Trypsin (tryp) Inhibition (Ki), in Vitro Anticoagulant
Potency (2xaPTT) and Dog Pharmacokinetic Parameters
Ki
IIa
tryp 2xaPTT Cmax
t1/2
compd
R
R1
R2
(nM) (µM)
(µM)
(µM)a (h)b
2
2
2
2
2
3
3
4
6
7
8
9
0
1
Cl
Cl
Cl
Cl
CN
Cl
Cl
F
Cl
H
F
F
H
H
Me
Me
Me
H
5.2
7.9
5.3
0.90
0.40
202
567
429
80
61
706
665
0.78
1.41
0.91
0.48
0.31
2.19
0.71
1.38
0.062 5.7
6.6
F igu r e 3. Synthetic approach to pyrazinone thrombin inhibi-
tors.
2.32
0.67
1.70
0.90
2.1
3.2
2.4
2.3
Sch em e 1a
OMe
H
15
3.6
OMe
0.53c 2.0
a
Dose ) 1 mg/kg unless otherwise noted. b po half-life. c Dose
)
0.5 mg/kg.
Ta ble 7. Pharmacokinetics Parameters of 24 in Dog, Rat,
Rhesus, and African Green Monkey
species
dose (mg/kg) po Cmax (µM)
0.65 1.16
10 1.06 ( 0.49 0.42 ( 0.23 23
0.310 0.73 ( 0.21 5.2
1.71 ( 1.75 1.6 ( 0.3 66 ( 54
iv t1/2 (h)
F (%)
dog
rat
rhesus
4.48
66
5
5
a
(a) KHMDS, MnBr2, -78 °C, N-fluorobenzenesulfonimide,
African green
THF; (b) NaBH4, EtOH, 0 °C; (c) Tf2O, 2,6-di-tert-butyl-4-meth-
ylpyridine, -78 °C, CH2Cl2; (d) NaN3, DMF, 60 °C; (e) 1 atm H2,
1
0% Pd/C, EtOAc.
analogues (27, Ki ) 5.3 nM; 28, Ki ) 0.9 nM); however,
neither modification enhanced the pharmacokinetic
duration. The 6-cyanopyrazinone analogue of 28 was
very potent (29, Ki ) 0.4 nM), but displayed an inferior
half-life (t1/2 ) 2.4 h) versus the parent 25. Similarly,
the 3- and 4-methoxypyridines 30 and 31 conferred no
pharmacokinetic advantage. Thus, the same S1 and S3
binding elements that proved to be optimal in terms of
potency and pharmacokinetics in the 6-methyl series
were also superior in the 6-chloro- and 6-cyanopyrazi-
none series.
As in the 6-methylpyrazinone series all compounds
in Table 6 exhibit minimal (>61 µM) activity versus
trypsin and against other proteases assayed: t-PA,
plasmin, factor Xa, plasma kallikrein, activated protein
C, urokinase, and chymotrypsin.
commenced with electrophilic gem-difluorination of the
potassium enolate of ethyl 2-pyridyl acetate 32 with
2
6
N-fluorobenzenesulfonimide (Scheme 1). The use of
manganese(II) bromide was critical to the success of this
transformation, as a significant amount of the mono-
fluorinated product was obtained in its absence. Elabo-
ration to the primary amine 35 was most effectively
accomplished through a four-step sequence. Sodium
borohydride reduction was followed by triflation of the
resultant primary alcohol 34 employing 2,6-di-tert-butyl-
2
2
4
-methylpyridine as the base. Displacement with azide
and subsequent hydrogenolysis afforded the amine 35
as a low-melting solid.
The substituted P1 2-aminomethylpyridines were
produced via a three-step sequence (Scheme 2). Pyridine
Due to its good Cmax, long t1/2 in dog and performance
in the rat FeCl3 efficacy assay, 24 was examined in
further detail (Table 7). This compound exhibits good
oral bioavailability in dog (66%, n ) 2), rat (23%, n )
2
7),
N-oxidation (MCPBA or H2O2 with MeReO3 catalysis
was followed by TMSCN-promoted cyanation,28 to pro-
duce the 2-cyanopyridines 37 with good regioselectivity.
Hydrogenation under acidic conditions afforded the
4
) and African green monkey (66%, n ) 4).23 Further-
more, 24 demonstrates good stability to human liver
microsome and hepatocyte preparations and, based on
in vitro metabolic scaling studies, is predicted to be a
low-clearance compound in man.24
Sch em e 2a
Ch em istr y. The general strategy employed for the
synthesis of these 3-aminopyrazinone acetamide throm-
1
0,25
bin inhibitors followed that established previously.
The modular nature of this series allows the indepen-
dent preparation of three individual subunits: a P3
2
1
-arylethylamine A, an ethyl 3-halopyrazin(1H)-2-one-
-acetate B, and a P1 benzylamine C (Figure 3). In
practice, installation of the P3 amine A onto the
pyrazinone B was followed by amide bond formation
with the P1 amine C to deliver the final products.
Synthesis of 2,2-difluoro-2-(2-pyridyl)ethylamine 35
a
(
a) MCPBA, CH2Cl2 or H2O2, cat. MeReO3, CH2Cl2/H2O; (b)
TMSCN, Et3N, CH3CN, reflux; (c) 10% Pd/C, 35 psi H2, HCl, EtOH;
d) Zn(CN)2, Pd(PPh3)4, DMF, 95 °C; (e) Raney Ni, 1 atm H2, EtOH,
NH3.
(

4
66 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Burgey et al.
Sch em e 3a
Sch em e 5a
a
i
(
a) Aminoacetaldehyde dimethylacetal, PrOH; (b) AcOH, HCl,
reflux; (c) POBr3, DCE, reflux.
primary amines 38 as their hydrochloride salts. The
3
-chloropyridine 41 was prepared by a different route:
selective Pd-mediated cyanation of 2,3-dichloropyridine
9, followed by Raney-Ni-catalyzed reduction, delivered
3
the primary amine 41.
A modification of the Cheeseman pyrazinedione syn-
thesis was employed for P2 subunit production (Scheme
2
9,25
3
).
Addition of aminoacetaldehyde dimethylacetal to
2
5
oxamate 42 afforded the cyclization precursor 43.
Refluxing oxamide 43 in acetic acid, with a catalytic
amount of HCl, affected cyclization to the crude pyra-
zinedione 44. Crystallization of this black tar from hot
ethanol produced 44 as a tan solid; notably, this
sequence was conducted on a multigram scale without
chromatography. Bromination with POBr3 produced
bromopyrazinone 45, which was used in subsequent
reactions without purification. The scaleability of this
sequence would prove valuable, as substrate 45 would
additionally serve as the precursor for the 6-chloropy-
a (a) PhCH , EtOH, sealed tube, 60-125 °C; (b) KOH, MeOH;
3
(c) 2-aminomethylpyridine, EDC, HOAT, Et3N, DMF; (d) NCS,
DCE, 80 °C.
times; conversely, reaction of the activated 3-chloro-6-
cyanopyrazinone 50 was complete within 1 h at 60 °C.
Chlorination of 52c with NCS in DCE occurred with
complete regioselectivity to afford the 6-chloropyrazi-
none 52d . Stoichiometry was crucial, as any excess
chlorinating agent produced the inseparable 5,6-dichlo-
ropyrazinone. Hydrolysis of ethyl esters 52a -d afforded
the corresponding acids, which underwent EDC-medi-
ated amide coupling with the P1 amines to afford the
final products 53.
azinone thrombin inhibitors (vide infra, Scheme 5).
The 6-methylpyrazinone intermediate 4625 was uti-
lized for the preparation of the 6-cyanopyrazinone 50
(Scheme 4). Oxidation to the aldehyde 47 was effected
with selenium dioxide, and subsequent condensation
with hydroxylamine produced the oxime 48. Direct
conversion to 3-chloro-6-cyanopyrazinone 50 via a one-
pot dehydration/chlorination sequence with POCl3 proved
unreliable; alternatively, the two-step PPh3-CCl4-medi-
ated dehydration and POCl3 chlorination afforded the
P2 6-cyanopyrazinone 50 in good yield.
Con clu sion
Metabolism-directed optimization of the 3-aminopy-
razinone acetamide template (e.g., 1) has been achieved
through modification of each of the three principal
components (i.e., P1, P2, P3) comprising this series.
Consequently, several potent thrombin inhibitors con-
taining weakly basic P1 and P3 pyridines (e.g., 20, 24,
25) were identified which demonstrate good oral bio-
availability and plasma half-lives. It remains to be
determined if clinical candidates emerging from this
structural class of inhibitors will possess the essential
safety and pharmacokinetic profile to supplant heparin
and warfarin anticoagulant therapy.
Sch em e 4a
Exp er im en ta l Section
Unless otherwise noted, all nonaqueous reactions were
carried out under a N atmosphere with commercial grade
2
reagents and solvents. Melting points were determined in open
capillary tubes in a Thomas-Hoover apparatus and are uncor-
1
rected. The H NMR were recorded on a Varian Unity Inova
4
00 MHz spectrometer. Chemical shifts are reported in ppm
relative to tetramethylsilane. Flash column chromatography
was performed using EM silica gel 60 (230-400 mesh). Reverse
phase preparative HPLC were performed using a Waters Prep
LC 2000 and a Waters C18 PrepPak 500 column. Analytical
HPLC was performed using an Agilent Zorbax SD-C8 4.6 ×
75 mm 3.5 µm column with a 4 min linear gradient from 95:5
a
(
a) SeO2, dioxane, reflux; (b) NH2OH‚HCl, pyridine, EtOH,
reflux; (c) PPh3, DCE, CCl4, reflux; (d) POCl3, NH4Cl, reflux.
Addition of the P3 2,2-difluoro-2-pyridylethylamine
5 to the 3-bromopyrazinones 45 and 51 (Scheme 5)
required elevated temperature and extended reaction
3
3 4 3
to 1:99 0.1% H PO :CH CN at a flow rate of 3 mL/min (system

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 467
A) or a Waters Xterra RP18 4.6 × 50 mm 3.5 µm column with
a 4 min linear gradient from 95:5 to 5:95 0.1% H PO :CH CN
at a flow rate of 4 mL/min (system B) with UV detection at
N-(Eth yl ca r boxym eth yl)-N′-(2,2-d im eth oxyeth yl)oxa -
m id e (43). To a solution of oxamate 4225 (84.0 g, 414 mmol)
in 2-propanol (500 mL) was added aminoacetaldehyde dim-
ethyl acetal (45.7 g, 435 mmol) in one portion. After being
stirred overnight at room temperature, the reaction mixture
was concentrated to a thick orange oil. This thick slurry was
diluted with 2-propanol (300 mL), and the solid was broken
up with a spatula. Filtration afforded a solid which was further
rinsed with an additional portion of 2-propanol. Removal of
3
4
3
2
2
15 and 254 nm. Procedures for protease inhibition and
xaPTT assays as well as animal pharmacokinetic studies
1
0
have been previously described.
