Discovery of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-2′-(methylsulfonyl)-[1,1′-b iphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa

Article abstract of DOI:10.1021/jm000409z

Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca²⁺ on the phospholipid surface of platelets or endotheli

Full text of DOI:10.1021/jm000409z

566
J . Med. Chem. 2001, 44, 566-578
Discover y of 1-[3-(Am in om eth yl)p h en yl]-N-[3-flu or o-2′-(m eth ylsu lfon yl)-
[1,1′-bip h en yl]-4-yl]-3-(tr iflu or om eth yl)-1H-p yr a zole-5-ca r boxa m id e (DP C423),
a High ly P oten t, Selective, a n d Or a lly Bioa va ila ble In h ibitor of Blood
Coa gu la tion F a ctor Xa 1
Donald J . P. Pinto,* Michael J . Orwat, Shuaige Wang, J ohn M. Fevig, Mimi L. Quan, Eugene Amparo,
J oseph Cacciola, Karen A. Rossi, Richard S. Alexander, Angela M. Smallwood, J oseph M. Luettgen, Li Liang,
Bruce J . Aungst, Matthew R. Wright, Robert M. Knabb, Pancras C. Wong, Ruth R. Wexler, and
Patrick Y. S. Lam
DuPont Pharmaceuticals Company, Experimental Station, P.O. Box 80500, Wilmington, Delaware 19880-0500
Received September 19, 2000
Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of
convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va
and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the
prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically
active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating
a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline
and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal
models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic
core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa Ki ) 13 pM). We
also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of
this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved
by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further
optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery
of DPC423 (17h ), a highly potent, selective, and orally active factor Xa inhibitor which was
chosen for clinical development.
In tr od u ction
specifically affect coagulation, but not platelet function,
this mechanism may be anticipated to have less poten-
tial to increase the risk of abnormal bleeding relative
to thrombin inhibitors and antiplatelet agents. A com-
parison between hirudin (a thrombin inhibitor) and tick
anticoagulant peptide (TAP, an fXa inhibitor) suggests
that inhibition of fXa may result in less bleeding risk,
leading to a more favorable safety/efficacy ratio.12
Several potent monobasic inhibitors of fXa have been
published (from our laboratories as well as by others).13
Our efforts have recently led to the discovery of potent
isoxazoline analogues such as SF30314 and, more re-
cently, include potent isoxazoles such as SA862 (1, fXa
Ki ) 0.15 nM, Figure 1).15 The lack of chirality of the
isoxazole and its high affinity for fXa made it an
attractive template for further optimization. In this
paper, we wish to discuss the SAR of other five-
membered heterocyclic templates in which the point of
attachment to the P1 substituent is through a nitrogen
atom on the heterocycle.
Anticoagulants currently available for the treatment
and prevention of thromboembolic diseases include
warfarin (Coumadin), heparins, hirudin, hirulog, and
argatroban.2-7 Coumadin, a vitamin K-dependent in-
hibitor, has a slow onset, high oral efficacy, and long
duration of action. Heparins and direct thrombin inhibi-
tors have a rapid onset of action, but they must be
administered parenterally. The normal protocol for
patients on these therapies requires careful monitoring
of clotting times to achieve efficacy and dose titration
to minimize excessive bleeding.5 Therefore, there is a
serious effort to identify orally active anticoagulants
that are clinically safe and which require less monitor-
ing.
Factor Xa (fXa), a trypsin-like serine protease, holds
the central position that links the intrinsic and extrinsic
mechanisms in the blood coagulation cascade.8 The
physiological role of activated fXa is to proteolytically
activate thrombin.9 Thrombin has several procoagulant
functions that include the activation of platelets, the
feedback activation of other coagulation factors, and the
conversion of fibrinogen to insoluble fibrin clots. Small
molecule thrombin inhibitors have been intensely in-
vestigated.10 More recently, direct inhibition of fXa has
emerged as an attractive strategy for the discovery of
novel antithrombotic agents.10,11 Since fXa inhibitors
Ch em istr y
The synthesis of the 3-unsubstituted pyrazole 2a , a
compound chosen to best mimic the isoxazole core, was
accomplished quite readily starting with the condensa-
tion of 3-cyanophenylhydrazine and 4-(dimethylamino)-
2-oxo-but-3-enoic acid ethyl ester in acetic acid (Scheme
1).16 The condensation afforded a 1:1 regioisomeric
mixture of 3- and 5-substituted pyrazole esters. The
desired pyrazole ester 4a was obtained following puri-
* Corresponding author. E-mail address: Donald.J .Pinto@
dupontpharma.com. Phone: (302)695-1429. Fax: (302)695-1502.
10.1021/jm000409z CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/24/2001

