Structure-based drug design of pyrazinone antithrombotics as selective inhibitors of the tissue factor VIIa complex

Article abstract of DOI:10.1016/S0960-894X(03)00410-4

Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole complexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P₂ surrogate for the tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attachment of a variety of P₁ and P₃ moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC₅₀ against TF/VIIa with >6250× selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a primate model of thrombosis.

Full text of DOI:10.1016/S0960-894X(03)00410-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2319–2325
Structure-Based Drug Design of Pyrazinone Antithrombotics as
Selective Inhibitors of the Tissue Factor VIIa Complex
Michael S. South,a,* Brenda L. Case,a Rhonda S. Wood,a Darin E. Jones,a
Michael J. Hayes,a Thomas J. Girard,b Rhonda M. Lachance,b Nancy S. Nicholson,c
Michael Clare,d Anna M. Stevens,e Roderick A. Stegeman,e William C. Stallings,e
Ravi G. Kurumbaile and John J. Parlowa
aDepartment of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Blvd., St. Louis, MO 63167, USA
bDepartment of Cardiovascular and Metabolic Disease, Pharmacia Corporation, 800 North Lindbergh Blvd., St. Louis, MO 63167, USA
cDepartment of Cardiovascular Pharmacology, Pharmacia Corporation, 4901 Searle Parkway, Skokie, IL 60077, USA
dStructure and Computational Chemistry, Pharmacia Corporation, 4901 Searle Parkway, Skokie, IL 60077, USA
eStructure and Computational Chemisty, Pharmacia Corporation, 700 Chesterfield Village Pkwy., St. Louis, MO 63198, USA
Received 7 March 2003; revised 16 April 2003; accepted 22 April 2003
Abstract—Structure-based drug design coupled with polymer-assisted solution-phase library synthesis was utilized to develop a
series of pyrazinone inhibitors of the tissue factor/Factor VIIa complex. The crystal structure of a tri-peptide ketothiazole com-
plexed with TF/VIIa was utilized in a docking experiment that identified a benzyl-substituted pyrazinone as a P2 surrogate for the
tri-peptide. A 5-step PASP library synthesis of these aryl-substituted pyrazinones was developed. The sequence allows for attach-
ment of a variety of P1 and P3 moieties, which led to synthesis pyrazinone 23. Compound 23 exhibited 16 nM IC50 against TF/VIIa
with >6250ꢀ selectivity versus Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for pre-
clinical intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side
effects in a primate model of thrombosis.
# 2003 Elsevier Science Ltd. All rights reserved.
Cardiovascular disease is the most common cause of
mortality in the western world. The disease is char-
acterized by the acute coronary syndromes unstable
angina and myocardial infarction.1 This inappropriate
thrombus formation is initiated via the extrinsic coagu-
lation cascade by a plaque rupture. The plaque rupture
exposes tissue factor (TF) to the serine protease Factor
VIIa (VIIa) in circulating blood, which forms the TF/
VIIa complex. This complex then further activates the
serine proteases Factors IX to IXa and X to Xa, which
in turn activate prothrombin to thrombin and fibrino-
gen to fibrin. Upon combination with activated plate-
lets, a fibrin clot may result in a life threatening
thrombus formation.2 There is evidence from our own
laboratories as well as others that selective inhibition
of the TF/VIIa complex may provide effective anti-
coagulation while lessening the risk of bleeding side
effects. as compared to other antithrombotic mechan-
isms (Glycoprotein IIb/IIIa antagonists and inhibitors
of Factor Xa and thrombin).3 Warfarin is currently the
only approved oral antithrombotic therapy, which is
complicated by frequent monitoring due to bleeding
side effects, drug-drug interactions, and food effects.4
Therefore, we sought to develop a series of potent and
selective TF/VIIa inhibitors in order to address the large
unmet medical need for safe and effective oral antic-
oagulants.
We recently described the synthesis and SAR of a series
of tripeptide-a-ketothiazoles.5 These reversible-covalent
serine protease inhibitors were found to have nanomo-
lar potency against TF/VIIa and exhibited 500ꢀ selec-
tivity versus thrombin. These compounds demonstrated
that it is possible utilizing structure-based drug design
(SBDD) techniques to achieve potent inhibition of the
TF/VIIa complex while maintaining selectivity versus
the other highly homologous serine proteases in the
coagulation cascade. Utilizing the crystal structure of
*Corresponding author. Tel.: +314-274-5104; fax: +314-274-1621;
e-mail: michael.s.south@pharmacia.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00410-4