Eth yl 2,2-Diflu or o-2-p yr id yl Aceta te (33). To a -78 °C
solution of 5.99 g (30.0 mmol) of potassium bis(trimethylsilyl)-
amide in 90 mL of THF was added 1.52 mL (10.0 mmol) of
ethyl 2-pyridyl acetate via syringe. The off-white slurry was
residual 2-propanol was accomplished via high vacuum to
1
afford a light orange solid (89.8 g): H NMR (CDCl
(
3
) δ 7.82
stirred for 45 min, then 4.30 g (20.0 mmol) of MnBr
in one portion. The tan slurry was stirred for 30 min, then
.90 g (28.2 mmol) of N-fluorobenzenesulfonimide was added
2
was added
br s, 1H), 7.50 (br s, 1H), 4.41 (t, 1H, 5.3 Hz), 4.24 (q, 2H, 7.1
Hz), 4.09 (d, 2H, 5.9 Hz), 3.47 (dd, 2H, 5.3, 6.2 Hz), 3.40 (s,
8
6
H), 1.30 (t, 3 H, 7.1 Hz).
in one portion. After a further 30 min at -78 °C, the reaction
was allowed to warm to room-temperature overnight. The thick
Eth yl 3-Hyd r oxyp yr a zin -(1H)-2-on e-1-a ceta te (44). A
solution of the oxamide 43 (89.8 g, 343 mmol), acetic acid (400
mL), and concentrated HCl (2 mL) was heated to reflux. After
slurry was poured into saturated aqueous NaHCO
and CH Cl (300 mL), the layers were separated, and the
aqueous layer extracted with CH Cl
(3 × 150 mL). The
combined organic layers were washed and dried over MgSO
3
(200 mL)
2
2
1
h the black reaction was concentrated to a thick oil (high
2
2
vacuum employed to ensure complete removal of AcOH) which
was diluted with EtOH (150 mL) and MeOH (150 mL).
Scraping the thick black oil with a spatula induced precipita-
tion of the product. The MeOH was removed via rotary
evaporation, and the remaining slurry was filtered and rinsed
with EtOH (200 mL) to deliver a tan solid. Recrystallization
from refluxing EtOH (300 mL) afforded 33.0 g of an off-white
4
,
and the solvent was evaporated. The residue was flash
chromatographed using 20 to 50% EtOAc-hexanes to give 1.19
1
g of the title compound: H NMR (CDCl
4
(
3
) δ 1.35 (t, 3H), 4.35-
.4 (m, 2H), 7.4-7.45 (m, 1H), 7.75 (d, 1H), 7.95 (d, 1H), 8.45
d, 1H).
,2-Diflu or o-2-(2-p yr id yl)eth a n ol (34). To a 0 °C stirred
2
powder: 1H NMR (CD
.9 Hz), 4.58 (s, 2H), 4.23 (q, 2H, 7.1 Hz), 1.28 (t, 3 H, 7.1 Hz).
OD) δ 6.50 (d, 1H, 5.9 Hz), 6.36 (d, 1H,
3
solution of 3.97 g (19.7 mmol) of ethyl 2,2-difluoro-2-pyridyl
acetate 33 in 40 mL of methanol was added 757 mg (20 mmol)
of sodium borohydride in several portions. After stirring for
5
Further crude dione could be obtained upon concentration of
the mother liquor.
Eth yl 3-Br om op yr a zin -(1H)-2-on e-1-a ceta te (45). A so-
lution of the hydroxypyrazinone 44 (25.0 g, 126 mmol) and
phosphorus oxybromide (37.9 g, 132 mmol) in 1,2-dichloro-
ethane (250 mL) was heated to reflux. After 8 h the reaction
0
.5 h, the cold bath was removed and stirring continued
another 0.5 h. The reaction was quenched with 2 M HCl, and
the solvents removed under reduced pressure. The residue was
partitioned between ether and 10% Na
was extracted with several portions of ether, and the combined
organic layers were washed with brine and dried over MgSO
Concentration gave 2.97 g of a yellow oil that was flash
chromatographed using 99:1 to 97:3 CH Cl -CH OH to give
.02 g of the title compound as solid: 1H NMR (CDCl
) δ 8.61
d, 1H, 4.5 Hz), 7.88 (td, 1H, 8.0, 1.7 Hz), 7.73 (d, 1H, 7.8 Hz),
.4-7.45 (m, 1H), 4.25 (td, 2H, 12.5, 7.1 Hz), 3.46 (t, 1H, 6.6
Hz).
,2-Diflu or o-2-(2-p yr id yl)et h yl
su lfon a te. To a stirred solution of 5 g (31.4 mmol) of
2 3
CO , the aqueous layer
2 3
mixture was treated with saturated aqueous Na CO (250 mL)
4
.
and stirred for 1 h. The mixture was diluted with water (100
mL) and dichloromethane (100 mL), the layers were separated,
and the aqueous layer was backwashed with EtOAc (3 × 200
2
2
3
1
3
4
mL). The combined organic layers were dried (MgSO ), and
(
7
concentrated to give an oil which was stored on a high vacuum
1
line overnight to afford 28.0 g of a brown solid: H NMR
(
2
CDCl
H), 4.27 (q, 2H, 7.2 Hz), 1.31 (t, 3 H, 7.2 Hz).
Eth yl 6-For m yl-3-h ydr oxypyr azin -(1H)-2-on e-1-acetate
3
) δ 7.17 (d, 1H, 4.2 Hz), 7.07 (d, 1H, 4.2 Hz), 4.65 (s,
2
Tr iflu or om et h a n e-
2
2
,2-difluoro-2-(2-pyridyl)ethanol 34 and 9.69 g (47.2 mmol) of
,6-di-tert-butyl-4-methylpyridine in 110 mL of methylene
25
(
47). A suspension of the hydroxypyrazinone 46 (5.0 g, 23.6
mmol) and selenium(IV) oxide (2.62 g, 23.6 mmol) in 1,4-
dioxane (100 mL) was heated to reflux for 24 h. The dark
reaction mixture was cooled and filtered through a pad of
Celite with MeOH. Concentration and flash chromatography
chloride at -78 °C was added 7.93 mL (47.2 mmol) of triflic
anhydride dropwise. After 1 h, the reaction was diluted with
1
00 mL of pentane and filtered. The filtrate was concentrated
and treated again with pentane and filtered. Concentration
of the filtrate gave 10.5 g of the title compound as a brown
with 3:97 to 10:90 MeOH:CH
compound as an orange solid: H NMR (CD
H), 7.39 (s, 1H), 5.12 (s, 2H), 4.22 (q, 2H, 7.1 Hz), 1.29 (t, 3
H, 7.1 Hz).
2
Cl
2
afforded 2.5 g of the title
1
3
OD) δ 9.11 (s,
oil, contaminated with 2,6-di-tert-butyl-4-methylpyridine: 1
H
1
NMR (CDCl
.76 (d, 1H, 7.9 Hz), 7.45-7.49 (m, 1H), 5.12 (t, 2 H, 11.9 Hz).
,2-Diflu or o-2-(2-p yr id yl)eth yl Azid e. A solution of 105
3
) δ 8.66 (d, 1H, 4.9 Hz), 7.89 (td, 1H, 7.7, 1.7 Hz),
7
Eth yl 6-F or m oxim yl-3-h yd r oxyp yr a zin -(1H)-2-on e-1-
a ceta te (48). To a suspension of the formylpyrazinone 47 (5.43
g, 24.0 mmol) and hydroxylamine hydrochloride (1.67 g, 24.0
mmol) in ethanol (100 mL) was added pyridine (1.90 mL, 24.0
mmol). After 2 h at reflux, the reaction mixture was cooled
and concentrated. This crude solid was recrystallized from
ethanol (100 mL) to deliver 2.80 g of the title compound as a
solid. An additional 1.90 g of product was obtained by
2
mg (0.31 mmol) of 2,2-difluoro-2-(2-pyridyl)ethyl trifluo-
romethanesulfonate and 43 mg (0.66 mmol) of sodium azide
in 1.0 mL of DMF was heated at 60 °C under Ar. After 1.5 h,
the mixture was cooled to room temperature, diluted with
water, and extracted with two portions of ether. The combined
organic layers were washed twice with water, brine, and dried
over MgSO
4
. The solvents were removed at reduced pressure
concentration of the filtrate and trituration with water (50
and a bath temperature of 20 °C to give 50 mg of a brown oil:
mL): 1H NMR (DMSO) δ 11.85 (d, 1H), 11.19 (s, 1H), 7.82 (s,
1
H NMR (CDCl
3
) δ 8.68 (d, 1H, 4.2 Hz), 7.86 (td, 1H, 7.8, 1.5
1H), 6.79 (d, 1H, 5.9 Hz), 5.05 (s, 2H), 4.12 (q, 2H, 7.1 Hz),
1.20 (t, 3 H, 7.1 Hz).
Hz), 7.72 (d, 1H, 7.8 Hz), 7.40-7.45 (m, 1H), 4.03 (t, 2 H, 13.2
Hz).
Eth yl 6-Cya n o-3-h yd r oxyp yr a zin -(1H)-2-on e-1-a ceta te
(49). A slurry of the hydroxypyrazinone 48 (2.70 g, 11.2 mmol)
and polymer-bound triphenylphosphine (1.55 mmol/g resin:
15.1 g, 23.5 mmol) in 1,2-dichloroethane (90 mL) and carbon
tetrachloride (9 mL) was heated to reflux for 1.5 h. The
reaction mixture was cooled and filtered and the resin rinsed
2
,2-Diflu or o-2-(2-p yr id yl)eth yla m in e (35). A stirred so-
lution of 1.8 g (9.7 mmol) of 2,2-difluoro-2-(2-pyridyl)ethyl azide
and 500 mg of 10% Pd/C in 100 mL of ethyl acetate was
hydrogenated under a balloon for 1 h. The catalyst was
removed by filtration, and the solvents were removed at
reduced pressure to give 1.2 g of the title compound as a yellow
with of 1:1 MeOH:CH
filtrate yielded 2.70 g of the product as a tan solid: H NMR
(CDCl ) δ 7.06 (s, 1H), 4.73 (s, 2H), 4.29 (q, 2H, 7.1 Hz), 1.33
(t, 3 H, 7.1 Hz).
2 2
Cl (200 mL). Concentration of the
1
1
oil: H NMR (CDCl
.7 Hz), 7.68 (d, 1H, 8.1 Hz), 7.37-7.40 (m, 1H), 3.44 (t, 2 H,
3
) δ 8.66 (d, 1H, 4.2 Hz), 7.82 (td, 1H, 7.7,
1
1
3
4.3 Hz), 1.41 (br s, 2H).

4
68 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
E t h yl 3-Ch lor o-6-cya n op yr a zin -(1H )-2-on e-1-a cet a t e
Burgey et al.
between CH
aqueous layer was backwashed with CH
bined organic layers were dried over MgSO
2
Cl
2
and saturated aqueous NaHCO
Cl
(4×). The com-
and the solvents
3
, and the
(
50). A suspension of the hydroxypyrazinone 49 (450 mg, 2.02
2
2
mmol) and ammonium chloride (237 mg, 4.44 mmol) in
phosphorus oxychloride (2 mL) was heated at reflux for 1.5 h.
The reaction mixture was cooled, and the volatiles were
removed via rotary evaporation. The residue was quenched
4
removed at reduced pressure to give an oil that was flash
chromatographed using 60:40 to 40:60 hexanes-EtOAc to give
6.81 g of the title compound as a yellow solid: 1H NMR (CDCl
)
3
with water, and solid Na
was basic. This aqueous mixture was extracted with dichlo-
2
CO
3
was added until the mixture
δ 8.67 (dd, 1H, 4.8, 0.7 Hz), 7.81 (ddd, 1H, 7.8, 7.8, 1.7 Hz),
7.69 (dd, 1H, 7.8, 1 Hz), 7.38 (dd, 1H, 5.1, 7.0 Hz), 6.86 (d, 1H,
4.8 Hz), 6.54 (br t, 1H, 5.9 Hz), 6.40 (d, 1H, 4.6 Hz), 4.54 (s,
2H), 4.38 (td, 2H, 14.0, 6.4 Hz), 4.24 (q, 2H, 7.1 Hz), 1.29 (t, 3
H, 7.1 Hz).
