Design, Synthesis, and Crystal Structure of Selective 2-Pyridone Tissue Factor VIIa Inhibitors

Article abstract of DOI:10.1021/jm0301686

Targeted 2-pyridones were selected as tissue Factor VIIa inhibitors and prepared from 2,6-dibromopyridine via a multistep synthesis. A variety of chemical transformations, including regioselective nucleophilic addition, selective nitrogen alkylation, and

Full text of DOI:10.1021/jm0301686

4696
J . Med. Chem. 2003, 46, 4696-4701
Design , Syn th esis, a n d Cr ysta l Str u ctu r e of Selective 2-P yr id on e Tissu e F a ctor
VIIa In h ibitor s
J ohn J . Parlow,*,† Ravi G. Kurumbail,‡ Roderick A. Stegeman,‡ Anna M. Stevens,‡ William C. Stallings,‡ and
Michael S. South†
Department of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Boulevard,
St. Louis, Missouri 63167, and Structure and Computational Chemistry, Pharmacia Corporation,
700 Chesterfield Village Parkway, St. Louis, Missouri 63198
Received April 15, 2003
Targeted 2-pyridones were selected as tissue Factor VIIa inhibitors and prepared from 2,6-
dibromopyridine via a multistep synthesis. A variety of chemical transformations, including
regioselective nucleophilic addition, selective nitrogen alkylation, and a Suzuki coupling,
afforded the targeted tissue Factor VIIa inhibitors. The pyridone core was selected as a
replacement for the pyrazinone core of noncovalent tissue Factor VIIa inhibitors and designed
such that their substitution pattern would occupy and interact with the S1, S2, and S3 pockets
of the tissue Factor VIIa enzyme. These compounds were tested in several serine protease
enzyme assays involved in the coagulation cascade exhibiting modest activity on tissue Factor
VIIa with excellent selectivity over thrombin and Factor Xa. Finally, an X-ray crystal structure
of inhibitor 14a bound to tissue Factor VIIa was obtained and will be described.
In tr od u ction
Acute coronary syndromes (ACS) consisting of un-
stable angina, myocardial infarction, and sudden death
are the most frequent cause of mortality in the United
States and Western countries.1 ACS is associated with
acute thrombus formation, often as the result of a
plaque rupture. Thrombosis also occurs in transient
ischemic attack, stroke, peripheral occlusive arterial
disease, deep vein thrombosis, pulmonary embolism,
abrupt closure following angioplasty, and the dis-
F igu r e 1. Pyrazinone core structure.
seminated intravascular coagulation associated with
These pyrazinone compounds are active-site inhibitors
sepsis and certain cancers. Effective and safe anti-
for Factor VIIa exhibiting potency at the single-digit
thrombotics are needed to combat these diseases. Most
nanomolar level (IC50) with excellent selectivity over
research has focused on thrombin and Factor Xa inhibi-
thrombin (IIa) and Factor Xa. In an effort to increase
tors as potentially valuable therapeutic agents for these
the potency and influence the pharmocokinetic proper-
diseases.2 More recently, small molecule inhibitors of
ties, other heterocyclic ring systems were evaluated.
tissue Factor (TF) VIIa have been the point of much
Depicted in Figure 1 is the lead pyrazinone core
research effort because of their potential to inhibit the
structure I. Focusing on the central ring, one of the
coagulation cascade while lessening the risk of bleeding
exercises was to replace the nitrogen at the 4-position
side effects.3 The extrinsic coagulation cascade is trig-
in the pyrazinone ring with carbon, resulting in a
gered by the binding of Factor VIIa to cell surface TF.