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M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
TF/VIIa bound to these tripeptide-a-ketothiazoles, we
sought to develop a series of highly selective small
molecule inhibitors of TF/VIIa that are non-peptidic
and do not interact covalently with the catalytic appa-
ratus of the enzyme.6 Several reports of small molecule
TF/VIIa inhibitors have appeared describing varying
degrees of selectivity versus the other plasma proteases
in the coagulation cascade.7 We theorized that a wider
selectivity margin over Factor Xa and thrombin was
necessary to unequivocally demonstrate that inhibition
of the TF/VIIa complex would lead to a lower bleeding
side effect in-vivo. These highly selective TF/VIIa inhib-
itors should then be suitable for pre-clinical intravenous
proof-of-concept studies demonstrating the separation
between antithrombotic efficacy and bleeding side
effects in a primate model of thrombosis.3a
3-yl-ethyl}-3-phenyl-2-phenylmethanesulfonylamino-
propionamide) bound to the TF/VIIa enzyme is
shown.5,8
The key feature that is responsible for the good
selectivity on TF/VIIa versus thrombin is the inter-
action of the 3-pyridylphenylalanine at P2 with the S2
pocket of TF/VIIa. This S2 pocket has an aspartic
acid residue (Asp 60) that is not present in the other
coagulation enzymes and is much larger in size than
the corresponding pocket of thrombin, which has an
insertion loop in this region.9 Utilizing these key fea-
tures of the TF/VIIa crystal structure, we docked
(FlexX was used) a series of 6-membered heterocyclic
cores with pendant functionality at P2 that mimicked
the structure of the pyridylphenylalanine. We settled
on the pyrazinone core with a benzyl functionality at P2
as a prototype inhibitor to begin our SBDD studies.
The benzyl group seemed to occupy much of the space
of the pyridylphenylalanine in the peptide series, Figure
1. Also, critical hydrogen bonds between the pyrazinone
carbonyl, the NH of the phenethylamine and the NH of
the glycine unit on the pyrazinone to Gly 216 and Ser
214 were maintained similar to those in the tripeptide.
The pyrazinone core was previously utilized in the
design of inhibitors for thrombin10 and the pyrimidine
core for human leukocyte elastase.11 Both cores are
bound in these serine protease enzymes in a similar
fashion along the peptide backbone. However, it is
clear from Figure 1 that the requirements for P1, P2,
and P3 would likely be very different for the TF/VIIa
enzyme.
Our first attempts at developing small molecule, non-
peptidic inhibitors of TF/VIIa centered on the docking
experiment shown in Figure 1. Here, an X-ray crystal
structure of a tripeptide ketothiazole (N-{1-[4-guani-
dino-1-(thiazole-2-carbonyl)-butylcarbamoyl]-2-pyridin-
The next step was to prepare the prototype pyrazinone
inhibitor in Figure 1 for testing. The phenyl substituted
P2 was concurrently prepared for comparison purposes
to probe the depth of the S2 pocket of the TF/VIIa
enzyme. A variation of the Strecker synthesis was uti-
lized for the preparation of the pyrazinone skeleton,
Scheme 1.10d,e,12 Glycine benzyl ester was converted to
the pyrazinones via incubation of the aldehyde cyano-
hydrin with oxalyl chloride in 1,2-dichlorobenzene. This
reaction afforded the substituted dichloropyrazinones 1
and 2 in 38% and 60% yields, respectively. Compounds
1 and 2 were converted to the P3 phenethylamine sub-
stituted pyrazinones 3 and 4 in 72% and 96% yields
respectively via heating the core and amine in ethyl
acetate.
Figure 1. Docking of a pyrazinone inhibitor (compound 10, cyan) in
the active site of TF/VIIa based on the crystal structure of a tripeptide
ketothiazole inhibitor (C: gold, N: dark blue; O: red and S: green).
Crystals of TF/VIIa complex were obtained by slight modification of
the procedure described by Banner et al.8 Diffraction data of the
ketothiazole complex were measured at the Advanced Photon Source
to 2.6 A resolution and the structure has been refined with good
agreement between the data and the model (Rfree of 28.6% and R-
Factor: 22.2%). Some of the key side chains of factor VIIa are dis-
played (C: green, N: dark blue, O: red, S: yellow and H: orange). The
carbon atoms of the inhibitor are shown in gold color. The active site
serine, Ser 195, forms a covalent bond (thin solid line) with the acti-
vated carbon of the inhibitor. This results in the formation of a tran-
sition-state analogue with the negatively charged oxygen forming two
hydrogen bonds with the peptide nitrogens of Ser 195 and Gly 193 in
the oxy-anion hole. The nitrogen atom of the ketothiazole moiety
forms a hydrogen bond to the active site histidine, His 57. The loca-
tions of P1/S1, P2/S2 and P3/S3 sites are labeled.
Pyazinones 3 and 4 were then reacted with LiOH to
cleave the benzyl ester, followed by coupling with the
aminoketothiazole 11 to give 7 and 8, Scheme 2. After
removal of the protecting group with TFA and thioani-
sole, the target compounds 9 and 10 were isolated in
57% and 63% yields, respectively, after reverse phase
chromatography.
Compounds 9 and 10 were tested on the TF/VIIa
enzyme as well as Factor Xa, thrombin, and trypsin and
the IC50 results are shown in Table 1. The benzyl-sub-
stituted pyrazinone 10 inhibited TF/VIIa with an IC50
of 290 nm and was 2ꢀ less potent against the TF/VIIa
enzyme than the phenyl substituted pyrazinone 9. Both