Eth yl 3-(2,2-Diflu or o-2-(2-p yr id yleth yla m in o)-6-ch lo-
r op yr a zin -(1H)-2-on e-1-a ceta te (52d ). To a stirred solution
of 6.81 g (20.1 mmol) of ethyl 3-(2,2-difluoro-2-(2-pyridylethy-
lamino)pyrazin-(1H)-2-one-1-acetate 52c in 100 mL of 1,2-
dichloroethane was added 2.42 g (18.1 mmol) of N-chlorosuc-
cinimide. An additional 242 mg (1.81 mmol) and 75 mg (0.56
mmol) of NCS were added to the reaction mixture after 1 and
1.5 h, respectively. After 2.5 h total, the solution was cooled
romethane (3×), and the combined organic layers were dried
(
2 4
Na SO ) and concentrated to give 415 mg of the product as
1
an amber oil: H NMR (CDCl
.32 (q, 2H, 7.1 Hz), 1.31 (t, 3 H, 7.1 Hz).
Eth yl 3-(2,2-Diflu or o-2-(2-p yr id yleth yla m in o)-6-m eth -
ylp yr a zin -(1H)-2-on e-1-a ceta te (52a ). A solution of 7.13 g
45.1 mmol) of 2,2-difluoro-2-(2-pyridyl)ethylamine 35 and 12.4
g (45.1 mmol) of ethyl 3-bromo-6-methylpyrazin-(1H)-2-one-
3
) δ 7.60 (s, 1H), 4.87 (s, 2H),
4
(
2
5
1
-acetate 51 in 15 mL of toluene and 15 mL of ethanol was
heated to 125 °C in a sealed tube overnight. The reaction was
concentrated, and the residue was diluted with ethyl acetate,
washed with 15% NaHCO
with ethyl acetate (3×). The combined organic layers were
dried over MgSO and the solvents removed at reduced
pressure to give an oil that was flash chromatographed using
0:50 hexanes-EtOAc to give 9.67 g of the title compound as
3
and the aqueous layer backwashed
to room temperature and partitioned between CH
and saturated aqueous NaHCO (200 mL). The layers were
separated, and the aqueous phase was backwashed with CH
Cl
(2 × 200 mL). The combined organic layers were dried over
MgSO
, and the solution concentrated to a volume of ∼10 mL.
This liquid was directly loaded onto a SiO column and eluted
with 65:35 to 55:45 hexanes-EtOAc to give 6.50 g of the title
compound as a yellow solid: 1H NMR (CDCl
) δ 8.68 (d, 1H,
4.8, Hz), 7.83 (ddd, 1H, 7.7, 7.7, 1.6 Hz), 7.9 (dd, 1H, 7.9 Hz),
2 2
Cl (150 mL)
3
4
2
-
2
5
4
1
a pale yellow solid: H NMR (CDCl
3
) δ 8.67 (d, 1H, 4.8 Hz),
2
7
6
6
.80 (t, 1H, 7.9 Hz), 7.68 (d, 1H, 7.9 Hz), 7.36-7.39 (m, 1H),
.71 (s, 1H), 6.31 (br t, 1H), 4.69 (s, 2H), 4.35 (td, 2H, 14.1,
.6 Hz), 4.24 (q, 2H, 7.1 Hz), 2.11 (s, 3H), 1.29 (t, 3 H, 6.8 Hz).
3
7
4
1
.40 (dd, 1H, 4.9, 7.3 Hz), 6.96 (s, 1H), 6.49 (br t, 1H, 5.9 Hz),
.89 (s, 2H), 4.38 (td, 2H, 13.9, 6.5 Hz), 4.26 (q, 2H, 7.1 Hz),
.30 (t, 3 H, 7.1 Hz).
3
-(2,2-D i flu o r o -2-(2-p y r i d y le t h y la m i n o )-6-m e t h -
ylp yr a zin -(1H)-2-on e-1-a cetic Acid (52a a cid ). To a stirred
solution of 9.67 g (27.5 mmol) of ethyl 3-(2,2-difluoro-2-(2-
pyridylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetate 52a
in 100 mL of methanol was added 8.58 g (153.0 mmol) of
potassium hydroxide in 20 mL of water. After 1 h, the solution
was concentrated at reduced pressure, and the residue was
dissolved in 25 mL of water. This solution was neutralized (pH
7) using 1.3 M HCl and concentrated at reduced pressure
to give 19.8 g of a yellow solid containing potassium chloride
and the title compound: H NMR (CD
Hz), 7.95 (td, 1H, 7.9, 1.8 Hz), 7.72-7.74 (m, 1H), 7.50-7.54
m, 1H), 6.64 (d, 1H, 1.09 Hz), 4.78 (s, 2H), 4.31 (t, 2H, 14.1
3-(2,2-Diflu or o-2-(2-pyr idyleth ylam in o)-6-ch lor opyr azin -
(1H)-2-on e-1-a cetic Acid (52d a cid ). To a stirred solution
of 7.27 g (19.5 mmol) of ethyl 3-(2,2-difluoro-2-(2-pyridylethy-
lamino)-6-chloropyrazin-(1H)-2-one-1-acetate 52d in 200 mL
of methanol was added 39 mL (39.0 mmol) of 1 M aq potassium
hydroxide. After 3 h the solution was neutralized (pH ∼ 7)
using concentrated HCl, and concentrated at reduced pressure
(azeotrope with PhCH
containing potassium chloride and the title compound:
NMR (CD OD) δ 8.64 (d, 1H, 4.8 Hz), 7.93 (ddd, 1H, 7.7, 7.7,
1.5 Hz), 7.70 (d, 1H, 8.0 Hz), 7.49 (dd, 1H, 5.2, 7.4 Hz), 6.80
∼
1
3
OD) δ 8.65 (d, 1H, 4.7
3
) to give 9.30 g of a white solid
1
H
(
3
Hz), 2.16 (s, 3H).
(s, 1H), 4.67 (s, 2H), 4.27 (t, 2H, 13.9 Hz).
Eth yl 3-(2,2-Diflu or o-2-(2-pyr idyl)eth ylam in o)-6-cyan o-
p yr a zin -(1H)-2-on e-1-a ceta te (52b). A mixture of 0.300 g
R/S-2-Am in o-6-m eth ylp yr id yl-3-(2-m eth yl-2-(p h en yl)-
(1.9 mmol) of 2,2-difluoro-2-(2-pyridyl)ethylamine 35, 0.35 mL
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e Dih y-
(
3
2.5 mmol) of triethylamine, and 0.42 g (1.75 mmol) of ethyl
-chloro-6-cyanopyrazin-(1H)-2-one-1-acetate 50 in 3 mL of
d r och lor id e (2). Prepared according to the previous procedure
for 1:10 mp > 200 C; H NMR (CD
1
OD) δ 8.86 (t, 1H, 5.6 Hz),
3
toluene and 0.5 mL of ethanol was heated to 60 °C for 1 h.
The reaction was concentrated and the residue partitioned
between CH
7.85 (d, 1H, 9.2 H), 7.21-7.34 (m, 5H), 6.83 (d, 1H, 9.2 Hz),
6.52 (d, 1H, 1.1 Hz), 4.74 (s, 2H), 4.33 (d, 2H, 5.5 Hz), 3.63 (m,
2H), 3.19 (m, 1H), 2.52 (s, 3H), 2.17 (d, 3H, 1.1 Hz), 1.37 (d,
2
Cl
2
and saturated aqueous NaHCO
Cl
3
. The aqueous
(2×), dried, and concen-
phase was backwashed with CH
2
2
3H, 7.0 Hz); HRMS (FAB) calcd C23
found 421.2355. Anal. (C23
53.04, 5.95, 16.14. Found 53.44, 5.56, 15.95.
-Am in o-6-m et h ylp yr id yl-3-(2,2-d im et h yl-2-(p h en yl)-
H
30
N
6
O
2
(M + 1) 421.2352,
trated. Flash chromatography using 25-50% EtOAc/hexanes
29 6 2
H N O ‚2.75HCl) C, H, N. Calcd
afforded 0.38 g of the title compound as a tan powder: 1H NMR
(
7
CDCl
3
) δ 8.67 (d, 1H, 4.8 Hz), 7.86 (ddd, 1H, 1.6, 7.8, 7.8 Hz),
2
.71 (dd, 1H, 0.9, 8.0 Hz), 7.43 (m, 2H), 7.35 (br t, 1H), 4.79
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (3). Pre-
(
(
s, 2H), 4.62 (dt, 2H, 6.5, 13.5 Hz), 4.29 (q, 2H, 7.1 Hz), 1.32
t, 3 H, 7.1 Hz).
10
pared according to the previous procedure for 1: mp 179-
1
1
81 C; H NMR (CDCl
3
) δ 7.35 (m, 4H), 7.21 (tt, 1H, 7.1, 1.4
3
-(2,2-Diflu or o-2-(2-p yr id yl)et h yla m in o)-6-cya n op y-
Hz), 7.13 (d, 1H, 8.2 Hz), 6.8 (t, 1H, 5.1 Hz), 6.71 (d, 1H, 0.9
Hz), 6.2 (d, 1H, 8.1), 5.64 (t, 1H, 5.9 Hz), 4.55 (s, 2H), 4.43 (s,
2H), 4.22 (d, 2H, 5.5 Hz), 3.56 (d, 2H, 6.0 Hz), 2.22 (s, 6H),
r a zin -(1H)-2-on e-1-a cetic Acid (52b a cid ). To a stirred
solution of 0.38 g (1.06 mmol) of ethyl 3-(2,2-difluoro-2-(2-
pyridyl)ethylamino)-6-cyanopyrazin-(1H)-2-one-1-acetate 52b
in dimethoxyethane (10 mL) was added 1.6 mL of lithium
hydroxide solution (1.0 M in water). After 1 h, the solution
was neutralized using 1 M HCl. Concentration at reduced
pressure (azeotrope with PhCH ) afforded 0.400 g of an off-
3
white solid containing lithium chloride and the title compound,
which was used directly in the amide couplings.
1.37 (s, 6H). Anal. (C24
6.97, 19.30. Found 66.24, 6.99, 18.93.
-(2-Am in o-6-m eth yl)-p yr id ylm eth yl 3-(2,2-Diflu or o-2-
p h e n yl)e t h yla m in o)-6-m e t h ylp yr a zin -2-on e -1-a ce t a -
H
30
N
6
O
2
‚0.05H
2
O) C, H, N. Calcd 66.20,
5
(
m id e (4). To a stirred solution of 350 mg (1.09 mmol) of 3-(2,2-
difluoro-2-(phenylethylamino)-6-methylpyrazin-(1H)-2-one-1-
30
acetic acid and 228 mg (1.09 mmol) 2-amino-5-aminomethyl-
Eth yl 3-(2,2-Diflu or o-2-(2-p yr id yleth yla m in o)p yr a zin -
1H)-2-on e-1-a ceta te (52c). A solution of 4.80 g (30.4 mmol)
6-methylpyridine dihydrochloride in 3 mL of DMF were added
209 mg (1.09 mmol) of EDC, 147 mg (1.09 mmol) of HOBT,
and 2 mL of triethylamine. After stirring for 1 d, the volatiles
were removed at reduced pressure. The residue was parti-
(
of 2,2-difluoro-2-(2-pyridyl)ethylamine 35, 4.24 mL (30.4 mmol)
of triethylamine, and 7.93 g (30.4 mmol) of ethyl 3-bromopy-
razin-(1H)-2-one-1-acetate 45 in 12 mL of toluene and 4 mL
of ethanol was heated to 120 °C in a sealed tube overnight.