pyridone heterocycle as the core ring.6 Other groups
This cascade is critical in normal hemostasis, but is also
have disclosed the pyridone template in the design of
involved in the pathogenesis of various thrombotic
human leukocyte elastase (HLC) inhibitors7 and throm-
diseases. Under normal conditions, TF expressed in the
bin inhibitors.8 We too were motivated by the at-
sub-endothelium of healthy blood vessels is not exposed
tractiveness of the pyridone template and the fact that
to blood. However, in disease or injury, vessel wall or
it has not been reported as a TF/VIIa inhibitor. Based
plaque TF is exposed and complexes with its cofactor
on the structure-activity relationship established with
Factor VIIa, leading to the activation of Factors IX and
the previously prepared pyrazinone analogues, two
X. The complex of Factor Xa and Factor Va on a
targeted pyridone analogues were selected for prepara-
membrane surface converts prothrombin to thrombin,
tion. Herein, we describe their synthesis, biological
leading to fibrin formation, deposition, and subsequent
activity, and X-ray crystal structure.
thrombus formation.4
We previously reported the preparation of pyrazinone
Resu lts a n d Discu ssion
analogues as noncovalent tissue Factor VIIa inhibitors.5
Several commercially available substituted pyridines
were evaluated in terms of potential starting material
for the synthesis. It was decided to proceed with the
synthesis using readily available, inexpensive 2,6-
* 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.
‡ Structure and Computational Chemistry.
10.1021/jm0301686 CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/13/2003

2-Pyridone Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4697
Sch em e 1a
a
Reagents: (a) NO2BF4, MeCN, reflux; (b) Fe, MeCO2H, 80 °C; (c) NaOMe, dioxane, reflux; (d) TiCl4, NaCNBH3, Me2CO, DCM; (e)
BBr3, DCM, -10 °C; (f) CaH2, THF; (g) Pd(PPh3)4, Cs2CO3, dioxane, 80 °C; (h) 4 N HCl/dioxane; (i) HOBt, NMM, PS-CD, DCM/DMF,
then PS-aldehyde and PS-polyamine; (j) H2, Pd/C, MeOH.
Ta ble 1. IC50 Values
dibromopyridine 1 as the starting material shown in
IC50 (uM)
Scheme 1. The dibromopyridine 1 was nitrated accord-
compd
VIIa
Xa
thrombin
ing to the literature procedure to afford 2,6-dibromo-3-
nitropyridine 2.9 The nitro group was reduced with iron
to afford the 3-amino-2,6-dibromopyridine 3. Reacting
the unsubstituted 3-amino-2,6-dibromopyridine 3 with
sodium methoxide in dioxane at reflux afforded exclu-
sively the desired regioisomer, 3-amino-6-bromo-2-
methoxypyridine 4. Several reductive amination condi-
tions were surveyed. The preformation of the imine
using titanium tetrachloride with acetone followed by
reduction with sodium cyanoborohydride afforded cleanly
the 6-bromo-2-methoxy-3-isopropylaminopyridine 5.10
The methoxy ether 5 was cleaved using boron tribro-
mide to afford the hydroxypyridine 6. Alkylation of the
hydroxypyridine 6 with tert-butylbromoacetate 7 using
calcium hydride as the base afforded a single isomer 8.
While alkylation could occur on either the nitrogen or
14a
14b
0.118
0.052
>30
>30
>30
>30
The target compounds 14a and 14b were screened for
potency on TF/VIIa and for other enzymes affecting
coagulation to determine specificity (Table 1). Each
enzyme assay consists of the specific enzyme and
chromogenic substrate for that enzyme. Enzyme activity
was determined by monitoring the increase in absor-
bance at 405 nm caused by the release of p-nitroaniline
when the substrate is hydrolyzed. All compounds were
assayed in duplicate at seven concentrations. Percent
inhibition at each concentration was calculated from the
OD405nm 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 dupli-
cates. Reported are the inhibition data as IC50 values
for TF/VIIa, Factor Xa, and thrombin. Compounds 14a
and 14b are both active against TF/VIIa with an IC50
of 118 nM and 52 nM, respectively. Both compounds are
selective over thrombin and Factor Xa exhibiting no
activity at 30 µM. However, both pyridones 14a and 14b
are approximately 5× less potent against TF/VIIa than
their corresponding pyrazinone analogues.5
the oxygen, only one regioisomer was isolated.11
A
Suzuki coupling of the N-alkylated bromopyridone 8
with the boronic acids 9 gave the biaryl compounds 10.
