Design, parallel synthesis, and crystal structures of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Article abstract of DOI:10.1021/jm030131l

Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-α-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S₁, S₂, and S₃ pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P₁, P₂, and P₃ moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC₅₀) activity on TF/VIIa with >6250x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.

Full text of DOI:10.1021/jm030131l

4050
J . Med. Chem. 2003, 46, 4050-4062
Design , P a r a llel Syn th esis, a n d Cr ysta l Str u ctu r es of P yr a zin on e
An tith r om botics a s Selective In h ibitor s of th e Tissu e F a ctor VIIa Com p lex
J ohn J . Parlow,*,† Brenda L. Case,† Thomas A. Dice,† Ricky L. Fenton,† Michael J . Hayes,† Darin E. J ones,†
William L. Neumann,† Rhonda S. Wood,† Rhonda M. Lachance,‡ Thomas J . Girard,‡ Nancy S. Nicholson,§
Michael Clare,| Roderick A. Stegeman, Anna M. Stevens, William C. Stallings, Ravi G. Kurumbail, and
Michael S. South†
Department of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Boulevard,
St. Louis, Missouri 63167, Department of Cardiovascular and Metabolic Disease, Pharmacia Corporation,
800 North Lindbergh Blvd., St. Louis, Missouri 63167, Department of Cardiovascular Pharmacology, Pharmacia Corporation,
4901 Searle Parkway, Skokie, Illinois 60077, Structure and Computational Chemistry, Pharmacia Corporation,
4901 Searle Parkway, Skokie, Illinois 60077, and Structure and Computational Chemistry, Pharmacia Corporation,
700 Chesterfield Village Parkway, St. Louis, Missouri 63198
Received March 20, 2003
Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library
synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor
VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-R-ketothiazole complexed with
TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting
scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement
to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was
designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds
of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme
assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/
VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity
relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of
selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately
led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with
>6250× selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of
TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the
separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate
model of electrolytic-induced arterial thrombosis.
In tr od u ction
been the subject of intense research because of their
potential to inhibit the coagulation cascade.19-32 There
is evidence from our own laboratories, as well as others,
that selective inhibition of the TF/VIIa complex may
provide effective anticoagulation while lessening the
risk of the bleeding side effects associated with other
antithrombotic mechanisms, such as glycoprotein IIb/
IIIa antagonists and inhibitors of Factor Xa and
thrombin.33-36 Therefore, structure-based drug design
(SBDD) and parallel medicinal chemistry were used to
develop a series of potent and selective TF/VIIa inhibi-
tors to address the large, unmet medical need for safe
and effective oral anticoagulants.
Blood coagulation is initiated upon vascular injury
when plasma Factor VIIa comes in contact with its
essential cofactor, Tissue Factor (TF), which is an
integral membrane protein not normally in contact with
blood. The TF/VIIa complex proteolytically activates its
substrates, Factors IX and X. This triggers a cascade of
reactions, ultimately resulting in the generation of
thrombin and a fibrin clot.1-10 Disruption of atheroscle-
rotic plaques, and the subsequent release of plasma
factor VIIa leading to occlusive thrombus formation, is
the main cause of the acute syndromes of unstable
angina, myocardial infarction, and sudden death.11,12
Effective and safe antithrombotics are needed to combat
these diseases. Most research has focused on thrombin
and Factor Xa inhibitors as potentially valuable thera-
peutic agents for these diseases.13-18 Since TF/VIIa
triggers the thrombotic process, this complex is a prime
target for intervention in these vascular diseases.
Recently, small molecule inhibitors of TF/VIIa have
We recently described the synthesis and structure-
activity relationship (SAR) of a series of tripeptide-R-
ketothiazoles.37,38 These reversible, covalent, serine
protease inhibitors had nanomolar potency against TF/
VIIa and exhibited 500× selectivity vs thrombin (Figure
1). This study demonstrated that it is possible to achieve
potent inhibition of the TF/VIIa complex while main-
taining selectivity against other highly homologous
serine proteases in the coagulation cascade. Several
reports of small molecule TF/VIIa inhibitors have ap-
peared, with varying degrees of selectivity vs the other
plasma proteases in the coagulation cascade.19,21,22,24,25,29
We hypothesized that a wider selectivity margin over
* To whom correspondence should be addressed. Phone: 314-274-
3494. Fax: 314-274-1621. e-mail: john.j.parlow@pfizer.com.
† Department of Medicinal and Combinatorial Chemistry.
‡ Department of Cardiovascular and Metabolic Disease.
§ Department of Cardiovascular Pharmacology.
| Structure and Computational Chemistry.
Structure and Computational Chemistry.
10.1021/jm030131l CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/09/2003

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4051
F igu r e 1. Ketothiazole TF/VIIa inhibitor.
Factor Xa and thrombin was necessary to unequivocally
demonstrate that inhibition of TF/VIIa would lead to a
lower bleeding side effect relative to that observed with
Factor Xa and thrombin inhibitors. These highly selec-
tive TF/VIIa inhibitors should then be suitable for
preclinical, intravenous proof-of-concept studies dem-
onstrating the separation between antithrombotic ef-
ficacy and bleeding side effects in a primate model of
thrombosis.39 The structural information obtained from
the tripeptide-R-ketothiazoles bound to TF/VIIa, com-
bined with the crystal structure of other related serine
proteases such as thrombin and Factor Xa, facilitated
the development of a series of highly selective, small
molecule inhibitors of TF/VIIa that were nonpeptidic
and did not interact with the catalytic apparatus of the
enzyme.40,41 Herein, we report the progression from a
peptidic lead to the development of noncovalent, highly
selective small molecule inhibitors of the TF/VIIa
complex utilizing SBDD coupled with PASP library
synthesis.42 The structure-activity relationship will be
discussed, and X-ray crystal structures of selected
compounds complexed with the TF/VIIa enzyme will be
described.
F igu r e 2. Crystal structure of ketothiazole 1 bound in the
active site of TF/VIIa complex. Some of the key side chains of
Factor VIIa are displayed (C: green, N: dark blue, O: red, S:
yellow, H: orange). The inhibitor is represented with carbon,
nitrogen, oxygen, and sulfur atoms displayed in yellow, blue,
red, and green, respectively. The hydrogen bonds formed by
the inhibitor and a bound water molecule in the S2 site are
shown as dotted white lines. The close interaction between the
bound solvent and the pyridyl nitrogen of the inhibitor is
shown as a dotted green line. The active site serine, Ser 195,
forms a covalent bond, shown as a thin solid line, with the
activated carbon of the inhibitor.
Resu lts a n d Discu ssion
Information obtained from various crystal structures
of TF/VIIa,37,38,43-46 thrombin,47,48 and Factor Xa49-53
was used to develop the initial small molecule inhibitors
of TF/VIIa. The X-ray crystal structure of the tripeptide-
R-ketothiazole 1 bound to the TF/VIIa enzyme is shown
in Figure 2. Excluding the catalytic apparatus, several
key interactions were crucial for both potency and
selectivity over thrombin and Factor Xa. The arginine
side chain at P1 forms four strong hydrogen bonds at
the bottom of the S1 pocket with the side chains of Asp
189 and Ser 190 as well as to the backbone carbonyl
oxygen of Gly 219. The inhibitor forms two other
hydrogen bonds with the peptide nitrogens of Gly 216
and Gly 219. The S2 pocket of Factor VIIa is relatively
open and has a negative potential due to the presence
of Asp 60. The pyridyl group of the inhibitor takes
advantage of these structural features to form strong
interactions in the S2 pocket of VIIa. Among the
coagulation proteases, only Factor VIIa has a negatively
charged residue at position 60. In contrast, Factor Xa
has a tyrosine at position 60. Moreover, the S2 pocket
of Factor Xa is occluded by the side chain of Tyr 99.
Thrombin has a large insertion in the S2 pocket with a
number of aromatic amino acid residues. These struc-
tural differences account for the enhanced selectivity
of compound 1 for TF/VIIa over thrombin and Factor
Xa. It was theorized that the ketothiazole moiety was
contributing to a loss of selectivity, since this moiety
captures the catalytic apparatus of all of the homologous
F igu r e 3. Pyrazinone core structure.
serine proteases. As a result, the intended inhibitors
were designed such that they did not interact with the
catalytic apparatus.
Using these key features of the TF/VIIa crystal
structure, a series of six-membered heterocyclic cores
were docked, which led to the pyrazinone scaffold as a
prototype inhibitor to begin our SBDD studies (Figure
3). The pyrazinone core orients the substituents in the
correct spatial arrangement to probe the S1, S2, and S3
pockets of the enzyme. In addition, the NH of the P3,
the NH of the glycine, and the carbonyl of the pyrazi-
none core provide potential hydrogen bonds to the
peptide backbone of the enzyme. The pyrazinone core
had previously been used in the design of inhibitors for
thrombin54-58 and is bound in the enzyme in a similar
fashion.
Several pyrazinone cores were prepared according to
a literature procedure to provide building blocks for
library synthesis.50,51,53,54 The synthesis involved a