The reaction was concentrated, the residue was partitioned
tioned between CH
the aqueous layer backwashed with CH
bined organic layers were dried over MgSO
2
Cl
2
and saturated aqueous NaHCO
Cl
(3×). The com-
and the solvents
3
and
2
2
4

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 469
removed at reduced pressure. This solid was purified by
reverse phase HPLC and lyophilized to give 130 mg of the TFA
to warm to room-temperature overnight. The solution was
concentrated and the residue triturated with EtOAc/MeOH to
give the title compound as a yellow solid (225 mg): H NMR
salt of the title compound as a white solid: 1H NMR (CD
OD)
δ 7.84 (d, 1H, 9.2 Hz), 7.57 (m, 2H), 7.46 (m, 3H), 6.82 (d, 1H,
1
3
(CD OD) δ 8.80 (d, 2H, 6.6 Hz), 8.11 (d, 2H, 6.6 Hz), 7.86 (d,
3
9
4
.0 Hz), 6.65 (d, 1H, 1.1 Hz), 4.72 (s, 2H), 4.31 (d, 2H, 4.0 Hz),
.10 (t 2H, 14.5 Hz), 2.51 (s, 3H), 2.16 (d, 3H, 0.9 Hz); Anal.
1H, 9.0 Hz), 6.83 (d, 1H, 9.1 Hz), 6.66 (d, 1H, 1.1 Hz), 4.78 (s,
2H), 4.33 (d, 2H, 5.5 Hz), 3.91 (t, 2H, 7.2 Hz), 3.65 (t, 2H, 7.1
(
1
C
22
H
24
N
6
O
2
F
2
‚2.15TFA‚0.85H
1.96. Found 44.90, 3.96, 11.97; LRMS 443.2 (MH) .
-(2-Am in o-6-m eth yl)-p yr id ylm eth yl 3-(2-(2-P yr id yl)-
2
O) C, H, N. Calcd 44.94, 3.99,
Hz), 2.53 (s, 3H), 2.21 (d, 3H. 0.7 Hz); Anal. (C21
3.0HCl‚0.60H O) C, H, N. Calcd 47.80, 5.58, 18.58. Found
7.41, 5.81, 18.93; LRMS 408.4 (MH) .
5-(2-Am in o-6-m eth yl)-p yr id ylm eth yl 3-(2,2-Diflu or o-2-
(2-p yr id yl)et h yla m in o)-6-m et h ylp yr a zin -2-on e-1-a cet a -
m id e (8). To a stirred solution of 50 mg (0.15 mmol) of 52a
a cid and 32 mg (0.15 mmol) 2-amino-5-aminomethyl-6-meth-
ylpyridine dihydrochloride in 1 mL of DMF were added 35 mg
(0.18 mmol) of EDC, 2.0 mg (0.15 mmol) of HOAT, and 105
µL (0.75 mmol) of triethylamine. After stirring for 1 d, the
volatiles were removed at reduced pressure. The residue was
25 7 2
H N O ‚
+
2
+
4
5
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (5). To
a stirred solution of 189 mg (0.46 mmol) of 3-(2-(2-pyridyl-
ethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid (TFA
salt) and 110 mg (0.46 mmol) of 2-tert-butoxycarbonylamino-
5
1
1
-aminomethyl-6-methylpyridine in 7 mL of DMF were added
07 mg (0.55 mmol) of EDC, 75 mg (0.55 mmol) of HOBT, and
61 µL (1.15 mmol) of triethylamine. After stirring for 1 d,
the volatiles were removed at reduced pressure. The residue
was diluted with EtOAc and washed successively with satu-
diluted with saturated aqueous NaHCO
with H O. This solid was flash chromatographed using 2 to
15% MeOH:CH Cl to give 30 mg of the title compound as a
white solid: H NMR (CD OD) δ 8.63 (d, 1H, 4.8 Hz), 7.92
3
, filtered, and rinsed
rated aqueous NaHCO
layer was dried over MgSO
reduced pressure. This residue was triturated with EtOAc
with heating) to give 130 mg of the product as a white solid.
3
, H
2
O (4×), and brine. The organic
2
4
and the solvents removed at
2
2
1
3
(
(ddd, 1H, 1.6, 7.8, 7.8 Hz), 7.71 (dd, 1H, 1.0, 7.8 Hz), 7.49 (br
t, 1H, 6.3 Hz), 7.34 (dd, 1H, 8.2 Hz), 6.62 (d, 1H, 1.1 Hz), 6.38
Through a stirred solution of this material in EtOAc was
bubbled HCl(g) for 10 min at -78 °C. The reaction was allowed
to warm to room-temperature overnight. The solution was
concentrated and the residue triturated with EtOAc/MeOH to
(d, 1H, 8.4 Hz), 4.68 (s, 2H), 4.26 (m, 4H), 2.33 (s, 3H), 2.17
+
(d, 3H, 0.9 Hz); LRMS 444.3 (MH) ; t
R
) 1.47 min (99% @ 215
) 0.83 min (99% @ 215,
(M + 1) 444.1954,
and 98% @ 254 nm, system A) and t
R
1
give the title compound as a white solid (90 mg): H NMR
system B); HRMS (ES) calcd C21
found 444.1919.
H
23
N
7
O
2
F
2
(CD
3
OD) δ 8.78 (d, 1H, 4.9 Hz), 8.59 (dd, 1H, 7, 7 Hz), 8.12 (d,
1
6
3
3
5
H, 7.9 Hz), 8.00 (dd, 1H, 6.7, 6.7 Hz), 7.87 (d, 1H, 9.0 Hz),
.83 (d, 1H, 8.9 Hz), 6.70 (s, 1H), 4.78 (s, 2H), 4.33 (s, 2H),
.98 (t, 2H, 6.7 Hz), 3.49 (t, 2H, 6.6 Hz), 2.54 (s, 3H), 2.23 (s,
H); Anal. (C21
.53, 18.74. Found 48.22, 5.88, 18.55; LRMS 408.2 (MH) .
-(2-Am in o-6-m eth yl)-p yr id ylm eth yl 3-(2-(3-P yr id yl)-
3-(2-Meth yl)-p yr id ylm eth yl 3-(2-P h en yleth yla m in o)-6-
m eth ylp yr a zin -2-on e-1-a ceta m id e (9). To a stirred solution
of 442 mg (1.54 mmol) of 3-(2-phenylethylamino)-6-meth-
ylpyrazin-(1H)-2-one-1-acetic acid10 and 183 mg (1.50 mmol)
of 3-aminomethyl-2-methylpyridine in 5 mL of DMF were
added 288 mg (1.50 mmol) of EDC, 203 mg (1.50 mmol) of
HOBT, and 329 mg (3.25 mmol) of NMM. After stirring for 1
d, the volatiles were removed at reduced pressure. The residue
H
25
N
7
O
2
2
.2.0 HCl‚0.35H O) C, H, N. Calcd 48.21,
+
5
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (6). To
a stirred solution of 162 mg (0.28 mmol, 56% purity) of 3-(2-
(3-pyridylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid
was partitioned between EtOAc and H
layer backwashed with EtOAc. The combined organic layers
were dried over MgSO , and the solvents were removed at
2
O and the aqueous
(HCl salt) and 73 mg (0.31 mmol) of 2-tert-butoxycarbony-
lamino-5-aminomethyl-6-methylpyridine in 6 mL of DMF were
added 66 mg (0.34 mmol) of EDC, 44 mg (0.34 mmol) of HOBT,
and 97 µL (0.70 mmol) of triethylamine. After stirring for 1 d,
the volatiles were removed at reduced pressure. The residue
was diluted with EtOAc and washed successively with satu-
4
reduced pressure and flash chromatographed using EtOAc
(100 mg): 1H NMR (DMSO) δ 8.67 (br t, 1H), 8.32 (d, 1H, 4.6
Hz), 7.56 (d, 1H, 7.6 Hz), 7.28-7.15 (m, 6H), 6.84 (br t, 1H),
6.64 (s, 1H), 4.63 (s, 2H), 4.28 (d, 2H, 5.6 Hz), 3.46 (dt, 2H,
7.6, 7.6 Hz), 2.83 (t, 2H, 7.3 Hz), 2.49 (d, 3H, 1.1 Hz), 2.05 (s,
rated aqueous NaHCO
layer was dried over MgSO
at reduced pressure and flash chromatographed with 0.5 to
% MeOH:CH Cl to give 81 mg of a white solid. Through a
3
, H
2
O (4×), and brine. The organic
, and the solvents were removed
3H); Anal. (C22
17.65. Found 66.25, 6.56, 18.03. LRMS 392.2 (MH) .
3-P yr id ylm et h yl 3-(2-P h en ylet h yla m in o)-6-m et h -
H
25
N
5
O
2
2
‚0.30H O) C, H, N; Calcd 66.57, 6.50,
4
+
5
2
2
stirred solution of this material in EtOAc was bubbled HCl(g)
for 10 min at -78 °C. The reaction was allowed to warm to
room-temperature overnight. The solution was concentrated
ylp yr a zin -2-on e-1-a ceta m id e (10). To a stirred solution of
143 mg (0.50 mmol) of 3-(2-phenylethylamino)-6-methylpyrazin-
(1H)-2-one-1-acetic acid10 and 56 mg (0.52 mmol) of 3-ami-
nomethylpyridine in 10 mL of DMF were added 166 mg (0.87
mmol) of EDC, 94 mg (0.70 mmol) of HOBT, and 110 µL (1.0
mmol) of NMM. After stirring for 1 d, the volatiles were
removed at reduced pressure. The residue was diluted with
and the residue triturated with EtOAc/MeOH to give the title
1
compound as a tan solid (76 mg): H NMR (CD
3
OD) δ 8.95 (s,
1
5
1
3
H), 8.79 (d, 1H, 5.7 Hz), 8.66 (d, 1H, 8.1 Hz), 8.07 (dd, 1H,
.9, 8.1 Hz), 7.86 (d, 1H, 9.2 Hz), 6.83 (d, 1H, 9.2 Hz), 6.66 (d,
H, 0.9 Hz), 4.78 (s, 2H), 4.33 (s, 2H), 3.86 (t, 2H, 7.1 Hz),
.28 (t, 2H, 7.1 Hz), 2.53 (s, 3H), 2.21 (s, 3H); Anal.
H
H
2
O, filtered, and rinsed with saturated aqueous NaHCO
3
and
O to give 164 mg of the title compound as a solid: H NMR
) δ 8.50 (m, 2H), 7.56 (d, 1H, 7.9 Hz), 7.32-7.20 (m,
6H), 6.89 (br t, 1H), 6.78 (s, 1H), 5.92 (br t, 1H, 5.8 Hz), 4.64
s, 2H), 4.43 (d, 2H, 6.0 Hz), 3.65 (dt, 2H, 6.7, 6.7 Hz), 2.92 (t,
H, 7.0 Hz), 2.26 (s, 3H); Anal. (C21 ‚0.15H O) C, H,
N; Calcd 66.34, 6.18, 18.42. Found 66.34, 6.34, 18.59.