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 567
Sch em e 2. Synthesis of 3-Methyl Pyrazole Analogue
2ba
F igu r e 1. Optimization of the five-membered heterocyclic
template.
Sch em e 1. Synthesis of Pyrazole 2a a
a
(a ) Acetic acid reflux, 75%, separated via flash chromatogra-
phy; (b) LiOH/THF/H2O, 90%; (c) oxalyl chloride/CH2Cl2 rt; DMAP/
CH2Cl2/rt, 99%; (d ) HCl/MeOH/ammonium carbonate/MeOH, TFA
80 °C, 36%.
coupled to 4′-amino[1,1′-biphenyl]-2-tert-butylsulfon-
amide via the intermediate acid chloride (oxalyl chloride/
DMAP, dichloromethane) to afford 8. Alternatively, the
ester 6 can be coupled with 4′-amino[1,1′-biphenyl]-2-
tert-butylsulfonamide using the Weinreb trimethyl-
aluminum18 methodology to afford intermediate 8b in
good yield. Most of the amide P4 variants, the 3-n-butyl
analogue 2c, and the N-methylated amide analogue 2d
were also prepared via the methodology outlined for 2b.
The desired benzamidine pyrazole analogues 2b-n were
obtained from the corresponding benzonitrile precursors
via the Pinner amidine protocol described previously.
The synthesis of the trifluoromethylpyrazole analogue
14a is outlined in Scheme 3. Condensation of m-bromo-
phenylhydrazine with commercially available 1,1,1-
trifluoro-2,4-pentanedione (Aldrich) afforded an insepa-
rable regioisomeric mixture (8:2) of pyrazoles 9a and
9b. Treatment of 9a and 9b with copper cyanide in
N-methyl pyrrolidinone at reflux provided cyanophenyl
pyrazole intermediates 10a and 10b. Treatment of this
mixture with NBS in refluxing carbon tetrachloride
afforded the alkylhalides which were immediately con-
verted to the hydroxymethyl intermediates 11a and 11b
under basic conditions. At this stage the desired pyr-
azole intermediate 11a was easily separated and puri-
fied via flash chromatography. Oxidation of 11a with a
mixture of ruthenium trichloride and sodium periodate
then afforded the desired 3-trifluoromethylpyrazole-5-
carboxylic acid intermediate 12. Acid chloride coupling
of 12 to 4′-amino[1,1′-biphenyl]-2-tert-butylsulfonamide
provided benzonitrile precursor 13a , which was treated
under the Pinner amidine protocol to afford the desired
benzamidine analogue 14a . Trifluoromethylpyrazole
analogues 14b-f were prepared in a similar manner.
a
(a ) AcOH reflux, 17%; (b) LiOH/THF/water, 98%; (c) oxalyl
chloride/CH2Cl2; (d ) DMAP/CH2Cl2, 76%; (e) HCl, MeOH; (f)
ammonia/MeOH, 45%.
fication via flash chromatography. Hydrolysis of the
ester under basic conditions (LiOH/THF/water) gave the
requisite pyrazole carboxylic acid derivative 4b, which
was treated with oxalyl chloride to form the acid
chloride. This was then coupled with 4′-amino[1,1′-
biphenyl]-2-tert-butylsulfonamide14a to provide the key
intermediate 5. Treatment of 5 under the Pinner condi-
tions14,15 (HCl/MeOH) afforded the imidate which was
immediately reacted with excess ammonia in methanol
to afford pyrazole analogue 2a in moderate yield. The
tert-butylsulfonamide substituent is simultaneously
deprotected to the primary sulfonamide under these
conditions.
The synthesis of a 3-substituted pyrazole 2b is
outlined in Scheme 2. Condensation of 3-cyanophenyl-
hydrazine with methyl 2-methoxyimino-4-oxopentanoate
afforded pyrazole 6 regioselectively.17 Hydrolysis of 6
under basic conditions (LiOH/THF/water) gave the
requisite carboxylic acid 6a in good yield. This was then