M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
Table 1. Comparison of the biological activity of compounds 9 and 10
2321
Compd
n
IC50 (mM)
IIa
VIIa
Xa
Trypsin
9
10
0
1
0.15
0.29
1.41
2.90
<0.04
0.11
0.42
0.47
Scheme 1. Preparation of prototype pyrazinone inhibitors. Reagents
and conditions: (i) TMSCN, RCHO, CH2Cl2; (ii) (ClCO)2,1,2-
dichlorobenzene; (iii) Phenethylamine (3 equiv), EtOAc.
compounds showed some selectivity versus Factor Xa
(ꢁ10ꢀ), but no selectivity versus thrombin and trypsin.
The conclusion from this initial study was that the
phenyl substituent at P2 of the pyrazinone was preferred
over the benzyl substituent and would be utilized in
further SAR studies due to its enhanced potency.
Docking of compound 10 in the active site of the TF/
VIIa complex is illustrated in Figure 1.
format without chromatography in >95% purity.
Scheme 3 takes a slightly different course when R1 and
R2 were NO2 and CO2Me respectively since selective
deprotection of only the benzyl ester was desired for the
resin capture step. Pyrazinone 15 was treated with
hydrogen and Pd-C to simultaneously reduce the nitro
group to the aniline and remove the benzyl ester. The
synthesis then proceeds as described above to give
compound 18. The P1 moiety was then coupled to the
pyrazinone 18 by treatment with polymer bound carbo-
diimide and HOBt. Excess amine and starting acid 18
were removed from the solution by simultaneously uti-
lizing the polyamine and aldehyde resins leaving pure 19
in solution. The CBZ protecting group was then
removed via treatment with TMSCl and NaI followed
by a MeOH quench. Any excess TMSCl was scavenged
by polymer bound pyridine. Alternatively, a low-pres-
sure catalytic reduction will remove the CBZ protecting
group without concomitant reduction of the pyrazinone
chloro-group. In general, the 5-step PASP synthesis of
pyrazinones gave good yields (8–86% yields) and very
high purities (>95%) of final products 20, which were
suitable for in-vitro enzymatic testing. Scheme 3 allowed
for a variety of P1 and P3 substituents to rapidly be
incorporated into the molecules without chromato-
graphy. Three molecules were synthesized to illustrate
how SBDD was coupled with PASP synthesis to opti-
mize activity and selectivity for TF/VIIa complex versus
the other coagulation enzymes. A more detailed account
of this synthesis with SAR data will appear elsewhere.
The ultimate goal was to obtain high selectivity levels
for TF/VIIa versus Factor Xa and thrombin. We theo-
rized that the ketothiazole moiety was contributing to a
loss of selectivity since this moiety captures the catalytic
apparatus of all of the homologous serine proteases.
Therefore, our next study was to remove the ketothia-
zole and modify P1, P2, and P3 in a library format to
obtain the potency for TF/VIIa and selectivity levels
versus other proteases that were desired for our pre-
clinical studies.
A general Scheme for the 5-step PASP library synthesis
of pyrazinone TF/VIIa inhibitors is shown in Scheme
3.13 This library synthesis allows for the attachment of
any P1 and P3 for SAR studies. Core molecules 12, 13,
and 14 were prepared as shown in Scheme 1. The first
step was to incorporate the P3 moiety. Pyrazinones 15
were prepared by treatment of 12–14 with 3 equiv of a
primary amine in acetonitrile at 70 degrees centigrade.
Excess amine and amine hydrochloride were left in the
reaction mixture and carried on to the next step. The
next step involved the hydrolysis of the benzyl ester with
excess KOH followed by an acidification leaving the
crude pyrazinone acid 16 as a mixture with excess amine
hydrochloride and benzyl alcohol. Resin capture of the
pyrazinone acids 17 was accomplished by treatment
with a polyamine resin followed by repeated washings
with DMF, dichloromethane, MeOH and dichloro-
methane, which removed excess amine, benzyl alcohol
and any other non-acid impurities from the reaction
mixture. The pyrazinone acids 18 were then removed
from the resin with 4N HCl in dioxane. This sequence
allowed pyrazinone acids 18 to be prepared in a parallel
One of the first analogues that was prepared via the
5-step PASP library synthesis of pyrazinones (Scheme 3)
was compound 21 (65% overall yield). Compound 21
has a p-aminomethyl-benzamidine group at P1, a phenyl
group at P2 and a phenethyl group at P3. This com-
pound, which is shown in Table 2, was found to inhibit
TF/VIIa with an IC50 of 620 nM with no selectivity
versus Factor Xa and trypsin. A docking experiment
based on the X-ray crystal structure of the des-Cl ana-