4052 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Sch em e 1. Pyrazinone Core Structure Synthesisa
Parlow et al.
afforded desired products 6. Rather than purify at this
step, the crude reaction mixture was carried on to the
next step.
The second step of the synthesis involved a series of
manipulations in the same reaction vessel. The benzyl
ester 6 was hydrolyzed by adding a solution of potas-
sium hydroxide in methanol to each reaction vessel.
Upon completion of hydrolysis, the reaction mixture was
acidified with 4 N hydrochloric acid in dioxane and then
evaporated to dryness. Alternatively, when the P2
phenyl ring contained an ester functionality, the pyrazi-
none 6 was treated with hydrogen and palladium on
carbon to remove the benzyl ester. In the initial studies,
the resulting pyrazinone carboxylic acid was captured
onto polycarbonate resin. However, this resulted in
significant formation of byproducts. Polyamine resin 8
successfully captured the pyrazinone carboxylic acid
intermediate without causing the formation of the
byproducts. Each mixture was then poured directly onto
a disposable filter, and the product-containing resin 9
was washed sequentially with a variety of solvents to
remove the benzyl alcohol, amine hydrochloride, potas-
sium chloride, and other impurities that were present.
The resin was dried and the acids were released from
the resin with 4 N hydrochloric acid in dioxane. The
pyrazinone carboxylic acid intermediates 7 were ob-
tained in pure form after filtration and evaporation.
a
Reagents: (a) TMSCN, P2CHO 3, CH2Cl2; (b) (ClCO)2, 1,2-
dichlorobenzene.
modified Strecker reaction between glycine benzyl ester
2, trimethylsilylcyanide, and an aldehyde 3, followed by
cyclization of the intermediate with oxalyl chloride to
give the desired dichloropyrazinone 4 (Scheme 1). The
P2 diversity element was contained in the starting
aldehyde and thus was hardwired in place at the
beginning of the synthesis. The aldehyde inputs were
biased such that the final products would mimic the
phenylalanine P2 analogues of the ketothiazole series.
In an effort to rapidly prepare libraries of pyrazinones
in a parallel format, a solution-phase library synthesis
was developed using PASP technology.61-63 A summary
description of the library synthesis is depicted in
Scheme 2. The goal of the library synthesis was to use
the PASP synthesis technology in such a way as to
eliminate the need for chromatography and isolation of
intermediates. The synthesis was accomplished using
a combination of polymeric reagents and PASP purifica-
tion concepts, including reactant-sequestering resins
and resin capture-release. To maximize the efficiency
of the multistep sequence and to minimize manual
manipulation of the reaction vessels, multiple reaction
steps were carried out in the same vessel. Each of the
steps in the synthesis underwent independent valida-
tion to identify and optimize conditions such that each
transformation could be performed in a high-yielding,
parallel format. The synthesis is general and variations
of both the P3 and P1 inputs are easily allowed.
The next step of the synthesis incorporated the P1
input on the pyrazinone through an amide coupling
(Scheme 3). The pyrazinone acids 7 were incubated at
room temperature with the polymer-bound carbodiimide
11 and hydroxybenzotriazole 12 to afford the activated
1-hydroxybenzotriazole esters 13. The amine 14 and a
3-fold stoichiometric excess of N-methylmorpholine were
added to each reaction vessel to afford products 10.
Upon completion of the reaction, a mixed-resin bed of
the polyamine resin 8 and aldehyde resin 15 was added
to each reaction vessel to sequester the hydroxybenzo-
triazole 12, remaining activated ester 13, and any
unreacted acid 7 or amine 14. The sequestering resins
were agitated for a period of 2 h. If an incubation time
of an extended period was used, a chloromethyl adduct
of 1-hydroxybenzotriazole was observed as an impu-
rity.64,65 Simple filtration and rinsing with dichlo-
romethane and dimethylformamide, followed by evapo-
The first step of the synthesis was a nucleophilic
displacement of the 3-chloro of pyrazinone 4 with the
desired P3 amine 5. In the initial validation studies,
N-methylmorpholine was used as the base and dimeth-
ylformamide was used as the solvent. Byproducts
resulting from addition of morpholine and dimethyl-
amine at the P3 position were observed. Using excess
P3 amine 5 (3 equiv) as both reactant and base and
using acetonitrile as the solvent at 70 °C overnight
Sch em e 2. Polymer-Assisted Solution-Phase (PASP) Pyrazinone Synthesisa
a
Reagents: (a) 3 equiv of P3NH2 5, AcCN (b) (i) aq KOH, MeOH; (ii) 4 N HCl/dioxane; (c) H2, Pd/C.

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4053
Sch em e 3. Polymer-Assisted Solution-Phase (PASP) Coupling of P1 Amine
Sch em e 4. CBz Deprotection Protocola
or secondary amine was used as a sequestering resin
with products that contained an amidine group, the
resin reacted with the amidine group and sequestered
the desired product.
Several hundred compounds were prepared from the
synthesis described above. All of the inhibitors were
screened against several serine protease enzymes in-
volved in the coagulation cascade, including TF/VIIa,
thrombin (IIa), and Factor Xa. Each enzyme assay
consisted of the specific enzyme and the chromogenic
substrate for that enzyme. Reported are the inhibition
data as IC50 values for TF/VIIa and thrombin. In
general, these compounds were selective vs Factor Xa
with no activity observed at concentrations of 30 µM
and/or 100 µM. Factor Xa has a tyrosine at position 60,
and the S2 pocket of Factor Xa is occluded by the side
chain of Tyr 99. Thus, selectivity vs Factor Xa is
probably due to a collision of the P2 phenyl ring with
the Tyr 99 of Factor Xa (data not shown).
The structure-activity relationships of the pyrazi-
none series can be divided into three parts: the amidine
which occupies the S1 pocket and interacts with Asp 189,
the 3-amino substituent that occupies the S3 pocket, and
the substituent at the 6-position that occupies the S2
pocket (Figure 3). Chemistry efforts exploring all three
points of diversification occurred simultaneously in the
lab. Described first is the progress and structure-
activity relationships around S1, followed by S3, and
concluding with the S2 pocket.
Early in the program, libraries with P1 inputs con-
taining various basic or hydrogen bond-accepting groups
were prepared in an attempt to engage the Asp 189 at
the bottom of the S1 pocket. A representative sample of
analogues with various P1 amides is shown in Table 1.
Data from the first set of compounds established that
p-benzamidine as the P1 amide was required for any
type of biological activity (24a ,b).67 All other analogues,
including compounds containing p-phenethylamidine
24e or m-benzamidine 24f, were inactive. Replacing the
p-benzamidine with either pyridylamidine regioisomer,
compounds 24c and 24d , resulted in a loss of potency
on TF/VIIa.68
It was determined early in the project that p-benz-
amidine as the P1 moiety was crucial for potency. Thus,
chemistry efforts focused primarily on exploring the P2
and P3 groups. On the basis of crystal structure data,
the S3 pocket is wide-open and hydrophobic with resi-
dues Val 170E, Pro 170I, and Gln 217 from the peptide
segment. Initially, potential inhibitors with relatively
a
Reagents: (a) TMSCl, NaI, acetonitrile; (b) MeOH.
ration of the solvents, afforded pure products 10 from
each parallel reaction.
The P1 amine inputs 14 were biased toward contain-
ing a basic moiety to engage the Asp 189. As a result,
many of the basic groups of the P1 amines 14 were
protected with either a BOC or CBz protecting group.
The BOC protecting group was easily removed by
incubating the compound with 4 N hydrochloric acid in
dioxane, followed by evaporation to afford the desired
products. Two deprotection protocols could be used for
removal of the CBz group. The use of high-pressure
hydrogen with palladium on carbon resulted in both the
deprotection of the CBz group and the reduction of the
chloro at the 5-position to give 5-hydropyrazinone
analogues. The reductions were run in parallel using
an Argonaut Endeavor reactor. A second deprotection
protocol for the CBz protecting group was required
which allowed the chlorine to remain intact and the
reaction to be run in a parallel format using PASP
technology. In a modified solution-phase procedure that
generated trimethylsilyl iodide in situ,66 the CBz group
was removed by heating an acetonitrile solution of
compound 16 in the presence of excess sodium iodide
and trimethylsilyl chloride to afford product 21 (Scheme
4). The reaction was quenched with methanol to give
volatile byproducts 17, 18, and 19. Polyvinyl pyridine
23 was then added to sequester the benzyl iodide
byproduct 22. The polymer-bound benzyl iodide 22 was
filtered away along with the NaI(Cl) 20, the only other
nonvolatile byproduct generated in the reaction. It
should be noted that when a polymer-bound primary

4054 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Ta ble 1. Pyrazinone Analogs with Various P1 Amides
Parlow et al.
Ta ble 2. Pyrazinone Analogs with a P3 Phenyl Ring Varying
in the Length of the Carbon Chain Linker
IC50 (µM)
compd
R
VIIa
IIa
>30
7.63
0.16
23.6
7.1
25a
25b
24a
25c
25d
Ph
11.2
0.77
0.63
12.6
4.1
CH2Ph
(CH2)2Ph
(CH2)3Ph
(CH2)4Ph
Ta ble 3. Pyrazinone Analogs with a Substituted Benzyl or
Phenethyl P3 Substituent
IC50 (µM)
compd
n
X
Y
VIIa
IIa
7.63
18.9
>30
>30
>30
0.16
4.4
<0.04
<0.04
IIa/VIIa
25b
26a
26b
26c
26d
24a
26e
26f
26g
1
1
1
1
1
2
2
2
2
H
H
H
H
6-F
5-F
H
H
H
0.77
6.3
4.3
3.8
6
0.63
30
0.6
0.18
10
3
m-CF3
p-Cl
3-F
>7
>7
3-F
>5
H
0.24
0.1
m-CF3
p-Cl
m-Cl
<0.07
<0.22
H
Ta ble 4. Pyrazinone Analogues with Varying P3 Substituents
large substituents were synthesized to form hydrophobic
interactions with the S3 region. Table 2 shows the data
for the initial set of compounds synthesized. The com-
pounds contained P3 amines with a terminal phenyl
group that varied in the length of the carbon chain
linker. The phenethyl analogue 24a was the most potent
of the series on TF/VIIa, but it showed no selectivity vs
thrombin. The benzyl analogue 25b was the first
compound to show sub-micromolar activity on TF/VIIa
along with 10-fold selectivity vs thrombin.
A series of compounds with substituted benzyl and
phenethyl groups in the P3 were prepared. A represen-
tative set is depicted in Table 3. Analogues with a P3
benzyl group, regardless of substitution on the phenyl
ring, showed selectivity vs thrombin, whereas analogues
with a P3 phenethyl group showed no selectivity vs
thrombin. In fact, analogues with a P3 phenethyl group,
regardless of substitution, were more potent on throm-
bin than on TF/VIIa.