2-P yr id ylm et h yl 3-(2-P h en ylet h yla m in o)-6-m et h -
1
2
(
1
C
21
H
25
N
7
O
2
.3.0 HCl‚1.55H
8.00. Found 45.96, 5.38, 18.06; LRMS 408.3 (MH) .
-(2-Am in o-6-m eth yl)-p yr id ylm eth yl 3-(2-(4-P yr id yl)-
2
O) C, H, N. Calcd 46.30, 5.75,
(CDCl
3
+
(
5
2
H
23
N
5
O
2
2
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (7). To
a stirred solution of 503 mg (0.87 mmol, 50% purity) of 3-(2-
(4-pyridylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid
and 210 mg (0.87 mmol) 2-tert-butoxycarbonylamino-5-ami-
nomethyl-6-methylpyridine in 8 mL of DMF were added 211
mg (1.04 mmol) of EDC, 211 mg (1.04 mmol) of HOBT, and
ylp yr a zin -2-on e-1-a cet a m id e (11). Prepared according to
procedure for 10 [using 3-(2-phenylethylamino)-6-methylpyrazin-
(1H)-2-one-1-acetic acid10 (143 mg, 0.50 mmol); 2-aminometh-
ylpyridine (56 mg, 0.52 mmol); DMF (10 mL); EDC (166 mg,
1
50 µL (1.04 mmol) of triethylamine. After stirring for 1 d,
the volatiles were removed at reduced pressure. The residue
was diluted with EtOAc and washed successively with satu-
0.87 mmol), HOBT (94 mg, 0.70 mmol); NMM (110 µL, 1.0
1
mmol)] to give 170 mg of the title compound as a solid:
H
rated aqueous NaHCO
layer was dried over MgSO
at reduced pressure and flash chromatographed with 2 to 7%
MeOH:CH Cl to give 252 mg of the product as an off-white
solid. Through a stirred solution of this material in EtOAc was
bubbled HCl(g) for 10 min at -78 °C. The reaction was allowed
3
, H
2
O (4×), and brine. The organic
3
NMR (CDCl ) δ 8.49 (d, 1H, 4.0 Hz), 7.64 (ddd, 1H, 1.8, 7.6,
4
, and the solvents were removed
7.6 Hz), 7.31-7.17 (m, 8H), 6.76 (s, 1H), 6.78 (s, 1H), 5.97 (br
t, 1H), 4.71 (s, 2H), 4.55 (d, 2H, 5.1 Hz), 3.65 (dt, 2H, 6.7, 6.7
2
2
23 5 2
Hz), 2.93 (t, 2H, 7.1 Hz), 2.24 (s, 3H); Anal. (C21H N O ‚
0.20H
18.43.
2
O) C, H, N; Calcd 66.19, 6.19, 18.38. Found 66.11, 6.17,

4
70 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
-P yr id ylm et h yl 3-(2-P h en ylet h yla m in o)-6-m et h -
Burgey et al.
4
3-F lu or o-4-m et h yl-2-p yr id ylm et h yl 3-(2,2-Diflu or o-2-
(p h e n yl)e t h yla m in o)-6-m e t h ylp yr a zin -2-on e -1-a ce t a -
m id e (17). To a stirred solution of 76 mg (0.24 mmol) of 3-(2,2-
difluoro-2-(phenylethylamino)-6-methylpyrazin-(1H)-2-one-1-
acetic acid and 60 mg (0.28 mmol) of 2-aminomethyl-3-fluoro-
4-methylpyridine dihydrochloride salt in 4 mL of DMF were
added 46 mg (0.24 mmol) of EDC, 32 mg (0.24 mmol) of HOBT,
and 276 mg (2.0 mmol) of NMM. After stirring for 1 d, the
volatiles were removed at reduced pressure. The residue was
ylp yr a zin -2-on e-1-a cet a m id e (12). Prepared according to
procedure for 10 [using 3-(2-phenylethylamino)-6-methylpyrazin-
(
1H)-2-one-1-acetic acid (143 mg, 0.50 mmol); 4-aminometh-
ylpyridine (56 mg, 0.52 mmol); DMF (10 mL); EDC (166 mg,
.87 mmol), HOBT (94 mg, 0.70 mmol); NMM (110 µL, 1.0
0
1
mmol)] to give 178 mg of the title compound as a solid:
H
NMR (DMSO) δ 8.82 (br t, 1H, 6.0 Hz), 8.50 (m, 2H), 7.31-
7
4
.19 (m, 7H), 6.87 (br t, 1H, 5.9 Hz), 6.66 (s, 1H), 4.67 (s, 2H),
.33 (d, 2H, 5.9 Hz), 3.48 (dt, 2H, 6.9, 6.9 Hz), 2.85 (t, 2H, 7.4
flash chromatographed using 95:5 CHCl
title compound as a white solid: 1H (CDCl
Hz), 7.54 (m, 2H), 7.42 (m, 4H), 7.07 (t, 1H, 6.0 HzHz), 6.70
s, 1H), 6.16 (br t, 1H, 6.0 Hz), 4.73 (s, 2H), 4.60 (d, 2H, 4.8
3
-MeOH to give the
Hz), 2.08 (s, 3H); Anal. (C21
6.50, 6.17, 18.47. Found 66.49, 6.17, 18.36.
-P yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)eth yla m i-
H
23
N
5
O
2
‚0.10H
2
O) C, H, N; Calcd
3
) δ 8.15 (d, 1H, 4.8
6
(
2
Hz), 4.10 (td, 2H, 14.3, 6.4 Hz), 2.31 (s, 3H), 2.24 (s, 3H).
Conversion to the dihydrochloride salt can be carried out by
treating a EtOAc solution with 3 mL of 3.0 M HCl in EtOAc,
H N O F ‚2.40HCl) C,
22 5 2 3
H, N. Calcd 49.58, 4.61, 13.14. Found 49.53, 5.01, 12.90; LRMS
n o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (13). To a stirred
solution of 50 mg (0.15 mmol) of 52a a cid and 16 mg (0.15
mmol) 2-aminomethylpyridine in 1 mL of DMF was added 35
mg (0.18 mmol) of EDC, 2.0 mg (0.15 mmol) of HOAT and 105
µL (0.75 mmol) of triethylamine. After stirring for 1 d, the
volatiles were removed at reduced pressure. The residue was
followed by concentration: Anal. (C22
+
446.3 (MH) .
partitioned between CH
and the aqueous layer backwashed with CH
organic layers were dried over MgSO
removed at reduced pressure. This solid was rinsed with H
to give 40 mg of the title compound as a white solid: 1H NMR
CDCl ) δ 8.67 (d, 1H, 4.2 Hz), 8.50 (d, 1H, 4.2 Hz), 7.80 (ddd,
H, 1.5, 7.7, 7.7 Hz), 7.68 (d, 1H, 7.9 Hz), 7.64 (ddd, 1H, 1.7,
2
Cl
2
and saturated aqueous NaHCO
Cl . The combined
and the solvents
3
3-F lu or o-4-m et h yl-2-p yr id ylm et h yl 3-(2,2-Diflu or o-2-
(2-p yr id yl)et h yla m in o)-6-m et h ylp yr a zin -2-on e-1-a cet a -
m id e (18). To a stirred solution of 62 mg (0.17 mmol) of 52a
a cid and 63 mg (0.30 mmol) of 2-aminomethyl-3-fluoro-4-
methylpyridine dihydrochloride in 2 mL of DMF were added
50 mg (0.26 mmol) of EDC, 35 mg (0.26 mmol) of HOBT and
151 mg (1.5 mmol) of triethylamine. After stirring for 1 d, the
volatiles were removed at reduced pressure. The resulting dark
oil was diluted with ethyl acetate and washed with 5%
2
2
4
2
O
(
3
1
7
6
5
.7, 7.7 Hz), 7.38 (br t, 1H, 6.0), 7.32 (br s, 1H), 7.2 (m, 2H),
.75 (s, 1H), 6.33 (br t, 1H, 6.2 Hz), 4.70 (s, 2H), 4.55 (d, 2H,
.1 Hz), 4.36 (td, 2H, 14.1, 6.6 Hz), 2.23 (s, 3H); LRMS 415.3
NaHCO , and the aqueous layer was backwashed with EtOAc
3
+
(
t
(
MH) ; t
R
R
) 1.46 min (99% @ 215 and 254 nm, system A) and
(3×). The combined organic layers were dried over MgSO and
4
) 0.71 min (95% @ 215 and 97% @254 nm, system B); HRMS
ES) calcd C20 (M + 1) 415.1689, found 415.1652.
-Ch lor o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(p h en yl)-
the solvents removed at reduced pressure. This residue was
H
20
N
6
O
2
F
2
flash chromatographed using 95:5 CHCl -MeOH to give 42
3
4
mg of the title compound as a white solid. Conversion to the
hydrochloride salt can be carried out by treating a dioxane
eth yla m in o)-6-m eth ylp yr a zin -2-on e-1-a ceta m id e (14). To
a stirred solution of 100 mg (0.31 mmol) of 3-(2,2-difluoro-2-
solution with three equiv of 4.0 M HCl in dioxane, followed
phenylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid30
1
3
(
by concentration: H NMR (CD
OD) δ 8.63 (d, 1H, 4.8 Hz),
and 48 mg (0.34 mmol) of 2-aminomethyl-4-chloropyridine in
mL of DMF were added 60 mg (0.31 mmol) of EDC, 42 mg
0.31 mmol) of HOBT, and 51 mg (0.50 mmol) of triethylamine.
8.17 (d, 1H, 5.0 Hz), 7.93 (dd, 1H, 7.7, 7.7 Hz), 7.70 (dd, 1H,
0.9, 7.9 Hz), 7.49 (dd, 1H, ∼6, 6 Hz), 7.24 (dd, 1H, 5.2, 5.2
Hz), 6.62 (s, 1H), 4.78 (s, 2H), 4.57 (s, 2H), 4.26 (t, 2H, 14.0
5
(
+
After stirring for 1 d, the volatiles were removed at reduced
pressure. The residue was purified by reverse phase HPLC to
Hz), 2.38 (s, 3H), 2.33 (d, 3H, 0.9 Hz); LRMS 447.2 (MH) .
3-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(p h en yl)-
eth ylam in o)-6-m eth ylpyr azin -2-on e-1-acetam ide (19). Pre-
pared according to procedure for 18 [using 3-(2,2-difluoro-2-
(phenylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid
(80 mg, 0.24 mmol); 2-aminomethyl-3-fluoropyridine dihydro-
chloride salt (71 mg, 0.36 mmol); DMF (1 mL); EDC (46 mg,
0.24 mmol); HOBT (32 mg, 0.24 mmol); triethylamine (287 mg,
2.84 mmol)] to give 60 mg of the title compound as a white
solid. Conversion to the hydrochloride salt can be carried out
by treating a EtOAc solution with 3 mL of 3.0 M HCl in EtOAc,
1
give 53 mg of the TFA salt of the title compound: H NMR
3
(CD OD) δ 8.45 (d, 1H, 5.5 Hz), 7.60 (m, 2H), 7.53 (d, 1H, 1.8
Hz), 7.48 (m, 3H), 7.42 (dd, 1H, 2.1, 5.6 Hz), 6.64 (d, 1H, 1.1
Hz), 4.85 (s, 2H), 4.55 (s, 2H), 4.130 (t 2H, 14.5 Hz), 2.23 (d,
3
H, 0.9 Hz); Anal. (C21
H
20
N
5
O
2
ClF
2
‚2.30TFA‚0.60H
2
O) C, H,
N. Calcd 42.65, 3.29, 9.72. Found 42.66, 3.58, 9.33; LRMS 448.2
+
(MH) .