Selective hydrolysis of the tert-butyl ester with hydro-
chloric acid afforded the carboxylic acids 11. The car-
boxylic acids 11 were coupled with benzyl amino[4-
(aminomethyl)phenyl]methylenecarbamate 12 using
polymer-bound carbodiimide, hydroxybenzotriazole, and
N-methylmorpholine as base to afford the desired Cbz-
protected products 13. Concomitant deprotection of the
Cbz (and benzyl ester with compound 13a ) and reduc-
tion of the nitro group was accomplished using hydrogen
with palladium on carbon to afford the desired target
compounds 14.
The crystal structure of compound 14a bound to TF/
VIIa is shown in Figure 2. The benzamidine moiety of
the pyridone inhibitor forms an ion-pair with the

4698 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Parlow et al.
F igu r e 3. Overlap of the crystal structures of compound 14a
(multiccolored as shown in Figure 2) and a closely related
pyrazinone (cyan) inhibitor. The protein atoms of the pyrazi-
none complex are not shown for clarity. For superposition of
the two structures, only the R-carbon atoms of the catalytic
domain of VIIa were used. The second nitrogen atom of the
pyrazinone inhibitor forms an additional hydrogen bond to a
bound solvent molecule (red sphere).
F igu r e 2. Crystal structure of pyridone inhibitor 14a bound
in the active site of TF/VIIa comlex. The structure was refined
to an Rfree of 27.5% at 2.2 Å resolution (Rcrystal:21.3%). Some of
the key side chains of Factor VIIa are displayed (C: green, N:
dark blue, O: red, S: yellow and H: orange). The inhibitor is
represented with carbon, nitrogen, and oxygen atoms displayed
in gold, blue, and red, respectively. The hydrogen bonds formed
by the inhibitor are shown in dotted (magenta) lines.
in most of the pyrazinone structures that have been
determined. The bound solvent molecule appears to be
trapped between the side chain of Pro 170I and the
pyrazinone inhibitor. Thus, compared to pyrazinone, the
pyridone inhibitor forms one less hydrogen bond in the
active site of VIIa. While it is true that the extra
hydrogen bond in the case of pyrazinone inhibitor is
with a bound solvent molecule and is relatively weak,
it nevertheless could account for a slight increase in the
binding affinity of this class of inhibitors relative to
pyridones. Moreover, the extra nitrogen atom in the
pyrazinone ring (in comparison to pyridone core) could
also lead to better hydrogen bonding interactions of the
core with the atoms of VIIa. These factors together could
explain why the pyridones are slightly weaker inhibitors
of VIIa compared to the pyrazinones.
carboxylate of Asp 189 as observed in the structures of
pyrazinone complexes with TF/VIIa.30,31 The pyridone
carbonyl oxygen accepts a hydrogen bond (3.0 Å) from
the amide nitrogen of Gly 216. The amide nitrogen of
the glycine linker (between benzamidine and the pyri-
done core) forms a hydrogen bond to the carbonyl oxygen
of Ser 214 (3.3 Å). Moreover, the 3-amino group on the
pyridone core interacts with the carbonyl oxygen of Gly
216 (3.2 Å). The m-amino group on the phenyl ring at
P2 forms a four-centered H-bond network with the main
chain oxygen of Thr 98 and with the side chains of Tyr
94 and Asp 60.
Figure 3 shows the overlap of compound 14a with a
closely related pyrazinone inhibitor.5 As expected, most
of the functional groups of the pyrazinone and pyridone
inhibitors superimpose well in their bound conforma-
tions. The binding sites and the dihedral angles are well
conserved between the pyridone and the pyrazinone
inhibitors. Small differences are found in the conforma-
tions of the isopropyl groups at P3. There are two key
differences between the two inhibitors: (a) presence of
a chlorine atom on the pyrazinone ring, (b) replacement
of the second nitrogen atom by a carbon in the pyridone.