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4055
Ta ble 5. Pyrazinone Analogues with Varying P3 Substituents
Several analogues with a tertiary P3 amine were
prepared (27a -c), all of which resulted in a loss of
potency on TF/VIIa (Table 4). Replacing the P3 nitrogen
with a sulfur atom, as in 27d , resulted in a decrease in
potency. The P3 NH hydrogen-bonds to Gly 216 of the
peptide backbone in the S3 pocket. As shown by this
series of compounds, loss of this interaction results in
a decrease in potency on TF/VIIa.
Table 5 contains analogues with alkyl, branched alkyl,
and cyclic alkyl groups as the P3 substituents (28a -q).
With the exception of the analogues with larger cyclic
alkyl groups, these compounds were consistently as
active or more active than the benzyl analogues de-
scribed above. The majority of the analogues with small
alkyl and cyclic alkyl groups at P3 (28a -i) had sub-
micromolar potency with at least 10× selectivity vs
thrombin. Also contained in Table 5 are analogues with
P3 substituents possessing polar groups on the terminus
(28r -bb). The polar analogues were comparable in
activity to their alkyl analogues, with no significant
increase in potency or selectivity.
Efforts to optimize the P2 and P3 regions of the
inhibitor occurred simultaneously. It was shown that
an aniline group in the P2 region was significantly more
active than the unsubstituted phenyl group. Thus, the
more active P3 analogues were synthesized with aniline
in the P2 (29a -i) and the activity compared with the
data of the corresponding analogues with an unsubsti-
tuted phenyl group in the P2 position (Table 5). The
relative potency was analogous to that of the phenyl
series. The majority of the compounds were approxi-
mately 10 times more active on TF/VIIa and signifi-
cantly more selective over thrombin with the aniline in
place of the unsubstituted phenyl in the P2 position.

4056 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Ta ble 6. Pyrazinone Analogues with Various P2 Substituents
Parlow et al.
Ta ble 7. Pyrazinone Analogs with a Substituted P2 Phenyl
Ring
IC50 (µM)
IC50 (µM)
compd
R
VIIa
IIa
0.06
<0.04
0.16
4.0
compd
R
VIIa
0.63
3.0
1.98
8.88
1.8
IIa
30a
30b
24a
30c
30d
Me
Et
Ph
Bn
4.28
3.45
0.63
24a
31a
31b
31c
31d
31e
31f
31g
31h
31i
31j
31k
31l
31m
31n
31o
H
0.16
2.1
0.72
0.2
1.2
1.1
0.1
8.6
3.5
6.3
0.30
0.89
0.30
3.3
1.0
4.06
o-Cl
m-Cl
p-Cl
> 30
> 30
4-biphenyl
>30
o-F
m-F
2.3
8.1
2.7
2.9
In general, the SAR studies indicate that the S3
pocket of TF/VIIa prefers small alkyl groups for lipo-
philic interaction with Gln 217 and residues from the
peptide segment 170E-170I (including Pro 170I). The
NH is essential for potency as this hydrogen bonds to
the Gly 216 of the peptide backbone.
p-F
o-CF3
m-CF3
p-CF3
o-CH3
m-CH3
p-CH3
o-OCH3
m-OCH3
p-OCH3
>30
0.43
1.0
4.14
1.6
On the basis of crystal structure data, the S2 pocket
of TF/VIIa is relatively open and has a negative poten-
tial due to the presence of Asp 60. Among the coagula-
tion proteases, only Factor VIIa has a negatively
charged residue at position 60. Thrombin has a large
insertion in the S2 pocket with a number of aromatic
amino acid residues. From the docking experiments, a
benzyl group at P2 seemed to occupy much of the space
of the S2 pocket. To confirm this, and to probe the depth
of the S2 pocket of TF/VIIa, scaffolds were synthesized
from aldehydes containing R groups of various sizes.
The data for a set of compounds with methyl, ethyl,
phenyl, benzyl, and 4-biphenyl groups in the P2 is shown
in Table 6. This data set shows the phenyl group (24a )
as the optimal size for fitting in the S2 pocket. Larger
groups, such as the benzyl (30c) and biphenyl (30d ),
showed no activity on TF/VIIa at 30 µM. Smaller groups,
such as methyl (30a ) and ethyl (30b), showed some TF/
VIIa activity, but were more potent on thrombin. This
is consistent with the difference in the S2 pocket size of
the two enzymes.
The next step was to determine whether substitution
on the phenyl ring in the P2 was tolerated and, if so,
whether the size of the group or its position on the ring
affected potency. A representative set of data for these
studies is shown in Table 7. A variety of halogens and
small alkyl groups could be placed in both the ortho and
meta positions on the P2 phenyl ring while maintaining
potency vs TF/VIIa. However, activity on TF/VIIa
decreased considerably when the group was in the para
position. This trend held true when tested with a variety
of groups in the P3 position.
5.2
23.45
Ta ble 8. Pyrazinone Analogs with a Substituted P2 Phenyl
Ring
IC50 (µM)
compd
R′
R
VIIa
IIa
>30
0.98
32a
32b
32c
29d
29f
32d
32e
32f
32g
cBu
m-bromo
m-NO2
m-NH2
m-NH2
m-NH2
m-OH
o-OH
m-CO2CH3
m-CO2H
4.2
phenethyl
phenethyl
iPr
2.52
0.07
0.02
0.02
0.20
0.34
0.78
0.36
0.9
6.98
8.0
cBu
cBu
cBu
iPr
4.25
>30
2.0
2.8
iPr
to TF/VIIa, it would be possible to gain potency and
achieve selectivity vs thrombin by placing a proton
donor at the meta position of the P2 phenyl ring. The
m-amino analogue 29f was prepared and resulted in a
potent inhibitor on TF/VIIa (IC50 ) 20 nM) with
increased selectivity against thrombin (400×) and Fac-
tor Xa (>5000×).
An X-ray crystal structure of compound 29f bound to
TF/VIIa is shown in Figure 4. It is very clear from the
crystal structure that there is a four-centered hydrogen
bonding interaction between the m-amino P2 of 29f, Asp
60, Tyr 94, and Thr 98. These key hydrogen bonding
interactions with the S2 pocket of TF/VIIa are likely
responsible for the observed 20× boost in potency and
the increase in selectivity vs thrombin over the unsub-
stituted P2 phenyl analogue 28m .
Having established an ortho- or meta-substituted
phenyl as the optimal-sized P2 group to occupy the S2
region, the next objective was to determine which
substituents gave the most active and selective TF/VIIa
inhibitors. A variety of ortho- and meta-substituted P2
phenyl analogues were prepared with a representative
set shown in Table 8. Of particular interest is compound
29f, with a m-amino group on the P2 phenyl ring. It was
theorized that since the S2 pocket of TV/VIIa is larger
in size than that of thrombin, and since Asp 60 is unique
This theory was further confirmed by the data in
Table 9, which shows a drop in potency when the aniline