4
-Meth yl-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(p h en yl)-
eth ylam in o)-6-m eth ylpyr azin -2-on e-1-acetam ide (15). Pre-
pared according to procedure for 14 [using 3-(2,2-difluoro-2-
followed by concentration: 1H NMR (CD
OD) δ 8.56 (dd, 1H,
3
(
phenylethylamino)-6-methylpyrazin-(1H)-2-one-1-acetic acid
0.9, 5.3 Hz), 8.14 (dd, 1H, 9.0, 9.0 Hz), 7.80 (m, 1H), 7.62 (m,
2H), 7.54 (m, 3H), 6.64 (s, 1H), 4.89 (s, 2H), 4.75 (s, 2H), 4.19
(68 mg, 0.21 mmol); (38 mg, 0.32 mmol) 2-aminomethyl-4-
methylpyridine; DMF (5 mL); EDC (60 mg, 0.32 mmol); HOBT
43 mg, 0.32 mmol); triethylamine (202 mg, 2.0 mmol)] to give
2 mg of the TFA salt of the title compound as an off-white
(t 2H, 14.6 Hz), 2.25 (s, 3H); Anal. (C21
H, N. Calcd 49.83, 4.39, 13.84. Found 50.05 4.38, 13.47; HRMS
(FAB) calcd C21 (M + 1) 432.1642, found 432.1638;
LRMS 432.2 (MH) .
-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
20 5 2 3
H N O F ‚2.05HCl) C,
(
2
21 5 2 3
H N O F
1
+
solid: H NMR (CD
3
OD) δ 8.58 (d, 1H, 5.7 Hz), 7.85 (s, 1H),
.75 (d, 1H, 5.3 Hz), 7.56-7.47 (m, 5H), 6.67 (s, 1H), 4.83 (s,
H), 4.73 (s, 2H), 4.12 (t 2H, 14.5 Hz), 2.65 (s, 3H), 2.20 (s,
H); Anal. (C22 ‚2.05TFA‚1.35MeOH) C, H, N. Calcd
6.80, 4.36, 9.94. Found 46.99, 4.01, 9.55; LRMS 428.2 (MH) .
-Meth yl-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
7
2
3
4
3
eth ylam in o)-6-m eth ylpyr azin -2-on e-1-acetam ide (20). Pre-
pared according to procedure for 18 [using 52a a cid (200 mg,
23 5 2 2
H N O F
+
0
.60 mmol); 2-aminomethyl-3-fluoropyridine dihydrochloride
4
salt (143 mg, 0.72 mmol); DMF (3 mL); EDC (119 mg, 0.62
mmol); HOBT (84 mg, 0.62 mmol); triethylamine (581 mg, 5.7
eth ylam in o)-6-m eth ylpyr azin -2-on e-1-acetam ide (16). Pre-
pared according to procedure for 14 [using 52a a cid (0.49
mmol); 2-aminomethyl-4-methylpyridine (61 mg, 0.50 mmol);
DMF (4 mL); EDC (95 mg, 0.49 mmol); HOBT (67 mg, 0.49
mmol)] to give 150 mg of the title compound as a white solid:
1
H NMR (CDCl ) δ 8.67 (dd, 1H, 0.7, 4.8 Hz), 8.31 (ddd, 1H,
3
1.3, 1.3, 4.6 Hz), 7.81 (ddd, 1H, 1.7, 7.7, 7.7 Hz), 7.69 (ddd,
1H, 0.9, 0.9, 8.1 Hz), 7.20 (br t, 1H), 7.37 (m, 2H), 7.23 (ddd,
1H, 8.6, 4.3, 4.3 Hz), 6.75 (d, 1H, 0.9 Hz), 6.34 (br t, 1H, 6.3
Hz), 4.73 (s, 2H), 4.63 (dd, 2H, 4.7, 1.6 Hz), 4.37 (td, 2H, 14.2,
6.5 Hz), 2.25 (d, 3H, 0.9 Hz). Conversion to the dihydrochloride
mmol); NMM (202 mg, 2.0 mmol)] to give 158 mg of the TFA
1
salt of the title compound as a yellow solid: H NMR (CD
3
-
OD) δ 8.67 (d, 1H, 4.6 Hz), 8.60 (d, 1H, 6.0 Hz), 7.99 (ddd, 1H,
.5, 7.9, 7.9 Hz), 7.86 (s, 1H), 7.77 (app d, 2H, 7.5 Hz), 7.56
dd, 1H, 4.9, 7.3 Hz), 6.69 (s, 1H), 4.87 (s, 2H), 4.74 (s, 2H),
.39 (t, 2H, 14.4 Hz), 2.65 (s, 3H), 2.23 (s, 3H); Anal.
‚2.35TFA) C, H, N. Calcd 44.32, 3.52, 12.07.
1
(
4
salt can be carried out by treating a dioxane solution with 2
1
equiv of 4.0 M HCl in dioxane, followed by concentration:
NMR (CD
Hz), 8.15 (ddd, 1H, 0.9, 8.9, 8.9 Hz), 8.05 (ddd, 1H, 1.6, 7.8,
H
(
C
21
H
22
N
6
O
2
F
2
3
OD) δ 8.71 (br d, 1H, 4.6 Hz), 8.56 (dd, 1H, 0.9, 5.3
+
Found 44.20, 3.28, 12.37; LRMS 429.2 (MH) .

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 471
7
7
4
2
4
.8 Hz), 7.83 (d, 1H, 8.1 Hz), 7.81 (m, 1H), 7.61 (dd, 1H, 5.1,
.3 Hz), 6.68 (d, 1H, 0.9 Hz), 4.88 (s, 2H), 4.74 (d, 2H, 1.3 Hz),
(d, 2H, 1.1 Hz), 4.35 (t, 2H, 13.8 Hz); Anal. (C19
ClF ‚2.50HCl‚0.45CH Cl ) C, H, N. Calcd 40.12, 3.36, 14.44.
Found 40.12, 3.16, 14.53.
-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
eth yla m in o)-6-cya n op yr a zin -2-on e-1-a ceta m id e Hyd r o-
ch lor id e (25). Prepared according to procedure for 24 [using
52b a cid (24 mg, 0.08 mmol); 2-aminomethyl-3-fluoropyridine
dihydrochloride salt (24 mg, 0.12 mmol; 20 mg); DMF (1 mL);
EDC (23 mg, 0.12 mmol; 25 mg); HOAT (16 mg, 0.12 mmol);
16 6 2
H N O -
3
2
2
.44 (t, 2H, 14.4 Hz), 2.25 (d, 3H, 0.8 Hz); Anal. (C20
19 6 2 3
H N O F ‚
.50 HCl‚0.70 MeOH) C, H, N. Calcd 45.53, 4.49, 15.39. Found
3
+
5.22, 4.36, 15.39; LRMS 433.4 (MH) .
3
-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
eth yla m in o)p yr a zin -2-on e-1-a ceta m id e (22). Prepared ac-
cording to procedure for 18 [using 52c a cid (91 mg, 0.29
mmol); 2-aminomethyl-3-fluoropyridine dihydrochloride salt
(70 mg, 0.35 mmol); DMF (1 mL); EDC (59 mg, 0.31 mmol);
triethylamine (56 µL; 50 µL)] to give a yellow solid (19 mg):
1
HOBT (42 mg, 0.31 mmol); triethylamine (363 mg, 3.6 mmol)]
to give 40 mg of the title compound as a white solid. Conversion
to the hydrochloride salt can be carried out by treating an
EtOAc solution with 2.0 M HCl in EtOAc, followed by
concentration. Recrystallization from hot 2-propanol gave 18
mg of pure material: H NMR (CD
3
H NMR (CDCl ) δ 8.66 (d, 1H, 4.8 Hz), 8.34 (dd, 1H, 1, 4.8
Hz), 7.84 (ddd, 1H, 1.5, 7.8, 7.8 Hz), 7.70 (dd, 1H, 1, 7.9 Hz),
7.44-7.39 (m, 3H), 7.33 (m, 2H), 7.26 (m, 2H), 4.83 (s, 2H),
4.68 (d, 2H, 4.4 Hz), 4.44 (dt, 2H, 6.5, 13.1 Hz); LRMS 444.5
+
(MH) ; t
R
) 2.02 min (98% @ 215 and 254 nm, system A) and
1
OD) δ 8.71 (br d, 1H, 4.8
t
R
) 1.62 min (97% @ 215 and 96% @254 nm, system B); HRMS
(ES) calcd C20 (M + 1) 444.1390, found 444.1362.
3
Hz), 8.58 (dd, 1H, 1.1, 5.5 Hz), 8.12 (dd, 1H, 8.6, 8.6 Hz), 8.05
ddd, 1H, 1.7, 7.8, 7.8 Hz), 7.85 (m, 1H), 7.62 (dd, 1H, 4.9, 7.6
Hz), 7.10 (d, 1H, 5.7 Hz), 6.79 (d, 1H, 5.7 Hz), 4.79 (s, 2H),
.75 (s, 2H), 4.47 (t, 2H, 14.4 Hz); Anal. (C19 ‚2.50
HCl‚0.10 EtOAc) C, H, N. Calcd 45.07, 4.00, 16.26. Found
16 7 2 3
H N O F
(
3-Ch lor o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
eth yla m in o)-6-ch lor op yr a zin -2-on e-1-a ceta m id e (26). To
a stirred solution of 50 mg (0.19 mmol) 52d a cid and 62 mg
(0.29 mmol) of 2-aminomethyl-3-chloropyridine dihydrochlo-
ride in 1 mL of DMF were added 56 mg (0.29 mmol) of EDC,
4
17 6 2 3
H N O F
+
4
4.74, 4.21, 15.98; LRMS 419.1 (MH) .
3
-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
39 mg (0.29 mmol) of HOAT, and 0.50 mL Et
overnight, the volatiles were removed in vacuo. The residue
was diluted with saturated aqueous NaHCO , filtered, and
3
N. After stirring
e t h yla m in o)-6-t r iflu or om e t h ylp yr a zin -2-on e -1-a ce t a -
m id e (23). To a stirred solution of 50 mg (remainder LiCl,
3
rinsed with water to afford a solid. Conversion to the HCl salt
was accomplished by diluting the free base with MeOH (5 mL),
treating with 2.5 M HCl (2 mL), and concentrating to a yellow
0
6
(
.13 mmol) of 3-(2,2-difluoro-2-(2-pyridyl-N-oxide-ethylamino)-
-trifluoromethylpyrazin-(1H)-2-one-1-acetic acid31 and 25 mg
0.13 mmol) 2-aminomethyl-3-fluoropyridine dihydrochloride
solid (75 mg): 1H NMR (CD
OD) δ 8.77 (d, 1H, 5.0 Hz), 8.74
in 1 mL of DMF were added 24 mg (0.13 mmol) of EDC, 14
mg (0.10 mmol) of HOAT, and 42 µL (0.3 mmol) of triethy-
lamine. After stirring for 1 h, 25 mg of EDC was added and
the reaction was allowed to stir overnight. The volatiles were
removed at reduced pressure, the residue partitioned between
3
(d, 1H, 5.7 Hz), 8.57 (d, 1H, 8.3 Hz), 8.23 (dd, 1H, 7.8, 7.8 Hz),
7
5
4
.94 (m, 2H), 7.78 (dd, 1H, 6.4, 6.4 Hz), 6.89 (d, 1H, 1.6 Hz),
.0 (d, 2H, 1.1 Hz), 4.80 (s, 2H), 4.40 (t, 2H, 13.4 Hz); LRMS
68.8 (MH) .