The chlorine atom of the pyrazinone inhibitor is within
van der Waals distance (3.1 A) of the carbonyl oxygen
of Gly 97 (Figure 3). This might contribute to the
enhanced potency of the pyrazinone inhibitor for TF/
VIIa (∼5×). The second nitrogen atom (4-position) of the
pyrazinone ring forms a hydrogen bond to a bound water
molecule (3.2 Å). A similar solvent molecule is observed
In summary, targeted pyridones were successfully
prepared via multistep synthesis using various trans-
formations and regioselective additions with a pyridine
nucleus to afford novel pyridone derivatives. The ana-
logues were tested against TF/VIIa but were less potent
than their corresponding pyrazinone analogues. The
X-ray crystal structure of targeted pyridone 14a was
obtained and compared with a closely related pyrazin-
one inhibitor, suggesting that additional hydrogen
bonding and van der Waals interactions may be respon-
sible for the enhanced potency of the pyrazinone inhibi-
tor. With no improved potency and a lack of favorable
pharmacokinetics, further syntheses using the pyridone
as the core ring were not pursued.

2-Pyridone Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4699
columns and on a HPLC system using a 15um 100A, C18
column (25 mm ID × 100 mm L). Reported yields are not
optimized, with emphasis on purity of products rather than
quantity.
Exp er im en ta l Section
Assa ys for Biologica l Activity. Recombinant soluble TF,
consisting of amino acids 1-219 of the mature protein se-
quence was expressed in E. coli and purified using a Mono Q
Sepharose FPLC. Recombinant human VIIa was purchased
from American Diagnostica (Greenwich, CT) and 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 Chromogenix (Sweden).
TF -VIIa Assa y. Recombinant 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 in-
hibitor 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
mL, 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 inhibi-
tion at each concentration was calculated from OD405nm 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.
F a ct or Xa Assa y. Human Factor Xa (0.3 nM) and N-R-
benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroani-
line dihydrochloride (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
OD405nm 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.
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
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 mL,
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 OD405nm 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 dupli-
cates.
3-Am in o-2,6-d ibr om op yr id in e (3). The 2,6-dibromo-3-
nitropyridine 2 (6.8 g, 24.3 mmol) was stirred in glacial acetic
acid. Powdered iron (6.7 g, 119 mmol) was added, and the
solution was heated to 80 °C with vigorous stirring. The
solution was stirred at 80 °C for 15 min at which point the
iron had turned gray. The reaction mixture was filtered
through Celite, and the solid was washed with diethyl ether
and ethyl acetate. The resultant organic layer was washed
with water and brine, dried over magnesium sulfate, and
filtered. The solvent was removed to afford the crude product.
The product was purified by column chromatography (20%
ethyl acetate-hexane) to afford 5.26 g (87%) of a white solid
of product 3; 1H NMR ppm (deuteriochloroform): 3.89 (s, 2H),
6.89 (d, 1H, J ) 8.4 Hz), 7.10 (d, 1H, J ) 8.4 Hz); 13C NMR
ppm (deuteriochloroform): 124.8, 125.7, 126.9, 127.7, 141.8;
HPLC purity (retention time): >99% (2.95 min); HRMS calcd
for C4H4Br2N2 (M+ + H) 252.8799, found 252.8779.
6-Br om o-2-m et h oxyp yr id in -3-a m in e (4). A solution of
3-amino-2,6-dibromopyridine 3 (10.4 g, 41.5 mmol) and sodium
methoxide (15.7 g, 290 mmol) in dioxane was heated to reflux
for 48 h. The brown reaction was allowed to cool to room
temperature and diluted with a saturated solution of am-
monium chloride. The solution was extracted with diethyl
ether, and the organic layer was washed with water, brine,
dried over magnesium sulfate, and filtered. The solvent was
removed by evaporation to afford the crude product. The
product was purified by column chromatography (20% ethyl
acetate-hexane) to afford 7.1 g (84%) of a red solid of product
4; 1H NMR ppm (deuteriochloroform): 3.94 (s, 3H), 6.71 (d,
1H, J ) 6.0 Hz), 6.83 (d, 1H, J ) 6.0 Hz); 13C NMR ppm
(deuteriochloroform): 67.2, 120.4, 122.5, 124.1, 130.3, 152.4;
HPLC purity (retention time): 94% (2.92 min); HRMS calcd
for C6H7Br1N2O (M+ + H) 204.9800, found 204.9769.