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4057
F igu r e 5. Pyrazinone intravenous POC compound 34.
sible. Since the S2 pockets of the TF/VIIa and thrombin
enzymes are substantially different, multiple substitu-
tion on the P2 m-aminophenyl ring should lead to
increased selectivity over thrombin via additional in-
teractions with the S2 pocket. Specifically, the Lys 60A
residue was targeted as a potential site of interaction
in the TF/VIIa S2 pocket. Compound 34 was prepared
with a 3-amino-5-carboxyphenyl ring as the P2 substitu-
ent with the 5-carboxylic acid moiety intended to engage
Lys 60A (Figure 5). The activity of 34 on TF/VIIa was
relatively unchanged vs 29f. However, the selectivity
vs thrombin was dramatically increased from 400× to
> 6250×. An X-ray crystal structure of 34 revealed that
Lys 60A was engaged by the carboxyl moiety on P2,
which led to the superior selectivity profile for this
compound (Figure 6). Compound 34 was deemed selec-
tive enough vs Factor Xa and thrombin to proceed with
the preclinical intravenous proof-of-concept studies to
demonstrate the separation between antithrombotic
efficacy and bleeding side effects in a nonhuman pri-
mate model of electrolytic-induced arterial thrombosis.39
In conclusion, we used SBDD and multistep PASP
library synthesis of pyrazinones to demonstrate that
high levels of activity and selectivity for TF/VIIa are
possible. Starting from a tripeptide-R-ketothiazole that
was active on TF/VIIa, we were able to design a series
of active and selective pyrazinone inhibitors of TF/VIIa
that were reversible and did not capture the catalytic
apparatus of the enzyme. These inhibitors exhibited
sufficient levels of activity and selectivity for TF/VIIa
vs Factor Xa and thrombin to proceed with our preclini-
cal, intravenous proof-of-concept studies. These studies
were designed to demonstrate the separation of anti-
thrombotic efficacy and bleeding side effects in a non-
human primate model of electolytic-induced arterial
thrombosis. Further efforts to refine the SAR in the
pyrazinone series are in progress. In addition, a variety
of core molecules as alternative central templates for
the selective inhibition of TF/VIIa have been synthe-
sized and will be reported in due course.
F igu r e 4. Crystal structure of compound 29f bound in the
active site of TF/VIIa complex. The structure was refined to
an Rfree of 29.0% at 2.8 Å resolution. Some of the key side
chains of Factor VIIa are displayed (C: green, N: dark blue,
O: red, S: orange). The inhibitor is represented with carbon,
nitrogen, oxygen, and chlorine atoms displayed in yellow, blue,
red, and purple, respectively. The hydrogen bonds formed by
the inhibitor are shown as dotted white lines. The m-amino
group on the phenyl group of the inhibitor forms strong
interactions with Asp 60, Tyr 94, and the carbonyl oxygen of
Thr 98 in the S2 pocket.
Ta ble 9. Pyrazinone Analogs with a Substituted P2
m-Aminophenyl Ring
IC50 (µM)
compd
R
R′
VIIa
IIa
32c
33a
33b
33c
33d
33e
H
CH3
CH3
COCH3
COCF3
SO2CH3
H
H
CH3
H
H
0.07
0.60
1.2
1.61
0.45
0.6
0.9
0.58
1.1
0.11
2.0
0.10
Exp er im en ta l Section
H
Gen er a l. Solvents and chemicals were reagent grade or
better and were obtained from commercial sources. 1H and 13C
NMR spectra were recorded using a 300 or 400 MHz NMR
spectrometer. Sample purities were determined by HPLC
analysis equipped with a mass spectrometric detector using a
C18 3.5 µm, 30 × 2.1 mm column, eluting with a gradient
system of 5:95 to 95:5 acetonitrile:water with a buffer consist-
ing of 0.1% TFA over 4.5 min at 1 mL/min and detected by
DAD. HPLC purifications were conducted using a C18 15 mm,
100 Å, 25 × 100 mm column, eluting with a gradient system
of 5:95 to 95:5 acetonitrile:water with a buffer consisting of
0.1% TFA over 10 min at 30 mL/min, unless otherwise noted.
nitrogen was substituted with various groups. Func-
tionalizing the P2 aniline nitrogen affects the key
hydrogen bonding interactions with the S2 pocket.
Compound 29f was sufficiently potent on TF/VIIa and
selective enough on Factor Xa (>5000×) to proceed with
the intravenous proof-of-concept studies. However, a
more selective inhibitor vs thrombin was desired to
unequivocally prove that separation between anti-
thrombotic efficacy and bleeding side effects was pos-

4058 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Parlow et al.
P ASP Libr a r y Syn th esis. Gen er a l P r oced u r e A. Nu -
cleop h ilic Disp la cem en t of 3-Ch lor op yr a zin on e 4 w ith
Am in e 5 to Affor d P r od u ct 6. Individual reaction products
were prepared in multiple reaction block vessels in a spatially
addressed format. The desired dichloropyrazinone scaffold 4
(1.5 mL of a 0.2 M solution) in acetonitrile was added to the
reaction vessels followed by a 3-fold stoichiometric excess of
the amine 5 (4.5 mL of a 0.2 M solution) in acetonitrile. The
reaction mixtures were incubated at 70 °C for 16-20 h. After
being cooled to ambient temperature, the contents of each
reaction vessel were taken on to the next step without
purification.
Gen er a l P r oced u r e B. Hyd r olysis of th e Ben zyl Ester
6 t o Affor d P r od u ct 7. Each reaction vessel was charged
with methanol (3 mL) and a 3-4-fold stoichiometric excess of
aqueous potassium hydroxide (2.1 mL of a 0.5 M solution). The
reaction block was shaken vertically for 3 h on an orbital
shaker at ambient temperature. The contents of each reaction
vessel were acidified with 0.3 mL of 12 M aqueous hydrochloric
acid. Each reaction vessel was then opened, and the solutions
were evaporated to dryness under nitrogen and/or a Savant
apparatus. Alternatively, when the P2 phenyl ring contained
an ester functionality, the pyrazinone 6 was treated with 10%
palladium on carbon in methanol (2-4 mL) under 10 psi of
hydrogen for 16-20 h to remove the benzyl ester. Polyamine
resin 8 (1.5 g, ∼4.2 mmol, 10-15-fold stoichiometric excess)
was added to the solid carboxylic acid 7 followed by dichlo-
romethane (12 mL) and water (1.2 mL). The mixture was
shaken laterally for 6 h on an orbital shaker at ambient
temperature (rotating the vials at least once so each side of
the vial was agitated for a minimum of 2 h). The desired
product 7 was sequestered away from the reaction byproducts
and excess reactants as the insoluble adduct 9. Simple
filtration of the insoluble resin-adduct 9 and rinsing of the
resin cake with dimethylformamide (3 × 3 mL), dichlo-
romethane (3 × 3 mL), methanol (3 × 3 mL), and dichlo-
romethane (3 × 3 mL) afforded the desired resin-bound
product 9. After drying the resin under vacuum for 2 h, an
excess of hydrochloric acid in 1,4-dioxane (4.0 N, 8 mL) (7-
8-fold stoichiometric excess based on the loading of amine
functionality) along with dichloromethane (3 mL) was added
to each reaction vessel to cleave the desired product 7 from
the resin. The reaction block was shaken laterally for 2-20 h
on an orbital shaker at ambient temperature. Simple filtration
of the solution, rinsing of the resin cake with dimethylforma-
mide/dichloromethane, and evaporation of the solvents af-
forded the desired products 7 in purified form, which were
weighed and analyzed by LC/MS.
F igu r e 6. Crystal structure of compound 34 bound in the
active site of TF/VIIa complex. The structure was refined to
an Rfree of 31.1% at 2.1 Å resolution. The atoms are colored as
in Figure 4 with some of the key side chains of Factor VIIa
shown. The hydrogen bonds formed by the inhibitor are shown
as dotted white lines. Consistent with the structure of com-
pound 29f, the m-amino group forms close interactions in the
S2 pocket. As predicted, the carboxylate group at the 5-position
of the phenyl group forms interactions with the ꢀ-amino group
of Lys 60A. The side chain of Lys 60A, which is on the surface,
is disordered in most of the crystal structures of TF/VIIa
complexes.
Sch em e 5. Synthesis of Aldehyde 37a
Gen er a l P r oced u r e C. Cou p lin g of Ca r boxylic Acid s
7 w ith P 1 Am in e In p u ts to Affor d P r od u ct 10. The acid 7
was added as a solution in dichloromethane (3 mL) and
dimethylformamide (2 mL) to each reaction vessel. A solution
of hydroxybenzotriazole 12 (2 mg, 0.015 mmol, >0.05 equiv)
in dimethylformamide/dichloromethane was added to each
reaction vessel, followed by the polymer-bound carbodiimide
reagent 11 (1.2-1.5-fold stoichiometric excess). The parallel
reaction block was agitated vertically on an orbital shaker for
30 min to 1 h. The amine 14 (1 equiv) in dimethylformamide
(0.10 mM), along with a 3-fold stoichiometric excess of N-
methylmorpholine, if the amine 14 was a salt, was added to
the activated acid 13 of each vessel. The parallel reaction block
was then agitated vertically on an orbital shaker for 1-20 h
at ambient temperature. An excess of the polyamine resin 8
(250 mg, ∼0.7 mmol) and aldehyde resin 15 (100 mg, ∼0.3
mmol), along with dichloromethane (1-3 mL), as needed for
effective agitation, were added to each reaction vessel. The
resin-charged reaction block was shaken vertically for 2 h on
an orbital shaker at ambient temperature. Filtration of the
insoluble resins and resin adducts, subsequent rinsing of the
vessel resin-bed with dimethylformamide (3 × 1 mL), and
concentration of the filtrates afforded pure products 10, which
were weighed and analyzed by LC/MS.
a
Reagents: (a) BH3, THF; (b) (ClCO)2, DMSO, TEA, CH2Cl2.
Yields were not optimized, with emphasis on purity of products
rather than quantity. Recombinant soluble TF, consisting of
amino acids 1-219 of the mature protein sequence, was
expressed in E. coli and purified using a Mono Q Sepharose
FPLC. Recombinant human VIIa was purchased from Ameri-
can Diagnostica, Greenwich, CT. Chromogenic substrate N-
methylsulfonyl-D-Phe-Gly Arg-p-nitroaniline was prepared by
American Peptide Company, Inc., Sunnyvale, CA. Factor Xa
was obtained from Enzyme Research Laboratories, South Bend
IN, thrombin from Calbiochem, La J olla, CA, and trypsin and
L-BAPNA from Sigma, St. Louis, MO. The chromogenic
substrates S-2765 and S-2238 were purchased from Chro-
mogenix, Sweden.
Gen er a l P r oced u r e D. Dep r otection of th e BOC Gr ou p
fr om Com p ou n d s 10 t o Affor d P r od u ct s 21. For those