+
CH
backwashed with CH
were dried over MgSO
pressure. To a solution of this material in 5 mL of EtOH was
added 10 mg 10% Pd/C and exposed to a balloon atmosphere
of hydrogen. After 3 h, the reaction was filtered through Celite
with EtOH and the filtrate concentrated. The residue was flash
2
Cl
2
/saturated aqueous NaHCO
Cl
(2×). The combined organic layers
and the solvents removed at reduced
3
and the aqueous layer
4-Meth yl-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
2
2
eth ylam in o)-6-ch lor opyr azin -2-on e-1-acetam ide (27). Pre-
pared according to procedure for 18 [using 52d acid (75 mg,
0.21 mmol); 2-aminomethyl-4-methylpyridine dihydrochloride
salt (49 mg, 0.25 mmol); DMF (1 mL); EDC (41 mg, 0.22 mmol);
HOBT (30 mg, 0.22 mmol); triethylamine (213 mg, 2.1 mmol)]
to give 40 mg of the title compound as a yellow solid.
Conversion to the hydrochloride salt can be carried out by
treating an EtOAc solution with 1.2 M HCl in EtOAc, followed
by concentration. Recrystallization from hot 2-propanol gave
4
chromatographed using 1 to 10% MeOH:CH
2
Cl
) δ 8.67 (d, 1H, 4.8 Hz),
.32 (d, 1H, 4.6 Hz), 7.84 (ddd, 1H, 1.4, 7.7, 7.7 Hz), 7.70 (d,
H, 7.9 Hz), 7.4 (m, 3H), 7.26 (m, 1H), 7.19 (br t, 1H), 7.06 (br
2
to give 35 mg
1
of the title compound: H NMR (CDCl
8
1
3
30 mg of pure material: 1H NMR (CD
OD) δ 8.66 (d, 1H, 4.9
3
t, 1H, 6.1 Hz), 4.80 (s, 2H), 4.653 (d, 2H, 3.4 Hz), 4.46 (td, 2H,
Hz), 8.60 (d, 1H, 6.1 Hz), 7.98 (dd, 1H, 7.8, 7.8 Hz), 7.87 (br s,
1H), 7.81 (d, 1H, 6.0 Hz), 7.74 (dd, 1H, 0.9, 8.0 Hz), 7.55 (dd,
1H, 6.2, 6.2 Hz), 6.89 (d, 1H, 0.9 Hz), 4.98 (s, 2H), 4.72 (d, 2H,
+
1
3.7, 6.4 Hz); LRMS 487.3 (MH) ; t
R
) 2.34 min (97% @ 215
and 98% @ 254 nm, system A) and t
R
) 1.90 min (95% @ 215
and 98% @ 254 nm, system B); HRMS (ES) calcd C20
(M + 1) 482.1312, found 487.1280.
-F lu or o-2-p yr id ylm eth yl 3-(2,2-Diflu or o-2-(2-p yr id yl)-
H
16
N
6
O
2
3.5 Hz), 4.31 (t, 2H, 14.1 Hz), 2.67 (s, 3H); Anal. (C20
Cl‚2.40HCl‚2.10H O) C, H, N. Calcd 41.83, 4.49, 14.64. Found
2.23, 4.27, 14.25; LRMS 449.1 (MH) .
3-F lu or o-4-m et h yl-2-p yr id ylm et h yl 3-(2,2-Diflu or o-2-
19 6 2 2
H N O F -
F
6
2
+
4
3
eth yla m in o)-6-ch lor op yr a zin -2-on e-1-a ceta m id e (24). To
a stirred solution of 330 mg (0.536 mmol) of 52d a cid and
(2-p yr id yl)et h yla m in o)-6-ch lor op yr a zin -2-on e-1-a cet a -
m id e (28). To a stirred solution of 80 mg (0.197 mmol,
remainder KCl) of 52d a cid and 63 mg (0.296 mmol) of
2-aminomethyl-3-fluoro-4-methylpyridine dihydrochloride in
1 mL of DMF were added 77 mg (0.40 mmol) of EDC, 54 mg
(0.40 mmol) of HOAT, and 0.13 mL (0.89 mmol) TEA. After
stirring overnight, the volatiles were removed in vacuo. The
1
60 mg (0.805 mmol) of 2-aminomethyl-3-fluoropyridine di-
hydrochloride in 2 mL of DMF were added 128 mg (0.67 mmol)
of EDC, 91 mg (0.67 mmol) of HOAT, and 0.30 mL (2.1 mmol)
triethylamine. After stirring overnight, 80 mg of 2-amino-
methyl-3-fluoropyridine dihydrochloride, 128 mg of EDC, 91
mg of HOAT, and 0.08 mL (2.1 mmol) triethylamine were
added. The reaction was stirred for an additional 24 h, and
the volatiles were removed en vacuo. The residue was diluted
residue was diluted with saturated aqueous NaHCO
and rinsed with water to afford a brown solid. This material
was flash chromatographed using 2-4% CH OH: CH Cl to
3
, filtered,
with saturated aqueous NaHCO
and filtered to afford a tan solid. This material was flash
chromatographed using 1:99 to 5:95 MeOH-CH Cl to give a
) δ 8.67 (d, 1H, 4.6 Hz), 8.32 (d,
H, 4.6 Hz), 7.81 (dd, 1H, 7.8, 6.4 Hz), 7.69 (d, 1H, 7.8 Hz),
3
(10 mL) and water (15 mL)
3
2
2
afford the title compound as a white solid. Conversion to the
HCl salt was accomplished by diluting the free base with
MeOH (5 mL), treating with 2.5 M HCl (2 mL), and concen-
2
2
1
yellow solid: H NMR (CDCl
3
trating to a solid (57.5 mg): 1H NMR (CD
OD) δ 8.82 (d, 1H,
4.1 Hz), 8.56 (d, 1H, 5.1 Hz), 8.33 (dd, 1H, 7.4, 7.4 Hz), 8.04
d, 1H, 7.8 Hz), 8.00 (br t, 1H), 7.87 (br t, 1H, 5.6 Hz), 6.92 (s,
1
7
1
1
3
.41-7.37 (m, 2H), 7.27-7.22 (m, 2H), 6.97 (s, 1H), 6.49 (br t,
H, 6.0 Hz), 4.93 (s, 2H), 4.66 (d, 2H, 4.0 Hz), 4.38 (td, 2H,
3.9, 6.5 Hz). Conversion to the hydrochloride salt can be
(
1
3
H), 5.0 (s, 2H), 4.80 (s, 2H), 4.44 (t, 2H, 13.6 Hz), 2.621 (s,
H).
carried out by diluting the product with ethyl acetate (2 mL)
and treating with 5 mL of 1.5 M HCl in ethyl acetate, followed
3-F lu or o-4-m et h yl-2-p yr id ylm et h yl 3-(2,2-Diflu or o-2-
by filtration to afford a white powder (221 mg): 1H NMR (CD
-
(2-p yr id yl)et h yla m in o)-6-cya n op yr a zin -2-on e-1-a cet a -
m id e Hyd r och lor id e (29). Prepared according to procedure
for 24 [using 52b a cid (60 mg, 0.16 mmol, remainder LiCl);
2-aminomethyl-3-fluoro-4-methylpyridine dihydrochloride (45
3
OD) δ 8.72 (d, 1H, 4.6 Hz), 8.56 (dd, 1H, 0.9, 5.3 Hz), 8.16-
.11 (m, 2H), 7.87 (d, 1H, 8.1 Hz), 7.80 (ddd, 1H, 4.8, 4.8, 9.0
Hz), 7.68 (dd, 1H, 5.4, 7.2 Hz), 6.87 (s, 1H), 4.95 (s, 2H), 4.74
8

4
72 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Burgey et al.
mg, 0.21 mmol; 45 mg); DMF (1 mL); EDC (40 mg, 0.21 mmol;
0 mg); HOAT (22 mg, 0.16 mmol); triethylamine (112 µL, 0.80
(9) Hirsh, J . Synthetic Oral Thrombin Inhibitors: New Promises
for Cardiovascular Therapy. Presented at the VII Congress
International Society of Thrombosis and Haemostasis, J uly
4
1
mmol; 60 µL)] to give a yellow foam (41 mg): H NMR (CDCl
δ 8.67 (d, 1H, 4.8 Hz), 8.18 (d, 1H, 1, 4.9 Hz), 7.84 (ddd, 1H,
.7, 7.7, 7.7 Hz), 7.70 (dd, 1H, 1.0, 8.0 Hz), 7.44 (s, 1H), 7.42
m, 1H), 7.35 (br t, 1H), 7.32 (br t, 1H, 6.6 Hz), 7.10 (dd, 1H,
3
)
6
-12, 2001, Paris, France; Abstract SY111. J ohansson, L. C.;
Eriksson, L.; Frison, L.; Fager, G. A Comparison of the Phar-
macokinetics of Ximelagatran in Young and Elderly Healthy
Subjects. Presented at the VII Congress International Society
of Thrombosis and Haemostasis, J uly 6-12, 2001, Paris, France;
Abstract P782.
1
(
5
6
.4, 5.4 Hz), 4.82 (s, 2H), 4.65 (d, 2H, 4.3 Hz), 4.45 (dt, 2H,
.5, 13.7 Hz), 2.32 (d, 3H, 1.3 Hz); LRMS 458.1 (MH) .
+
(
10) Sanderson, P. E. J .; Lyle, T. A.; Cutrona, K. J .; Dyer, D. L.;
Dorsey, B. D.; McDonough, C. M.; Naylor-Olsen, A. M.; Chen,
I-W.; Chen, Z.; Cook, J . J .; Cooper, C. M.; Gardell, S. J .; Hare,
T. R.; Krueger, J . A.; Lewis, S. D.; Lin, J . H.; Lucas, B. J .; Lyle,
E. A.; Lynch, J . J .; Stranieri, M. T.; Vastag, K.; Yan, Y.; Shafer,
J . A.; Vacca, J . P. Efficacious, Orally Bioavailable Thrombin
Inhibitors Based on 3-Aminopyridinone or 3-Aminopyrazinone
Acetamide Peptidomimetic Templates. J . Med. Chem. 1998, 41,
3
-Meth oxy-2-pyr idylm eth yl 3-(2,2-Diflu or o-2-(2-pyr idyl)-
eth ylam in o)-6-ch lor opyr azin -2-on e-1-acetam ide (30). Pre-
pared according to procedure for 24 [using 52d a cid (62 mg,
0
.18 mmol); 2-aminomethyl-3-methoxypyridine dihydrochlo-
ride (52 mg, 0.27 mmol); DMF (1 mL); EDC (52 mg, 0.27
mmol); HOAT (37 mg, 0.27 mmol); NMM (200 µL)]. Conversion
to the HCl salt was accomplished by diluting the free base with
4
466-4474.