2322
M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
Scheme 2. Preparation of protoype pyrazinone inhibitors. Reagents and conditions: (i) LiOH, THF:MeOH:H2O (3:3:1); (ii) 11, EDC, HOBt, DIEA,
THF; (iii) TFA, thioanisole.
Figure 3. Crystal structure of compound 22 bound in the active site of
TF/VIIa complex. The structure was refined to an Rfree of 29.0% at
2.8 A resolution. The atoms are colored as in Figure 1. Some of the
key side chains of Factor VIIa are displayed. The hydrogen bonds
formed by the inhibitor are shown in dotted white line. The meta-
amino group on the phenyl group of the inhibitor forms strong inter-
actions with Asp 60, Tyr 94, and the carbonyl oxygen of Thr 98 in the
S2 pocket.
Figure 2. Docking of compound 21 in the active site of TF/VIIa based
on the crystal structure of the complex with des-Cl analogue of
21(structure not shown). The crystal structure of the des-Cl analogue
was refined to an Rfree of 28.2% at 2.3 A resolution. The atoms are
colored as in Figure 1. Some of the key side chains of Factor VIIa are
displayed. The hydrogen bonds formed by the inhibitor are shown in
dotted white line.

M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
2323
logue of 21 (data not shown) is depicted in Figure 2.
The benzamidine P1 interacts with the Asp 189 at the
bottom of the S1 pocket. Key hydrophobic inter-
actions of the P2 phenyl group are noted with the S2
pocket of TF/VIIa, which is bounded by Asp 60, Tyr
94, Gly 97, and Thr 98. The phenethylamine P3
forms a hydrophobic interaction with the S3 pocket,
which includes residues Val 170E, Pro 170I, and Gln
217. We theorized that since the P2 pocket is larger
in size and Asp 60 is unique to TF/VIIa, we might
potentially be able to gain potency and selectivity
versus the other enzymes in the coagulation cascade
by placing a proton donor at the meta-position of the P2
phenyl ring. This SBDD hypothesis led to the synthesis
of compound 22, which was prepared according to the
library synthesis in Scheme 3 in 40% overall yield. We
were pleased to learn that compound 22 exhibited 20
nM activity on TF/VIIa, which is a 10ꢀ boost in
potency over 21. Also note that compound 22 exhibited
400ꢀ selectivity versus thrombin and >5000ꢀ selectiv-
ity versus Factor Xa, which is an improvement over the
unsubstituted phenyl P2 present in 21. An X-ray crystal
structure of compound 22 bound to TF/VIIa is shown
in Figure 3. It is very clear from the crystal structure of
22 that there is a 3-centered H-bonding interaction
between the m-amino P2 of 22 and Asp 60, Tyr 94, and
Thr 98. These key H-bonding interactions with the S2
pocket of TF/VIIa are likely responsible for the
observed 10ꢀ boost in potency and the increases in
selectivity versus thrombin. Selectivity versus Factor Xa
is probably due to a collision of the P2 phenyl ring with
Tyr 99 of Factor Xa (data not shown).
In general, our SAR studies indicate that the S3 pocket
of TF/VIIa seems to prefer small alkyl groups for lipo-
philic interaction with Gln 217 and residues from the
peptide segment 170E-170I (including Pro 170I). We felt
that the 400ꢀ selectivity versus thrombin exhibited by
compound 22 would not be sufficient for our intravei-
nous proof-of-concept study in primates. Further dock-
ing experiments indicated that placement of a carboxylic
acid moiety as a second meta-substituent on the P2