568 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pinto et al.
Sch em e 3. Synthesis of 3-Trifluoromethylpyrazole Analogue 14a
a
(a ) AcOH/reflux/quant; (b) CuCN/NMP, reflux, 40%; (c) NBS/CCl4, quant; (d ) CaCO3/dioxane/water, 44%; (e) NaIO4/RuCl3/CH3CN/
H2O, 90%; (f) oxalyl chloride/CH2Cl2, quant; DMAP/CH2Cl2, 58%; (g) MeOH/HCl, ammonium carbonate/MeOH, TFA, 80 °C, 46%.
The synthesis of the pyrrole analogues 3a and 3b is
shown in Scheme 4. Condensation of 3-aminobenzo-
nitrile with 2,5-dimethoxytetrahydrofuran afforded the
pyrrole intermediate 15a .24 Pyrrole 15a was formylated
(POCl3/DMF) regioselectively at the 2-position to de-
rivative 15b which was then oxidized (potassium per-
manganate/acetone/water) to afford the desired carbox-
ylic acid 15c in good yield. To prepare the 3-bromo-
pyrrole intermediate 15d , the 2-formyl intermediate 15b
was first brominated regioselectively at the 3-position
with NBS and then oxidized to the carboxylic acid as
described above. Acid chloride coupling of either 15c or
15d with 4′-amino[1,1′-biphenyl]-2-tert-butylsulfon-
amide afforded intermediates 16a and 16b which after
the Pinner reactions provided pyrrole analogues 3a ,b.
The benzonitrile precursors obtained as per Scheme
2 or 3 were subjected to reduction in a methanol-acetic
acid medium using a Parr Shaker apparatus with
catalytic palladium on carbon (5%) at 40 psi to afford
benzylamine analogues 17a -h (Scheme 5). Deprotection
of the tert-butylsulfonamide protecting group with tri-
fluoroacetic acid at 80 °C for 15 min afforded the
requisite primary benzylamine analogue 17a . Deriva-
tives not containing a sulfonamide functionality were
purified directly after reduction with palladium. Com-
pound 17h was dissolved in absolute methanol, and dry
HCl gas was bubbled through the mixture for 15 min.
DPC423 was obtained from this mixture by precipitation
with diethyl ether followed by recrystallization with
methanol and ethyl acetate. Alternatively, 17h was
neutralized with aqueous sodium hydroxide (1 N) and
subjected to the methodology described above to obtain
DPC423.
Resu lts a n d Discu ssion
Based on modeling experiments,19 a pyrazole template
was chosen to mimic the isoxazole core in compound 1.
Figure 2 shows an overlap of pyrazole 2a with isoxazole
1 in the fXa active site. Pyrazole 2a with a fXa Ki of
0.16 nM is equipotent to isoxazole 1 (fXa Ki ) 0.15nM).
On the basis of these data, it appears that the isoxazole
oxygen atom does not offer any binding advantages.
Additionally, pyrazole 2a offers the potential of substi-
tution at the 3-position, which is not possible with the
isoxazole core.
The 3-methylpyrazole analogue 2b is an order of
magnitude more potent than pyrazole 2a (fXa Ki ) 13
pM compared to 0.16 nM). Furthermore, the potency of
2b compares favorably to the very potent and naturally
occurring tick anticoagulant protein (TAP, fXa Ki ) 100
pM).20 Compound 2b shows >1000-fold selectivity for
fXa compared to thrombin and trypsin (Table 1). The
X-ray structure of pyrazole 2b complexed to bovine
trypsin was determined (Figure 3).21-23 The structure
has been refined to a crystallographic R-factor of 19.2%
at 1.80 Å resolution. As expected, the benzamidine P1

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 569
Sch em e 4. Synthesis of Pyrrole Analogues 3a and 3ba
F igu r e 2. Overlay of pyrazole 2a and SA862 in factor Xa.
interacts with Gly-218 (3.1 Å) through a bridging water
molecule. The P4 o-biphenylsulfonamide substituent is
situated in the S4 region with the terminal phenyl-
sulfonamide ring forming an edge to face interaction
with Trp-215. Overall, the conformations adopted by the
trypsin bound inhibitor and that predicted by the
binding of pyrazole analogue 2a in the active site of fXa
are similar.
To address the importance of substitution on the core
template, we examined the fXa activity of pyrrole
analogues 3a and 3b. The 5-fold loss in fXa potency for
pyrrole analogue 3a compared to pyrazole 2a confirms
that the N-2 nitrogen in the pyrazole ring is indeed
important for potency. The 3-bromo pyrrole 3b analogue
while more potent than 3a is still significantly less
potent than the 3-alkyl substituted pyrazoles 2b and
2c.
The N-methylation of the central amide linker as in
2d resulted in >800-fold loss of factor Xa potency
compared to the secondary amide analogue 2b. It is
possible that the loss in fXa activity is due to a change
in the amide bond conformation brought about by the
N-methyl substitution. Further work is currently being
pursued to determine the importance of the amide NH
and will be reported in due course.
a
(a ) AcOH reflux, 93%; (b) DMF/POCl3, 0 °C-reflux, 83%; (c)
KMnO4, acetone/water, 74%; (d ) oxalyl chloride, quant, DMAP/
CH2Cl2, 80%; (e) NBS/THF, rt, 53%; (f) HCl/MeOH, ammonium
carbonate/MeOH, 60%; TFA, 80 °C, 70%.
forms a bidentate interaction with Asp-189 (2.9 Å and
3.0 Å in the S1 specificity pocket). The dihedral angle
between the pyrazole and the benzamidine P1 substitu-
ent is 70°, which enables the pyrazole N-2 nitrogen to
interact with the backbone NH of Gln-192 (3.2 Å) and
form a Van der Waal interaction with Cys-220 (3.3 Å).
The 3-methyl substituent on the pyrazole is near the
solvent accessible surface at the outer ridge of the active
site and within contact distance of Gly-218 and Cys-
220. The amide carbonyl oxygen interacts with the NH
of Gly-216 (3.2 Å) and also with Ser-214 (3.0 Å) through
a bridging water molecule. Similarly, the amide NH
Sch em e 5. Synthesis of Benzylamine P1 Analogues
a
(a ) MeOH, 0 °C, HCl(gas) 0.15 min; (b) diethyl ether; (c) recrystallized, MeOH/EtOAc or (d ) neutralize, NaOH (1 N).