MeOH (5 mL), treating with 2.5 M HCl (2 mL), and concen-
(
11) Sanderson, P. E.J ; Stanton, M. G.; Balani, S. K. U.S. Patent No.
6,147,078, 2000. Riffel, K. A.; Hengchang, S.; Gu, X.; Yan, K.;
Lo, M.-W. Simultaneous Determination of a Novel Thrombin
Inhibitor and its Two Metabolites in Human Plasma by Liquid
Chromatography/Tandem Mass Spectrometry. J . Pharm. Biomed.
Anal. 2000, 23, 607-616.
1
trating to an off-white solid (45 mg): H NMR (CD
(
3
OD) δ 8.79
d, 1H, 4.8 Hz), 8.33 (d, 1H, 5.5 Hz), 8.27 (m, 2H), 8.00 (m,
2
H), 7.83 (dd, 1H, 5.5, 7.1 Hz), 6.91 (s, 1H), 4.99 (s 2H), 4.73
+
(s, 2H), 4.42 (t, 2H, 13.2 Hz), 4.13 (s, 3H); LRMS 465.1 (MH) .
4
-Meth oxy-2-pyr idylm eth yl 3-(2,2-Diflu or o-2-(2-pyr idyl)-
(
12) Lewis, S. D.; Ng, A. S.; Lyle, E. A.; Mellott, M. J .; Appelby, S.
D.; Brady, S. F.; Stauffer, K. S.; Sisko, J . T.; Mao, S.-S.; Veber,
D. F.; Nutt, R. F.; Lynch, J . J .; Cook, J . J .; Gardell, S. J .; Shafer,
J . A. Inhibition of Thrombin by Peptides Containing Lysyl-R-
Keto Carbonyl Derivatives. Thromb. Haemost. 1995, 74, 1107-
eth ylam in o)-6-ch lor opyr azin -2-on e-1-acetam ide (31). Pre-
pared according to procedure for 24 [using 52d a cid (86 mg,
0
.25 mmol); 2-aminomethyl-4-methoxypyridine dihydrochlo-
ride (66 mg, 0.31 mmol); DMF (2 mL); EDC (80 mg, 0.26
mmol); HOAT (35 mg, 0.26 mmol); NMM (200 µL)]. Conversion
to the HCl salt was accomplished by diluting the free base with
1
112.
(
13) For a discussion of the general criteria for thrombin inhibitor
development see: Tucker, T.J ; Isaacs, R. C. A. The Development
of Novel Noncovalent Thrombin Inhibitors. Adv. Amino Acid
Mimetics Peptidomimetics 1999, 2, 53-87.
14) Lumma, W. C.; Witherup, K. M.; Tucker, T. J .; Brady, S. F.;
Sisko, J . T.; Naylor-Olsen, A. M.; Lewis, S. D.; Lucas, B. J .;
Vacca, J . P. Design of Novel, Potent, Noncovalent Inhibitors of
Thrombin with Nonbasic P-1 Substructures: Rapid Structure-
Activity Studies by Solid-Phase Synthesis. J . Med. Chem. 1998,
MeOH (5 mL), treating with 2.5 M HCl (2 mL), and concen-
1
trating to a white solid (18 mg): H NMR (CD
1
3
OD) δ 8.74 (d,
(
H, 5.3 Hz), 8.55 (d, 1H, 7.7 Hz), 8.17 (ddd, 1H, 3.7, 7.7, 7.7
Hz), 7.90 (d, 1H, 8.0 Hz), 7.72 (dd, 1H, 5.4, 7.4 Hz), 7.42 (m,
H), 6.91 (s, 1H), 5.00 (s 2H), 4.70 (s, 2H), 4.37 (t, 2H, 13.7
2
+
Hz), 4.15 (s, 3H); LRMS 465.1 (MH) .
4
1, 1011-1013.
Ack n ow led gm en t. We thank the analytical chem-
istry, mass spectrometry, and NMR analysis groups for
their assisatance, as well as Carl Homnick for his
skillful HPLC separations and Cathy Wiscount for
optimization of the synthesis of 49.
(15) Tucker, T. J .; Lumma, W. C.; Lewis, S. D.; Gardell, S. J .; Lucas,
B. J .; Baskin, E. P.; Woltmann, R.; Lynch, J . J .; Lyle, E. A.;
Appleby, S. D.; Chen, I.-W.; Dancheck, K. B.; Vacca, J . P. Potent
Noncovalent Thrombin Inhibitors That Utilize the Unique
Amino Acid D-Dicyclohexylalanine in the P3 Position. Implica-
tions on Oral Bioavailability and Antithrombotic Efficacy. J .
Med. Chem. 1997, 40, 1565-1569.
16) Reported as number of carotid artery vesssels (n ) 6) occluding
after a 10 µg/kg/min 180 min infusion (arterial injury is initiated
after 120 min, followed by a 60 min observation period). Reflow
indicates that the vessel occluded then subsequently reopened.
Kurz, M. D.; Main, B. W.; Sandusky, G. E. Rat Model of Arterial
Thrombsosis Induced by Ferric Chloride. Thromb. Res. 1990, 60,
(
Su p p or tin g In for m a tion Ava ila ble: Procedures for the
preparation of the P1 pyridines (36-41, Scheme 2) and the
X-ray crystallography. This material is available via the
Internet at http://pubs.acs.org.
2
69-280. See also ref 12.
Refer en ces
(
(
(
17) Altuntas, T. G.; Gorrod, J . W. Factors Involved in the N-
Oxidation of Isomeric Aromatic Diazines by Microsomal Prepa-
rations. Drug Metab. Drug Interact. 1995, 12, 117-130.
18) Park, B. K.; Kitteringham, N. R. Effects of Fluorine Substitution
on Drug Metabolism: Pharmacological and Toxicological Impli-
cations. Drug Metab. Rev. 1994, 26, 605-643.
19) Hunter, C. A.; Sanders, J . K. M. The Nature of π-π Interactions.
J . Am. Chem. Soc. 1990, 112, 5525-5534.
20) Singh, R.; Wong, B. Unpublished results.
21) Isaacs, R. C. A.; Cutrona, K. J .; Newton, C. L.; Sanderson, P. E.
J .; Solinsky, M. G.; Baskin, E. A.; Chen, I.-W.; Cooper, C. M.;
Cook, J . J .; Gardell, S. J .; Lewis, S. D.; Lucas, B. J .; Lyle, E. A.;
Lynch, J . J .; Naylor-Olsen, A. M.; Stranieri, M. T.; Vastag, K.;
Vacca, J . P. C6 Modification of the Pyridinone Core of Thrombin
Inhibitor L-374,087 as a Means of Enhancing Its Oral Absorp-
tion. Bioorg. Med. Chem. Lett. 1998, 8, 1719-1724.
(22) The X-ray structure of 24 bound to thrombin revealed that the
6-chloro group effectively occupies the lipophilic insertion loop
and all other enzyme-inhibitor interactions are essentially the
same as in the methylpyrazinone analogue 20 (see Supporting
Information).
(23) The low oral bioavailability observed in rhesus monkey was
determined to be due to extensive first-pass hepatic elimination.
(24) Wong, B. Unpublished results.
(25) Fleitz, F. J .; Lyle, T. A.; Zheng, N.; Armstrong, J . D., III; Volante,
R. P. Kilogram Scale Synthesis of the Pyrazinone Acetic Acid
Core of an Orally Efficacious Thrombin Inhibitor. Synth. Com-
mun. 2000, 30, 3171-3180.
(26) Matsumura, Y.; Nakano, T.; Morisawa, Y. Preparation of
Electron-withdrawing Group-containing R,R-difluoro Compounds.
Patent No. J P 09110729 A2 970428, 1995.
(1) Thrombosis in Cardiovascular Disorders; Fuster, V., Verstraete,
M., Eds.; W. B. Saunders Co.: Philadelphia, 1992.
(
2) Stein, P. D.; Henry, J . W. Prevalence of Acute Pulmonary
Embolism Among Patients in a General Hospital and at Autopsy.
Chest 1995, 108, 978-981.
(
3) Adang, A. E. P.; Rewinkel, J . B. M. A New Generation of Orally
Active Antithrombotics: Comparing Strategies in the GPIIb/IIIa,
Thrombin and Factor Xa Areas. Drugs Future 2000, 25, 369-
(
(
3
83.
(
(
(
4) Goldsack, N. R.; Chambers, R. C.; Dabbag, K.; Laurent, J .
Molecules in Focus: Thrombin. Int. J . Biochem. Cell Biol. 1998,
3
0, 641-646.
5) Rewinkel, J . B. M.; Adang, A. E. P. Strategies and Progress
Toward the Ideal Thrombin Inhibitor. Curr. Pharm. Design
1
999, 5, 1043-1075.
6) Coburn, C. A. Small-molecule Direct Thrombin Inhibitors:
1
997-2000. Exp. Opin. Ther. Pat. 2001, 11, 1-18. Sanderson,
P. E. J . Chpater 8. Anticoagulants: Inhibitors of Thrombin and
Factor Xa. Annu. Reports Med. Chem. 2001, 36, 79-88.
7) For example p-aminobenzamidine has a K ) 300 nM against
i
(
thrombin: Hilpert, K.; Ackerman, J .; Banner, D. W.; Gast, A.;
Gubernator, K.; Hadvary, P.; Labler, L.; M u¨ ller; K.; Schmid, G.;
Tschopp, T. B.; Waterbeemd, H.v.d. Design and Synthesis of
Potent and Highly Selective Thrombin Inhibitors. J . Med. Chem.
1
994, 37, 3889-390
(
8) These groups have also been implicated in provoking untoward
cardiovascular effects (e.g. hypotension): Hauptmann, J .; Mark-
wardt, F. Pharmacologic Aspects of the Development of Selective
Synthetic Thrombin Inhibitors as Anticoagulants. Semin. Throm-
bosis Hemostasis 1992, 200-217. Schumacher, W. A.; Bala-
subramanian, N.; St.Laurent, D. R.; Seiler, S. M. Effect of a
Novel Thrombin Active-site Inhibitor on Arterial and Venous
Thrombosis. Eur. J . Pharmacol. 1994, 259, 165-171.
(27) Cop e´ ret, C.; Adolfsson, H.; Khuong, T-A. V.; Yudin, A. K.;
Sharpless, K. B. A Simple and Efficient Method for the Prepara-
tion of Pyridine N-Oxides. J . Org. Chem. 1998, 63, 1740-1741.

3
-Aminopyrazinone Acetamide Thrombin Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 473
(
28) Sakamoto, T.; Kaneda, S-I.; Nishimura, S.; Yamanaka, H. Site-
Selectivity in the Cyanation of 3-Substituted Pyridine 1-Oxides
with Trimethylsilanecarbonitrile. Chem. Pharm. Bull. 1985, 33,
(30) Sanderson, P. E.J ; Lyle, T. A.; Dorsey, B. D. WO 99/11267, 1999.
(31) Burgey, C. S.; Isaacs, R. C. A.; Dorsey, B. D.; Robinson, K. A.;
Staas, D. D.; Sanderson, P. E. J .; Barrow, J . C. WO 01/38323,
2001.
5
65-571.
(
29) Cheeseman, G. W. H.; Freestone, A. J .; Godwin, R. A.; Hough,
T. L. Experiments on the Synthesis of Pyrazine Nucleosides. J .
Chem. Soc., Perkin Trans. 1 1975, 1888-1891.
J M020311F