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4059
amines 14 which contained a BOC protecting group, hydro-
chloric acid in 1,4-dioxane (4.0 N, 3 mL) was added to the BOC
protected compound 10 (∼0.3 mmol) in a reaction vessel, and
the solution was agitated on an orbital shaker at room
temperature for 3 h. Evaporation of the solvents afforded the
products 21. If necessary, the products were purified by
reverse-phase HPLC.
and the volatiles were removed under reduced pressure to give
an orange oil. The crude oil was purified on 1 kg of silica gel
using ethyl acetate:hexanes (20:80, v:v, isocratic) as the eluent
to afford 15.4 g (78%) of 37 as an off-white solid: 1H NMR
(300 MHz, CDCl3) δ 4.06 (s, 3H), 8.86-8.87 (m, 1H), 8.90-
8.91 (m, 1H), 9.10-9.11 (m, 1H), 10.19 (s, 1H); LRMS m/z 210.0
(M+ + H).
Gen er a l P r oced u r e E. Dep r otection of th e CBz Gr ou p
fr om Com p ou n d s 16 Usin g TMSI to Affor d P r od u cts 21.
The residues 16 were dissolved in acetonitrile (1.5 mL). Sodium
iodide and trimethylsilyl chloride (5-fold stoichiometric excess
of each) were added, and the reaction mixtures were agitated
vertically at 55 °C for 14-20 h. After being cooled to room
temperature, the reactions were quenched with 0.1 mL of
methanol. Poly(4-vinylpyridine) or N,N-(dimethyl)amino-
methylpolystyrene resin (10-fold stoichiometric excess based
on the amount of trimethylsilyl chloride and sodium iodide
added) was added to each vessel to sequester the byproduct
22. The mixtures were agitated for another 3 h and then
filtered through Celite and rinsed with acetonitrile to remove
byproduct 20 and the polymer-bound byproduct 22. The filtrate
was concentrated to afford products 21. If necessary, the
products were purified by reverse-phase HPLC.
Gen er a l P r oced u r e F . Rem ova l of CBz P r otectin g
Gr ou p of P r od u cts 16 Usin g P d /C to Affor d P r od u cts 21.
The residues 16 were dissolved in methanol (2-4 mL). A
catalytic amount of 10% Pd/C was added, and the reaction
mixtures were stirred under 10 psi of hydrogen for 16-20 h.
The mixtures were filtered through Celite, rinsed with metha-
nol, and concentrated to afford pure products 21. If necessary,
the products were purified by reverse-phase HPLC.
Meth yl 3-{1-[2-(Ben zyloxy)-2-oxoeth yl]-3,5-d ich lor o-6-
oxo-1,6-d ih yd r op yr a zin -2-yl}-5-n itr oben zoa te 4′. A sepa-
ratory funnel was charged with glycine benzyl ester hydro-
chloride 2 (14.1 g, 69.7 mmol) and diluted with ethyl acetate
(200 mL). The solution was washed with 1:1 brine:aqueous
saturated sodium carbonate (2 × 250 mL). The organic portion
was dried over sodium sulfate, and the volatiles were removed
under reduced pressure to give the free base of 2. The free
base in dichloromethane (200 mL) was added to a glass reactor,
followed by the dropwise addition of 37 (14.6 g, 70 mmol) in
dichloromethane (100 mL) over 30 min. With caution, tri-
methylsilyl cyanide (26.0 mL, 195 mmol) was added via syringe
over 20 min. A slight exotherm of 4 °C was observed during
the addition. The reaction was stirred for 16 h at room
temperature. The volatiles were removed under reduced
pressure. The residue was diluted with ethyl acetate (400 mL)
and washed with water (1 × 250 mL) and brine (1 × 250 mL).
The organic phase was dried over magnesium sulfate and
filtered, and the solvent was removed under reduced pressure
to afford the intermediate as a yellow oil. The intermediate
was dissolved in chlorobenzene (150 mL) and added dropwise
over 1 h to a stirring, 50 °C solution of oxalyl chloride (24.3
mL, 279 mmol) in chlorobenzene (150 mL). The reaction was
warmed to 80 °C and stirred for 16 h. The reaction was
quenched with methanol (250 mL), and the volatiles were
removed under reduced pressure to give crude 4′ as a brown
oil: 1H NMR (300 MHz, CDCl3) δ 4.03 (s, 3H), 4.39-4.66 (ABq,
2H, J ) 17.0 Hz), 5.16 (d, 2H, J ) 3.6 Hz), 7.21-7.25 (m, 2H),
7.28-7.35 (m, 3H), 8.35-8.36 (m, 1H), 8.41-8.42 (m, 1H),
8.98-8.99 (m, 1H); LRMS m/z 514.0 (M+ + Na).
Meth yl 3-[1-[2-(Ben zyloxy)-2-oxoeth yl]-3-ch lor o-5-(iso-
p r op yla m in o)-6-oxo-1,6-d ih yd r op yr a zin -2-yl]-5-n itr oben -
zoa te 6′. Isopropylamine (11.9 mL, 140 mmol) was added to
a stirring solution of crude 4′ (17.2 g, 35 mmol) in ethyl acetate
(300 mL). The reaction turned black and was stirred at 55 °C
for 16 h. The reaction mixture was filtered through Celite 545,
and the volatiles were removed under reduced pressure. The
crude residue was purified on 500 g of silica gel using ethyl
acetate:hexanes (20:80, v:v, isocratic) as the eluent to afford
8.4 g (46%) of 6′ as a brown solid: 1H NMR (300 MHz, CDCl3)
δ 1.32 (d, 6H, J ) 6.6 Hz), 4.01 (s, 3H), 4.21-4.33 (m, 1H),
4.32-4.58 (ABq, 2H, J ) 17.0 Hz), 5.14 (d, 2H, J ) 0.9 Hz),
7.21-7.39 (m, 5H), 8.33-8.34 (m, 1H), 8.38-8.39 (m, 1H),
8.90-8.91 (m, 1H); LRMS m/z 515.2 (M+ + H).
[6-[3-Am in o-5-(m et h oxyca r b on yl)p h en yl]-5-ch lor o-3-
(isop r op yla m in o)-2-oxop yr a zin -1(2H)-yl]a cetic Acid 7′.
To a glass reactor were added 6′ (8.4 g, 16 mmol), 200 mL of
methanol, and hydrochloric acid (7.5 mL of a 3 M methanolic
solution). Under a blanket of nitrogen, 10% palladium on
carbon (0.8 g, 10 wt %) was carefully added in a methanol
slurry to the stirring solution. The vessel was sealed, purged
of its atmosphere, and flushed with nitrogen 3× and then
purged of its atmosphere and flushed with hydrogen 3×. The
reaction was stirred at room temperature under a balloon of
hydrogen for 16 h. The reaction was filtered through Celite
545, the volatiles were removed under reduced pressure, and
the residue was diluted with ethyl acetate (300 mL). The
organic phase was extracted with aqueous saturated sodium
bicarbonate (2 × 300 mL). The aqueous phase was acidified
to pH 4 with 1 M aqueous potassium hydrogensulfate and then
extracted with ethyl acetate (3 × 350 mL). The organic phase
was dried over magnesium sulfate and filtered, and the solvent
was removed under reduced pressure to give 4.1 g (76%) of 7′
as an orange solid: 1H NMR (300 MHz, CD3OD) δ 1.30 (d,
6H, J ) 6.9 Hz), 3.89 (s, 3H), 4.16-4.24 (m, 1H), 4.38-4.39
(m, 2H), 6.83 (s, 1H), 7.20 (s, 1H), 7.43 (s, 1H); LRMS m/z 395.1
(M+ + H).
The progress of the synthesis during production was fol-
lowed by LC/MS of the intermediates. The final products with
LC/MS purities of less than 85% were purified by reverse-
phase chromatography. All products were characterized by LC/
MS and HRMS, with several characterized by proton NMR.
Data for final compounds are tabulated in Table 10 in
Supporting Information.
Syn th esis of Com p ou n d 34 (Sch em e 5). Meth yl 3-(Hy-
d r oxym eth yl)-5-n itr oben zoa te 36. 3-(Methoxycarbonyl)-5-
nitrobenzoic acid 35 (30 g, 133 mmol) in tetrahydrofuran (100
mL) was cooled to -10 °C in a glass reactor equipped with a
reflux condenser and addition funnel. Borane-tetrahydrofuran
(260 mL, 1 M BH3-THF) was added dropwise over 2 h. The
reaction was slowly warmed to room temperature and then
heated to 64 °C. The reaction was stirred at reflux for 15 min,
at which time TLC analysis (20% ethyl acetate-hexane)
indicated the reaction was complete. After being cooled to room
temperature, the reaction was quenched with 8 mL of acetic
acid:water (1:1) and then neutralized to pH 7 using aqueous
saturated sodium bicarbonate. The volatiles were removed
under reduced pressure, and the residue was diluted with ethyl
acetate (500 mL). The solution was washed with water (1 ×
300 mL) and then brine (1 × 300 mL). The organic phase was
dried over magnesium sulfate and filtered, and the solvent was
removed under reduced pressure to give 19.8 g (70%) of 36 as
a yellow solid: 1H NMR (300 MHz, CDCl3) δ 4.00 (s, 3H), 4.89
(s, 2H), 8.35 (s, 1H), 8.43 (s, 1H), 8.77 (s, 1H); LRMS m/z 212.0
(M+ + H).
Meth yl 3-For m yl-5-n itr oben zoate 37 (Sch em e 5). Dichlo-
romethane (200 mL) was cooled to -78 °C in a glass reactor
equipped with an addition funnel. Oxalyl chloride (13.9 mL,
0.16 mol) was added in one portion. Dimethyl sulfoxide (18.9
mL) in dichloromethane (100 mL) was added dropwise over 1
h. Compound 36 (19.6 g, 93 mmol) in dichloromethane (200
mL) was added dropwise over 1.5 h. The reaction was stirred
at -78 °C for 45 min. Triethylamine (74.2 mL, 0.53 mol) was
added via addition funnel over 45 min, upon which time the
reaction turned yellow and thick. TLC analysis indicated the
reaction was complete. The reaction was quenched with
aqueous potassium hydrogensulfate (600 mL of a 1 M solution).
The organic phase was washed with aqueous saturated sodium
bicarbonate (1 × 600 mL), water (1 × 600 mL), and brine (1 ×
600 mL). The organic phase was dried over sodium sulfate,