phenyl ring should lead to a favorable interaction with
Lys 60A. Synthesis of this compound was achieved as
Scheme 3. Polymer-assisted solution-phase (PASP) pyrazinone synthesis. Reagents and conditions: (i) 3 equiv R0NH2, AcCN; (ii) aq KOH, MeOH;
(iii) 4 N HCl/dioxane; (iv) H2, Pd/C, MeOH; (v) TMSCl, NaI, acetonitrile; (vi) MeOH.

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M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
Table 2. IC50’s in mM of pyrazinone antithrombotics
Compd
IC50 (mM)
R
R3
R5
VIIa
Xa
IIa
Trypsin
21
22
23
PhCH2CH2
cyclo-Bu
i-Pr
H
NH2
NH2
H
H
CO2H
0.62
0.020
0.016
—
>100
>100
0.15
8.0
>100
0.24
0.032
0.064
shown in Scheme 3, which gave 23 in 25% overall yield.
The activity of 23 on TF/VIIa was relatively unchanged
versus 22, however the selectivity versus thrombin was
dramatically increased to >6250ꢀ. An X-ray crystal
structure of 23 revealed that the carboxylate group of
the inhibitor approaches the side chain of Lys 60A,
although the electron density for the lysine side chain is
not well defined, Figure 4. The dual substitution of the
P2 phenyl group and consequent steric clashes impart
excellent selectivity against both thrombin and Factor
Xa. Compound 23 was deemed selective enough versus
Factor Xa and thrombin to proceed with our pre-clin-
ical IV-proof of concept studies.
In conclusion, we have demonstrated that high levels
of activity and selectivity for TF/VIIa are possible by
utilizing SBDD coupled with a 5-step PASP library
synthesis of pyrazinones. Starting from a tripeptide
ketothiazole that was active on TF/VIIa, we were
able to design a series of active and selective pyr-
azinone inhibitors of TF/VIIa that were reversible
and did not covalently capture the catalytic apparatus
of the enzyme. These inhibitors exhibited sufficient
levels of activity and selectivity for TF/VIIa versus
Factor Xa and thrombin to proceed with our pre-clin-
ical intravenous proof-of-concept studies of the separa-
tion between antithrombotic efficacy and bleeding side
effects in a primate model of thrombosis. We are cur-
rently continuing to refine the SAR in the pyrazinone
series. We are also studying a large variety of core
molecules as alternative central templates for the selec-
tive inhibition of TF/VIIa, which will be reported in due
course.14
Acknowledgements
We thank Dr. Huey Shieh for some of the early
crystallographic refinements of the TF-VIIa structure.
Diffraction data of inhibitor complexes with TF-VIIa
were collected at beamline 17-ID in the facilities of
the Industrial Macromolecular Crystallography Asso-
ciation Collaborative 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.
Figure 4. Crystal structure of compound 23 complexed with TF/VIIa.
The structure was refined to an Rfree of 31.1% at 2.1 A resolution. The
atoms are colored as in Figure 1 with some of the key side chains of
Factor VIIa shown. The hydrogen bonds formed by the inhibitor are
shown in dotted white line. Consistent with the structure of compound
22, the meta-amino group forms close interactions in the S2 pocket. As
predicted, the carboxylate group at 5-position of the phenyl group
approaches the side chain of Lys 60A, although the electron density is
not well defined for the surface exposed lysine side chain.

M. S. South et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2319–2325
2325
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