6-Br om o-N-isop r op yl-2-m eth oxyp yr id in -3-a m in e (5). A
solution of 1 M titanium tetrachloride in dichloromethane (37
mL, 37 mmol) was added to a solution of 6-bromo-2-methoxy-
pyridin-3-amine 4 (6.8 g, 33.6 mmol) and acetone (3.3 mL,
44.9 mmol) in dichloromethane. After the mixture was
stirred at room temperature for 3 h, sodium cyanoborohy-
dride (6.3 g, 100 mmol) was added and the solution stirred at
room temperature for 14 h. The solution was diluted with
diethyl ether and water. The organic layer was washed with
brine, dried over magnesium sulfate, and filtered. The sol-
vent was removed by evaporation to afford the crude product.
The product was purified by column chromatography (5%
ethyl acetate-hexane) to afford 5.7 g (69%) of a red oil of
1
product 5; H NMR ppm (deuteriochloroform): 1.20 (d, 6H),
3.51 (septet, 1H), 3.95 (s, 3H), 6.62 (d, 1H, J ) 6.0 Hz), 6.88
(d, 1H, J ) 6.0 Hz); HPLC purity (retention time): 95% (3.97
min); HRMS calcd for C9H13Br1N2O (M+ + H) 245.0289, found
245.0240.
6-Br om o-3-(isop r op yla m in o)p yr id in -2-ol (6). A solution
of 1 M boron tribromide in dichloromethane (46.0 mL, 46
mmol) was added to a solution of 6-bromo-N-isopropyl-2-
methoxypyridin-3-amine 5 (5.6 g, 0.23 mmol) in dichlo-
romethane at -10 °C. The solution warmed to room temper-
ature and stirred for 16 h. The reaction mixture was diluted
with water, neutralized with a saturated sodium bicarbonate
solution, and extracted with diethyl ether. The organic layer
was washed with water and brine, dried over magnesium
sulfate, and filtered. The solvent was removed by evaporation
to afford the crude product. The product was purified by
column chromatography (40% ethyl acetate-hexane) to afford
4.12 g (78%) of a white solid of product 6; 1H NMR ppm
(deuteriochloroform): 1.22 (d, 6H), 3.49 (septet, 1H), 6.20 (d,
1H), 6.88 (d, 1H); 13C NMR ppm (deuteriochloroform): 22.3,
44.1, 110.0, 128.6, 132.2, 136.4, 160.0; HPLC purity (retention
Ch em istr y. Gen er a l. 1H and 13C NMR spectra were
recorded using a 300 and 400 MHz NMR spectrometer. Sample
purities were determined by HPLC analysis equipped with a
mass spec detector using a C18 3.5 um 30 × 2.1 mm column,
eluting with a gradient system of 5/95 to 95/5 acetonitrile/
water with a buffer consisting of 0.1% TFA over 4.5 min at 1
mL/min and detected by UV at 254 and 210 nm using a diode
array detector. Column chromatography was performed on a
preparative liquid chromatography instrument using silica gel
time): 94% (2.45 min); HRMS calcd for C8H11Br1N2O (M+
H) 231.0133, found 231.0130.
+

4700 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22
Parlow et al.
ter t-Bu tyl [6-Br om o-3-(isop r op yla m in o)-2-oxop yr id in -
1(2H)-yl]a ceta te (8). A suspension of calcium hydride (1.7 g,
40.3 mmol) in tetrahydrofuran was added to 6-bromo-3-
(isopropylamino)pyridin-2-ol 6 (4.2 g, 18 mmol) in tetrahydro-
furan dropwise via an addition funnel. The resulting suspen-
sion was heated to reflux for 30 min. To the mixture was then
added a solution of tert-butyl bromoacetate 7 (2.9 mL, 19.6
mmol) in tetrahydrofuran (2.3 M). Refluxing of the mixture
was continued for 18 h. The reaction mixture was allowed to
cool to room temperature and quenched with an ice-water
mixture. The aqueous layer was extracted with diethyl ether.