4060 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Parlow et al.
Met h yl 3-Am in o-5-[1-(2-{[4-((Z)-a m in o{[(b en zyloxy)-
car bon yl]im in o}m eth yl)ben zyl]am in o}-2-oxoeth yl)-3-ch lo-
r o-5-(isopr opylam in o)-6-oxo-1,6-dih ydr opyr azin -2-yl]ben -
zoa te 10′. To a glass bottle was added 7′ (4.0 g, 10 mmol),
hydroxybenzotriazole (0.41 g, 3 mmol), dichloromethane (40
mL), and dimethylformamide (5 mL). N-methylmorpholine (6.7
mL, 61 mmol) and benzyl amino[4-(aminomethyl)phenyl]-
methylenecarbamate (3.6 g, 10 mmol) in dimethylformamide
(15 mL) were added, followed by addition of the polymer-bound
carbodiimide 11 (19.0 g, 18 mmol). The reaction was agitated
on an orbital shaker for 3 h. Polyamine resin 8 (10.8 g, 30
mmol), aldehyde resin 15 (10.6 g, 30 mmol), and dichlo-
romethane (20 mL) were added to the bottle, and the mixture
was agitated for 1 h. The insoluble resins and resin adducts
were filtered, and the resin cake was rinsed with dimethyl-
formamide (2 × 50 mL) and dichloromethane (2 × 50 mL).
Concentration of the filtrate afforded 5.3 g (79%) of 10′ as an
orange solid: 1H NMR (300 MHz, CDCl3) δ 1.23 (d, 6H, J )
6.6 Hz), 3.78 (s, 3H), 4.10-4.15 (m, 1H), 4.20 (s, 2H), 4.27-
4.59 (ABq, 2H, J ) 15.9 Hz), 5.16 (s, 2H), 7.03-7.76 (m, 12H);
LRMS m/z 660.3 (M+ + H).
3-Am in o-5-[1-[2-({4-[a m in o(im in o)m e t h yl]b e n zyl}-
a m in o)-2-oxoet h yl]-3-ch lor o-5-(isop r op yla m in o)-6-oxo-
1,6-d ih yd r op yr a zin -2-yl]ben zoic Acid 34. Lithium hydrox-
ide monohydrate (1.64 g, 39 mmol) in water (15 mL) was added
to 10′ (5.3 g, 8 mmol) in methanol (100 mL). After stirring at
room temperature for 16 h, the reaction was neutralized to
pH 7 with 3 M aqueous hydrochloric acid. The volatiles were
removed under reduced pressure, and the crude residue was
purified by reverse-phase HPLC using a gradient of aceto-
nitrile:water (0.1% TFA) (1:99 to 60:40) as the eluent to afford
1.8 g (34%) of 34 as an off-white solid: 1H NMR (300 MHz,
CD3OD) δ 1.31 (d, 6H, J ) 6.6 Hz), 4.18-4.26 (m, 1H), 4.31-
4.63 (m, 4H), 7.46-7.49 (m, 2H), 7.71-7.78 (m, 3H), 8.12-
8.19 (m, 2H); HRMS (M + H): Calcd for C24H27ClN7O4:
512.1813; Found: 512.1819.
either inhibitor or buffer (50 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 0.1% BSA). The reaction, in a final volume of 100 µL,
was measured immediately at 405 nm to determine back-
ground absorbance. The plate was incubated at room temper-
ature for 60 min, at which time the rate of hydrolysis of the
substrate was measured by monitoring the reaction at 405 nm
for the release of p-nitroaniline. All compounds were assayed
in duplicate at seven concentrations. Percent inhibition at each
concentration was calculated from OD405
value from the
nm
experimental and control sample. IC50 values were calculated
from a four-parameter logistic regression equation. For each
compound the individual IC50 values were within 10% of each
other. The reported IC50 represents an average of the dupli-
cates.
Cr ysta l Str u ctu r e. Crystals of TF/VIIa complex were
obtained by slight modification of the procedure described by
Banner et al.46
Ackn owledgm en t. The authors wish to thank James
P. Doom, David Masters-Moore, and Ralph C. Scheibel
for HRMS analysis, and Martin P. McGrath for running
the hydrogenations. Diffraction data for the TF-VIIa
complex with the pyrazinone inhibitors were collected
at beamline 17-ID in the facilities of the Industrial
Macromolecular Crystallography Association Collabora-
tive Access Team (IMCA-CAT) at the Advanced Photon
Source. IMCA-CAT facilities are supported by the
corporate members of the IMCA and through a contract
with Illinois Institute of Technology (IIT), executed
through the IIT's Center for Synchrotron Radiation
Research and Instrumentation. Use of the Advanced
Photon Source was supported by the U.S. Department
of Energy, Basic Energy Sciences, Office of Science,
under Contract No. W-31-109-Eng-38.
Assa ys for Biologica l Activity. TF -VIIa Assa y. Recom-
binant soluble tissue factor (100 nM) and recombinant human
Factor VIIa (2 nM) were added to a 96-well assay plate
containing 0.4 mM of the substrate, N-methylsulfonyl-D-phe-
gly arg-p-nitroaniline and either inhibitor or buffer (5 mM
CaCl2, 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% BSA).
The reaction, in a final volume of 100 µL, was measured
immediately at 405 nm to determine background absorbance.
The plate was incubated at room temperature for 60 min, at
which time the rate of hydrolysis of the substrate was
measured by monitoring the reaction at 405 nm for the release
of p-nitroaniline. All compounds were assayed in duplicate at
seven concentrations. Percent inhibition at each concentration
was calculated from OD405 nm value from the experimental and
control sample. IC50 values were calculated from a four-
parameter logistic regression equation. For each compound the
individual IC50 values were within 10% of each other. The
reported IC50 represents an average of the duplicates.
Xa Assa y. Human Factor Xa (0.3 nM) and N-R-benzyloxy-
carbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroaniline-dihydro-
chloride (S-2765) (0.15 mM) were added to a 96-well assay
plate containing either inhibitor or buffer (50 mM Tris-HCl,
pH 8.0, 100 mM NaCl, 0.1% BSA). The reaction, in a final
volume of 100 µL, was measured immediately at 405 nm to
determine background absorbance. The plate was incubated
at room temperature for 60 min, at which time the rate of
hydrolysis of the substrate was measured by monitoring the
reaction at 405 nm for the release of p-nitroaniline. All
compounds were assayed in duplicate at seven concentrations.
Percent inhibition at each concentration was calculated from
OD405 nm value from the experimental and control sample. IC50
values were calculated from a four-parameter logistic regres-
sion equation. For each compound the individual IC50 values
were within 10% of each other. The reported IC50 represents
an average of the duplicates.
Su p p or tin g In for m a tion Ava ila ble: A table of charac-
terization data of final pyrazinone products. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Houston, D. S. Tissue Factor - a therapeutic target for throm-
botic disorders. Expert Opin. Ther. Targets 2002, 6, 159-174.
(2) Golino, P. The inhibitors of the tissue Factor: Factor VII
pathway. Thromb. Res. 2002, 106, V257-V265.
(3) Bajaj, M. S.; Birktoft, J . J .; Steer, S. A.; Bajaj, S. P. Structure
and biology of tissue factor pathway inhibitor. Thromb. Haemost.
2001, 86, 959-972.
(4) Kaiser, B.; Hoppensteadt, D. A.; Fareed, J . Tissue factor pathway
inhibitor: an update of potential implications in the treatment
of cardiovascular disorders. Expert Opin. Invest. Drugs 2001, 10,
1925-1935.
(5) Girard, T. J .; Nicholson, N. S. The role of tissue Factor/Factor
VIIa in the pathophysiology of acute thrombotic formation. Curr.
Opin. Pharmacol. 2001, 1, 159-163.
(6) Coughlin, S. R. Thrombin signaling and protease-activated
receptors. Nature 2000, 407, 258-264.
(7) Girard, T. J . Tissue Factor Pathway Inhibitor. In New Thera-
peutic Agents in Thrombosis and Thrombolysis; Sasahara, A. A.,
and Loscalzo, J ., Eds.; Marcel Dekker: New York, 1998.
(8) Banner, D. W. The Factor VIIa/tissue Factor complex. Thromb.
Haemost. 1997, 78, 512-515.
(9) Kirchhofer, D.; Nemerson, Y. Initiation of blood coagulation: the
tissue Factor/Factor VIIa complex. Curr. Opin. Chem. Biotech.
1996, 7, 386-391.
(10) Petersen, L. C.; Valentin, S.; Hedner, U. Regulation of the
extrinsic pathway system in health and disease: the role of
Factor VIIa and tissue factor pathway inhibitor. Thromb. Res.
1995, 79, 1-47.
(11) Moreno P. R.; Bernardi, V. H.; Lopez-Cuellar, J .; Murcia, A. M.;
Palacios, I. F.; Gold, H. K.; Mehran, R.; Sharma, S. K.; Nemer-
son, Y.; Fuster, V.; Fallon, J . T. Macrophages, smooth muscle
cells, and tissue factor in unstable angina: implications for cell-
mediated thrombogenicity in acute coronary syndromes. Circu-
lation 1996, 94, 3090-3097.
(12) Edgington, T. S.; Mackman, N.; Brand, K.; Ruf, W. The struc-
tural biology of expression and function of tissue factor. Thromb.
Haemost. 1991, 66, 67-79.
Th r om bin Assa y. Human thrombin (0.28 nM) and H-D-
phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochlo-
ride (0.06 mM) were added to a 96-well assay plate containing