The organic layer was washed with water and brine, dried over
magnesium sulfate, and filtered. The solvent was removed by
evaporation to afford the crude product. The product was
purified by column chromatography (10% ethyl acetate-
hexane) to afford 4.1 g (66%) of a white solid of product 8; 1H
NMR ppm (deuteriochloroform): 1.08 (H20,H15) (d, 6H, J )
6.4 Hz), 1.37 (H11,18,19) (s, 9H), 3.42 (H14) (septet, 1H, J )
6.4 Hz, 8.4 Hz), 4.81 (H7) (s, 2H), 5.05 (NH13) (d, 1H, J ) 8.4
Hz), 6.13 (H4) (d, 1H, J ) 7.9 Hz), 6.46 (H5) (d, 1H, J ) 7.9
Hz); 13C NMR ppm (DMSO): 22.3 (C15,C20), 28.2 (C11,18,-
19), 43.6 (C14), 50.7 (C7), 82.5 (C10), 107.0 (C4), 111.8 (C5),
136.8 (C3), 158.4 (C2), 167.1 (C8); HPLC purity (retention
procedure described for 11a , tert-butyl ester 10b (0.50 g, 0.10
mmol), was used to afford a yellow solid of product 11b; H
1
NMR ppm (deuteriomethanol): 1.38 (d, 6H), 3.87 (septet, 1H),
4.63 (s, 2H), 6.48 (d, 1H), 7.42 (m, 1H), 7.82 (m, 2H), 7.82 (m,
1H), 8.00 (m, 1H); HPLC purity (retention time): 90% (2.72
min); HRMS calcd for C16H18N4O5 (M+ + H) 347.1350, found
347.1334.
Ben zyl 3-[1-(2-{[4-((E)-Am in o{[(ben zyloxy)ca r bon yl]-
im in o}m et h yl)b en zyl]a m in o}-2-oxoet h yl)-5-(isop r op yl-
a m in o)-6-oxo-1,6-d ih yd r op yr id in -2-yl]-5-n it r ob en zoa t e
(13a ). Polystyrene-carbodiimide (2.3 g, 2.3 mmol) (1.00 mmol/
g) was added to a slurry of the acid 11a (0.53 g, 1.1 mmol),
1-hydroxybenzotriazole (0.16 g, 0.71 mmol), benzyl amino[4-
(aminomethyl)phenyl]methylcarbamate 12 (0.44 g, 1.3 mmol),
and N-methylmorpholine (1.3 mL, 11.8 mmol) in a dichlo-
romethane-N,N-dimethylformamide (3:1) solution, and the
suspension was agitated for 13 h. Upon completion of the
reaction, the polyamine resin (2.81 mmol/g) (5.6 mmol) and
polymer-bound aldehyde (2.3 mmol/g) (2.30 mmol) were added,
and the suspension was agitated for 1 h. The solution was
filtered, and the polymer was rinsed with N,N-dimethylfor-
mamide and dichloromethane until no more UV activity was
seen in the dichloromethane washing. The solvent was re-
moved by evaporation to afford the crude product. The product
was purified by reverse-phase chromatography to afford 0.73
g (87%) of an orange solid of product 13a ; 1H NMR ppm
(deuterioacetone): 1.19 (d, 6H,), 3.48 (septet, 1H), 4.41 (d, 2H),
4.63 (s, 2H), 5.18 (s, 2H), 5.48 (s, 2H), 6.30 (m, 2H), 7.40 (m,
11H), 7.99 (d, 1H), 8.18 (bt, 1H), 8.58 (m, 1H), 8.68 (m, 1H),
8.78 (m, 1H); HPLC purity (retention time): 90% (4.50 min);
time): 98% (4.63 min); HRMS calcd for C14H21Br1N2O3 (M+
H) 345.0814, found 345.0827.
+
Ben zyl 3-[1-(2-ter t-Bu toxy-2-oxoeth yl)-5-(isopr opylam i-
n o)-6-oxo-1,6-d ih yd r op yr id in -2-yl]-5-n itr oben zoa te (10a ).