Pyrazinone Inhibitors of Tissue Factor VIIa Complex
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4061
(13) Sanderson, P. E. J . Anticoagulants: Inhibitors of thrombin and
Factor Xa. Annu. Rep. Med. Chem. 2001, 36, 79-88.
(14) Betz, A. Recent advances in Factor Xa inhibitors. Expert Opin.
Ther. Pat. 2001, 11, 1007-1017.
(15) Zhu, B. Y.; Scarborough, R. M. Factor Xa inhibitors: Recent
advances in anticoagulant agents. Annu. Rep. Med. Chem. 2000,
35 83-102.
(16) Vacca, J . P. New Advances in the discovery of Thrombin and
Factor Xa inhibitors. Curr. Opin. Chem. Biol. 2000, 4, 394-400.
(17) Sanderson, P. E. J . Small, noncovalent serine protease inhibitors.
Med. Res. Rev. 1999, 19, 179-197.
(18) Ewing, W. R.; Pauls, H. W.; Spada, A. P. Progress in the design
of inhibitors of coagulation Factor Xa. Drugs Future 1999, 24,
771-787.
(19) Carroll, A. R.; Pierens, G. K.; Fechner, G.; de Almeida Leone,
P.; Ngo, A.; Simpson, M.; Hyde, E.; Hooper, J . N. A.; Bostro¨m,
S.-L.; Musil, D.; Quinn, R. J . Dysinosin A: A novel inhibitor of
Factor VIIa and thrombin from a new genus and species of
Australian sponge of the family Dysideidae. J . Am. Chem. Soc.
2002, 124, 13340-13341.
(20) Hanessian, S.; Margarita, R.; Hall, A.; J ohnstone, S.; Tremblay,
M.; Parlanti, L. Total synthesis and structural confirmation of
the marine natural product Dysinosin A: A novel inhibitor of
thrombin and Factor VIIa. J . Am. Chem. Soc. 2002, 124, 13342-
13343.
(21) Hanessian, S.; Therrien, E.; Granberg, K.; Nilsson, I. Targeting
thrombin and Factor VIIa: design, synthesis, and inhibitory
activity of functionally relevant indolizidinones. Bioorg. Med.
Chem. Lett. 2002, 12, 2907-2911.
(22) Young, W. B.; Kolesnikov, A.; Rai, R.; Sprengeler, P. A.; Leahy,
E. M.; Shrader, W. D.; Sangalang, J .; Burgess-Henry, J .;
Spencer, J .; Elrod, K.; Cregar, L. Optimization of a screening
lead for Factor VIIa/TF. Bioorg. Med. Chem. Lett. 2001, 11,
2253-2256.
(35) Himber, J .; Kirchhofer, D.; Riedereer, M.; Tschopp, T. B.; Steiner,
B.; Roux, S. P. Dissociation of antithrombotic effect and bleeding
time prolongation in rabbits by inhibiting tissue factor function.
Thromb. Haemost. 1997, 7, 1142-1149.
(36) Harker, L. A.; Hanson, S. R.; Wilcox, J . N.; Kelly, A. B.
Antithrombotic and antilesion benefits without hemorrhagic
risks by inhibiting tissue factor pathway. Haemostasis 1996, 26
(suppl 1), 76-82.
(37) South, M. S.; Dice, T. A.; Lachance, R. M.; Girard, T. J .; Stevens,
A. M.; Stegeman, R. A.; Stallings, W. C.; Kurumbail, R. G.;
Parlow, J . J . Polymer-assisted solution-phase (PASP) parallel
synthesis of an R-ketothiazole library as tissue Factor VIIa
inhibitors. Bioorg. Med. Chem. Lett. 2003, 13, 2363-2367.
(38) Parlow, J . J .; Dice, T. A.; Lachance, R. M.; Girard, T. J .; Stevens,
A. M.; Stegeman, R. A.; Stallings, W. C.; Kurumbail, R. G.;
South, M. S. Polymer-assisted solution-phase (PASP) parallel
synthesis of an R-ketothiazole library as tissue Factor VIIa
inhibitors. J . Med. Chem. 2003, 46, 4043-4049.
(39) Suleymanov, O. D.; Szalony, J . A.; Salyers, A. K.; Lachance, R.
M.; Parlow, J . J .; South, M. S.; Wood, R. S.; Nicholson, N. S.
Pharmacological interruption of acute thrombus formation with
minimal hemorrhagic complications by a small molecule tissue
Factor/Factor VIIa inhibitor: Comparison to Factor Xa and
thrombin inhibition in a nonhuman primate thrombosis model.
J . Pharmacol. Exp. Ther. 2003, in press.
(40) South, M. S.; Parlow, J . J .; J ones, D. E.; Case, B.; Dice, T.;
Lindmark, R.; Hayes, M. J .; Rueppel, M. L.; Fenton, R.; Franklin,
G. W.; Huang, H.-C.; Huang, W.; Kusturin, C.; Long, S. A.;
Neumann, W. L.; Reitz, D.; Trujillo, J . I.; Wang, C.-C.; Wood,
R.; Zeng, Q.; Mahoney, M. W. Preparation of arylpyrazinones
as coagulation cascade serine protease inhibitors. WO 0187854,
2001.
(41) South, M. S.; Parlow, J . J .; J ones, D. E.; Case, B.; Dice, T.;
Lindmark, R.; Hayes, M. J .; Rueppel, M. L.; Fenton, R. K.;
Franklin, G. W.; Huang, H.-C.; Huang, W.; Kusturin, C.; Long,
S. A.; Neumann, W. L.; Reitz, D. B.; Trujillo, J . I.; Wang, C.-C.;
Wood, R.; Zeng, Q. Preparation of arylpyrazinones as coagulation
cascade serine protease inhibitors. WO 0069834 2000.
(42) South, M. S.; Case, B. L.; Wood, R. S.; J ones, D. E.; Hayes, M.
J .; Girard, T. J .; Lachance, R. M.; Nicholson, N. S.; Clare, M.;
Stevens, A. M.; Stegeman, R. A.; Stallings, W. C.; Kurumbail,
R. G.; Parlow, J . J . Structure-based drug design of pyrazinone
antithrombotics as selective inhibitors of the tissue Factor VIIa
complex. Bioorg. Med. Chem. Lett. 2003, 13, 2319-2325.
(43) Kemball-Cook, G. J .; Daniel, J . D.; Tuddenham, E. G. D.; Harlos,
K. Crystal structure of active site-inhibited human coagulation
Factor VIIa (des-Gla). J . Struct. Biol. 1999, 127, 213-223.
(44) Pike, A. C. W.; Brzozowski, A. M.; Roberts, S. M.; Olsen, O. H.;
Persson, E. Structure of human Factor VIIa and its implications
for the triggering of blood coagulation. Proc. Natl. Acad. Sci. U.
S. A. 1999, 96, 8925-8930.
(23) Kohrt, J . T.; Filipski, K. J .; Rapundalo, S. T.; Cody, W. L.;
Edmunds, J . J . An efficient synthesis of 2-[3-(4-amidinophenyl-
carbamoyl)naphthalen-2-yl]-5-[(2,2-methylpropyl)carbamoyl]ben-
zoic acid:
a Factor VIIa inhibitor discovered by the Ono
Pharmaceutical Company. Tetrahedron Lett. 2000, 41, 6041-
6044.
(24) J akobsen, P.; Horneman, A. M.; Persson, E. Inhibitors of the
tissue Factor/Factor VIIa-induced coagulation: synthesis and
in vitro evaluation of novel 2-aryl substituted pyrido[3,4-d]-
[1,3]-, pyrido[2,3-d][1,3]-, pyrazino[2,3-d][1,3]-, pyrimido[4,5-d]-
[1,3]-, pyrazolo[3,4-d][1,3]-, thieno[3,2-d][1,3]- and thieno[2,3-d]-
[1,3]-oxazin-4-ones. Bioorg. Med. Chem. 2000, 8, 2803-2812.
(25) J akobsen, P.; Ritsmar Pedersen, B.; Persson, E. Inhibitors of
the tissue Factor/Factor VIIa-induced coagulation: synthesis and
in vitro evaluation of novel specific 2-aryl substituted 4H-3,1-
benzoxazin-4-ones. Bioorg. Med. Chem. 2000, 8, 2095-2103.
(26) Roussel, P.; Bradley, M.; Kane, P.; Bailey, C.; Arnold, R.; Cross,
A. Inhibition of the tissue Factor/Factor VIIa complex: lead
optimization using combinatorial chemistry. Tetrahedron 1999,
55, 6219-6230.
(45) Perera, L.; Darden, T. A.; Pedersen, L. G. Probing the structural
changes in the light chain of human coagulation Factor VIIa
due to tissue factor association. Biophys. J . 1999, 77, 99-113.
(46) Banner, D. W.; D’Arcy, A.; Chene, C.; Winkler, F. K.; Guha, A.;
Konigsberg, W. H.; Nemerson, Y.; Kirchhofer, D. The crystal
structure of the complex of blood coagulation Factor VIIa with
soluble tissue factor. Nature 1996, 380, 41-46.
(27) Gallagher, K. P.; Mertz, T. E.; Chi, L.; Rubin, J . R.; Uprichard,
A. C. G. Inhibitors of tissue Factor/Factor VIIa. Handbook Exp.
Pharmacol. 1999, 132, 421-445.
(47) Tulinsky, A. Molecular Interactions of Thrombin. Semin. Thromb.
Hemost. 1996, 117-124.
(28) Wang, D.; Girard, T. J .; Kasten, T. P.; LaChance, R. M.; Miller-
Wideman, M. A.; Durley, R. C. Inhibitory activity of unsaturated
fatty acids and anacardic acids toward soluble tissue Factor-
Factor VIIa complex. J . Nat. Prod. 1998, 61, 1352-1355.
(29) Semple, J . E.; Rowley, D. C.; Brunck, T. K.; Ripka, W. C.
Synthesis and biological activity of P2-P4 azapeptidomimetic
P1-argininal and P1-ketoargininamide derivatives: a novel class
of serine protease inhibitors. Bioorg. Med. Chem. Lett. 1997, 7,
315-320.
(30) Uchiba, M.; Okajima, K.; Abe, H.; Okabe, H.; Takatsuki, K. Effect
of nafamostat mesylate, a synthetic protease inhibitor, on tissue
Factor-Factor VIIa complex activity. Thromb. Res. 1994, 74,
155-161.
(31) Butenas, S.; Ribarik, N.; Mann, K. G. Synthetic substrates for
human Factor VIIa and Factor VIIa-tissue Factor. Biochemistry
1993, 32, 6531-6438.
(32) Van der Woerd-De Lange, J . A.; Van Dam-Mieras, M. C. E.;
Hemker, H. C. Inhibition of activated Factors II, VII, IX, and X
by synthetic organic compounds directed against the active-site
seryl residue. Haemostasis 1981, 10, 315-47.
(33) Szalony, J . A.; Taite, B. B.; Girard, T. J .; Nicholson, N. S.;
Lachance, R. M. Pharmacological intervention at disparate sites
in the coagulation cascade: Comparison of anti-thrombotic
efficacy vs bleeding propensity in a rat model of acute arterial
thrombosis. J . Thromb. Thrombolysis 2003, 14, 113-121.
(34) Zoldhelyi, P.; McNatt, J .; Shelat, H. S.; Yamamoto, Y.; Chen,
Z.-Q.; Willerson, J . T. Thromboresistance of balloon-injured
porcine carotid arteries after local gene transfer of human tissue
factor pathway inhibitor. Circulation 2000, 101, 289-295.
(48) Stubbs, M. T.; Bode, W. The clot thickens: clues provided by
thrombin structure. Trends Biochem. Sci. 1995, 20, 23-28.
(49) Adler, M.; Davey, D. D.; Phillips, G. B.; Kim, S. H.; J ancarik, J .;
Rumennik, G.; Light, D. R.; Whitlow, M. Preparation, charac-
terization, and the crystal structure of the inhibitor ZK-807834
(CI-1031) complexed with Factor Xa. Biochemistry 2000, 39,
12534-12542.
(50) Maignan, S.; Guilloteau, J . P.; Pouzieux, S.; Choi-Sledeski, Y.
M.; Becker, M. R.; Klein, S. I.; Ewing, W. R.; Pauls, H. W.; Spada,
A. P.; Mikol, V. Crystal structures of human Factor Xa com-
plexed with potent inhibitors. J . Med. Chem. 2000, 43, 3226-
3232.
(51) Kamata, K.; Kawamoto, H.; Honma, T.; Iwama, T.; Kim, S. H.
Structural basis for chemical inhibition of human blood coagula-
tion Factor Xa. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 6630-
6635.
(52) Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.; Park, C.
H.; Bode, W.; Huber, R.; Blankenship, D. T.; Cardin, A. D.; Kisiel,
W. Structure of human des(1-45) Factor Xa at 2.2 A resolution.
J . Mol. Biol. 1993, 232, 947-966.
(53) Brandstetter, H.; Kuhne, A.; Bode, W.; Huber, R.; von der Saal,
W.; Wirthensohn, K.; Engh, R. A. X-ray structure of active site-
inhibited clotting Factor Xa. Implications for drug design and
substrate recognition. J . Biol. Chem. 1996, 271, 29988-29992.
(54) Burgey, C. S.; Robinson, K. A.; Lyle, T. A.; Sanderson, P. E. J .;
Lewis, S. D.; Lucas, B. J .; Krueger, J . A.; Singh, R.; Miller-Stein,
C.; White, R. B.; Wong, B.; Lyle, E. A.; W., Peter, D.; Coburn, C.
A.; Dorsey, B. D.; Barrow, J . C.; Stranieri, M. T.; Holahan, M.