Tetrakis(triphenylphosphine)palladium(0) (0.20 g, 0.17 mmol)
was added to a solution of compound 8 (0.60 g, 1.7 mmol),
boronic acid 9a (0.68 g, 2.3 mmol), and cesium carbonate (2.2
g, 6.7 mmol) in 18 mL of anhydrous dioxane. The resulting
mixture stirred for at 80 °C for 16 h. The reaction mixture
was allowed to cool to room temperature and was diluted with
water and extracted with diethyl ether. The organic layer was
washed with water and brine, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation to afford
the crude product. The product was purified by column
chromatography (25% ethyl acetate-hexane) to afford 0.70 g
HRMS calcd for
731.2853.
C
40H38N6O8 (M+ + H) 731.2829, found
Ben zyl (1E)-Am in o{4-[({[6-(3-a m in o-5-n itr op h en yl)-3-
(isop r op yla m in o)-2-oxop yr id in -1(2H)-yl]a cetyl}a m in o)-
m eth yl]p h en yl}m eth ylid en eca r ba m a te (13b). Following
the same procedure described for 13a , polystyrene-carbodiim-
ide (2.0 g, 2.0 mmol) (1.00 mmol/g), the acid 11b (0.34 g, 3.4
mmol), 1-hydroxybenzotriazole (0.14 g, 1.0 mmol), benzyl
amino[4-(aminomethyl)phenyl]methylcarbamate 12 (0.38 g, 1.2
mmol), and N-methylmorpholine (1.1 mL, 10 mmol) was used
to afford 0.24 g (39%) of a yellow solid of product 13b; 1H NMR
ppm (deuteriodimethylformamide): 1.36 (d, 6H), 3.70 (septet,
1H), 4.56 (d, 2H), 4.75 (s, 2H), 5.33 (s, 2H), 6.25 (d, 1H), 6.43
(d, 1H), 7.53 (m, 8H), 8.06 (m, 4H), 8.57 (bt, 1H), 11.16 (bs,
1H); HPLC purity (retention time): 80% (3.41 min); HRMS
calcd for C32H33N7O6 (M+ + H) 612.2565, found 612.2521.
1
(78%) of a red solid of product 10a ; H NMR ppm (deuterio-
chloroform): 1.24 (d, 6H,), 1.39 (s, 9H), 3.57 (septet, 1H), 4.41
(s, 2H), 5.39 (s, 2H), 6.14 (d, 1H), 6.40 (d, 1H), 7.40 (m, 6H),
8.35 (m, 1H), 8.42 (m, 1H), 8.85 (m, 1H); HPLC purity
(retention time): >99% (5.70 min); HRMS calcd for C28H31N3O7
(M+ + H) 522.2240, found 522.2242.
ter t-Bu tyl [6-{3-[(ter t-Bu toxyca r bon yl)a m in o]-5-n itr o-
p h en yl}-3-(isop r op yla m in o)-2-oxop yr id in -1(2H )-yl]a ce-
ta te (10b). Following the same procedure described for 10a ,
tetrakis(triphenylphosphine)palladium(0) (0.33 g, 0.28 mmol),
compound 8 (1.0 g, 2.9 mmol), boronic acid 9b (1.3 g, 4.6 mmol),
and cesium carbonate (3.7 g, 11.3 mmol) were used to give 0.57
g (39%) of an orange solid of product 10b; 1H NMR ppm
(deuteriochloroform): 1.24 (d, 6H,), 1.40 (s, 9H), 1.49 (s, 9H),
3.67 (septet, 1H), 4.43 (s, 2H), 6.14 (m, 2H), 7.20 (m, 1H), 7.64
(m, 1H), 7.82 (m, 6H), 8.35 (m, 1H); HPLC purity (retention
time): 96% (5.37 min); LRMS for C25H34N4O7 (ES, m/z) 503
(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]-5-(isop r op yla m in o)-6-oxo-1,6-d ih y-
d r op yr id in -2-yl]ben zoic Acid (14a ). A catalytic amount of
palladium on carbon (10%) in methanol was added to com-
pound 13a (0.33 g, 0.45 mmol) in methanol, and the mixture
was stirred under a balloon of hydrogen at room temperature
for 6 h. The mixture was filtered through Celite, and the
solvent was evaporated to afford the product. The product was
purified by reverse-phase chromatography to afford 0.28 g
1
(88%) of a yellow solid of product 14a ; H NMR ppm (deuter-
[6-{3-[(Ben zyloxy)ca r bon yl]-5-n itr op h en yl}-3-(isop r o-
p yla m in o)-2-oxop yr id in -1(2H)-yl]a cetic Acid (11a ). The
tert-butyl ester 10a (0.60 g, 1.1 mmol) was dissolved into 4 N
hydrochloric acid in dioxane and stirred at room temperature
for 22 h. The solvent was removed under a stream of nitrogen
to afford a red gum of product 11a [Note: Using heat during
the evaporation of the solvent in the workup of acid 11a led
to decarboxylation to afford the N-methyl derivative (benzyl
3-[5-(isopropylamino)-1-methyl-6-oxo-1,6-dihydropyridin-2-yl]-
5-nitrobenzoate). As a result, evaporation of the solvent was
conducted using a stream of nitrogen.]; 1H NMR ppm (deuter-
iochloroform): 1.53 (d, 6H,), 3.86 (septet, 1H), 4.55 (s, 2H), 5.39
(s, 2H), 6.42 (d, 1H), 7.39 (m, 5H), 8.29 (d, 1H), 8.37 (m, 1H),
8.44 (m, 1H), 8.94 (m, 1H); HPLC purity (retention time): 78%
(4.67 min); HRMS calcd for C24H23N3O7 (M+ + H) 466.1614,
found 466.1625.
iomethanol): 1.26 (d, 6H), 3.63 (septet, 1H), 4.44 (d, 2H), 4.65
(d, 2H), 6.27 (dd, 1H), 6.78 (dd, 1H), 7.62 (m, 9H); HPLC purity
(retention time): >99% (2.39 min); HRMS calcd for C25H28N6O4
(M+ + H) 477.2250, found 477.2253.
N -{4-[Am in o(im in o)m e t h yl]b e n zyl}-2-[6-(3,5-d ia m i-
n oph en yl)-3-(isopr opylam in o)-2-oxopyr idin -1(2H)-yl]acet-
a m id e (14b). A catalytic amount of palladium on carbon (10%)
in methanol was added to compound 13b (0.20 g, 0.32 mmol)
in methanol, and the mixture was stirred under a balloon of
hydrogen at room temperature for 12 h. The mixture was
filtered through Celite, and the solvent was evaporated to
afford the product. The product was purified by reverse-phase
chromatography to afford 0.24 g (39%) of a brown solid of
product 14b; 1H NMR ppm (deuteriomethanol): 1.26 (d, 6H),
3.63 (septet, 1H), 4.44 (d, 2H), 4.65 (d, 2H), 6.27 (dd, 1H), 6.78
(dd, 1H), 7.62 (m, 9H); HPLC purity (retention time): >99%
(2.39 min); HRMS calcd for C24H29N7O2 (M+ + H) 448.2456,
found 448.2447.
[6-(3-Am in o-5-n it r op h en yl)-3-(isop r op yla m in o)-2-ox-
op yr id in -1(2H)-yl]a cet ic Acid (11b ). Following the same

2-Pyridone Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 22 4701
Parlow, J . J .; Case, B. L.; Dice, T. A.; Hayes, M. J .; J ones, D. E.;
Neumann, W. L.; Wood, R. S.; Lachance, R. M.; Girard, T. J .;
Nicholson, N. S.; Clare, M.; Stegeman, R. A.; Stevens, A. M.;
Stallings, W. C.; Kurumbail, R. G.; South, M. S. Design, parallel
synthesis, and crystal structures of pyrazinone antithrombotics
as selective inhibitors of the tissue Factor VIIa complex. J . Med.
Chem. 2003, 46, 4050-4062.
Ack n ow led gm en t. The authors thank Rhonda M.
Lachance for the biological evaluation and Dr. Huey
Shieh for some of the early crystallographic refinements
of the TF/VIIa structure. Diffraction data for the TF/
VIIa complex with the different 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.
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J M0301686