4062 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Parlow et al.
A.; Sitko, G. R.; Cook, J . J .; McMasters, D. R.; McDonough, C.
M.; Sanders, W. M.; Wallace, A. A.; Clayton, F. C.; Bohn, D.;
Leonard, Y. M.; Detwiler, T. J ., J r.; Lynch, J . J ., J r.; Yan, Y.;
Chen, Z.; Kuo, L.; Gardell, S. J .; Shafer, J . A.; Vacca, J . P.
Metabolism-directed optimization of 3-aminopyrazinone aceta-
mide thrombin inhibitors. Development of an orally bioavailable
series containing P1 and P3 pyridines. J . Med. Chem. 2003, 46,
461-473.
(59) Vekemans, J .; Pollers-Wieers, C.; Hoornaert, G.; A new synthesis
of substituted 2(1H)-pyrazinones. J . Heterocycl. Chem. 1983, 20,
919-923.
(60) Gloanec, P.; Herve, Y.; Bremand, N.; Lecouve, J .; Breard, F.;
De Nanteuil, G. Synthesis of benzyl (6S)-1,3-dichloro-4-oxo-
4,6,7,8-tetrahydro-pyrrolo[1,2-a]pyrazine-6-carboxylic ester, a
new conformationally constrained peptidomimetic derivative.
Tetrahedron Lett. 2002, 43, 3499-3501.
(61) Flynn, D. L.; Devraj, R. V.; Parlow, J . J . Polymer-Assisted
Solution-Phase Methods for Chemical Library Synthesis; Bur-
gess, K., Ed.; Solid-Phase Organic Synthesis; J ohn Wiley & Sons:
New York, 2000; Chapter 5.
(62) Parlow, J . J .; Devraj, R. V.; South, M. S. Solution-phase chemical
library synthesis using polymer-assisted purification techniques.
Curr. Opin. Chem. Biol. 1999, 3, 320-336.
(63) Flynn, D. L.; Devraj, R. V.; Naing, W.; Parlow, J . J .; Weidner,
J . J .; Yang, S. Polymer-assisted solution phase (PASP) chemical
library synthesis. Med. Chem. Res. 1998, 8, 219-243.
(64) J i, J .; Zhang, D.; Ye, Y.; Xing, Q. Studies on the reactions of
HOBt, HOOBt, HOSu with dichloroalkane solvents. Tetrahedron
Lett. 1998, 39, 6515-6516.
(65) South, M. S.; Dice, T. A.; Parlow, J . J . Polymer-assisted solution-
phase (PASP) library synthesis of R-Ketoamides. Biotechnol.
Bioeng. 2000, 61, 51-57.
(66) Olah, G. A.; Narang, S. C.; Gupta, B. G. B.; Malhotra, R.
Synthetic methods and reactions. 62. Transformations with
chlorotrimethylsilane/sodium iodide, a convenient in situ iodot-
rimethylsilane reagent. J . Org. Chem. 1979, 44, 1247-1251.
(67) The TF/VIIa enzyme potency for the 5-hydropyrazinones vs the
corresponding 5-chloropyrazinone analogues are essentially
equal with no more than a 2× difference in IC50 values.
(68) A full detail of compounds with P1 arginine mimetic amides will
be the topic of a future publication.
(55) Singh, R.; Silva Elipe, M. V.; Pearson, P. G.; Arison, B. H.; Wong,
B. K.; White, R.; Yu, X. O.; Burgey, C. S.; Lin, J . H.; Baillie, T.
A. Metabolic activation of a pyrazinone-containing thrombin
inhibitor. evidence for novel biotransformation involving pyrazi-
none ring oxidation, rearrangement, and covalent binding to
proteins. Chem. Res. Toxicol. 2003, 16, 198-207.
(56) Sanderson, P. E. J .; Cutrona, K. J .; Dyer, D. L.; Krueger, J . A.;
Kuo, L. C.; Lewis, S. D.; Lucas, B. J .; Yan, Y. Small, low
nanomolar, noncovalent thrombin inhibitors lacking a group to
fill the distal binding pocket. Bioorg. Med. Chem. Lett. 2003,
13, 161-164.
(57) Reiner, J . E.; Siev, D. V.; Araldi, G.; Cui, J . J .; Ho, J . Z.; Reddy,
K. M.; Mamedova, L.; Vu, P. H.; Lee, K. S.; Minami, N. K.;
Gibson, T. S.; Anderson, S. M.; Bradbury, A. E.; Nolan, T. G.;
Semple, J . E. Noncovalent thrombin inhibitors featuring P3-
heterocycles with P1-monocylic arginine surrogates. Bioorg. Med.
Chem. Lett. 2002, 12, 1203-1208.
(58) 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.; 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 ., J r.; Lyle,
E. A.; Lynch, J . J ., J r.; Stranieri, M. T.; Vastag, K.; Yan, Y.;
Shafer, J . A.; Vacca, J . P. Efficacious, orally bioavailable
thrombin inhibitors based on 3-aminopyridinone or 3-aminopy-
razinone acetamide peptidomimetic templates. J . Med. Chem.
1998, 41, 4466-4474.
J M030131L