570 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pinto et al.
Ta ble 1. Comparison of SA862 with Pyrazole and Pyrrole
Analogues
Ta ble 2. In Vitro Activity Profile of P4 Variants in the
3-Methylpyrazole Series
human
in vitro
FXaa
human
in vitro
human
in vitro
thrombina trypsina
compd
X
Y
R
Ki, nM
Ki, nM
Ki, nM
2b
2e
2f
2g
2h
2i
2j
2k
2l
CH
CH SO2NH2
CH SO2CH3
CH CF3
0.013
0.008
0.040
0.460
1.300
0.007
0.041
0.005
0.010
0.009
0.020
300
180
300
450
600
1400
7400
210
200
300
230
16
13
49
100
87
59
150
4.6
17
CH
CH
CH
CH
N
N
C-F
CH
CH
F
H
CH SO2NH2
SO2NH2
CH SO2NH2
N
C-Br CH SO2NH2
C-Cl CH SO2NH2
C-F
2m
2n
15
human
in vitro
FXaa
human
in vitro
thrombina
Ki, nM
human
in vitro
trypsina
Ki, nM
CH SO2CH3
a,b
Refer to Table 1.
compdb
isoxazole
SA862 (1)
pyrazoles
R
Ki, nM
is orthogonal to the inner phenyl ring, forming an edge-
to-face interaction with Trp-215. This orthogonal ar-
rangement places the sulfonamide or the sulfone sub-
stituent in close proximity to the OH group of Tyr-99.
In the case of the isoxazole and pyrazole series, the P1
and the P4 substituents are 1,2-substituted on the
heterocycle. The rigid framework adopted by these
inhibitors are complementary to the fXa active site and
places the P4 substituent in a highly optimized fashion
in the S4 region. Table 2 summarizes the fXa activity
of additional substituted P4 biaryl, pyridylphenyl, and
pyrimidylphenyl pyrazole analogues. In the biaryl se-
ries, the order of potency for the 2′-substituted biphenyl
P4 substituents is SO2CH3 g SO2NH2 > CF3 > H. While
the rank order of potency for these compounds is similar
to that previously determined for the isoxazoline se-
ries,14 the pyrazoles are significantly more potent.
Substitution at the 3-biphenyl position (proximal phen-
yl) was clearly shown to affect the potency of the
isoxazoline and isoxazole analogues. In the pyrazole
series, the introduction of a 3-fluoro (2k ) substituent on
the proximal phenyl ring or the replacement of this ring
with a 2-pyridyl ring (2i) results in a 2-3-fold enhance-
ment in potency. In general, the 3-halogen biphenyl
substituents improve potency in the order of F > Cl g
Br > H. The pyrimidyl analogue (2j) shows a slight loss
in activity when compared to 2b or 2i, but it is still
considerably more potent than pyrazole 2a . The sub-
stituted biaryl analogues show good selectivity for fXa
compared to thrombin but are less selective over trypsin.
However, improved trypsin selectivity is observed for
the aza P4 analogues 2i and 2j. Thus, in the 3-methyl-
pyrazole series, the combination of either a 3-fluoro
substitution on the proximal phenyl ring or the 3-pyridyl
substitution, with either an o-methylsulfone or o-sulfon-
amide substituent on the terminal P4 phenyl ring, is
considered optimal for potency or selectivity or both.
Su bstitu tion on P yr a zole Cor e. The replacement
of the 3-methyl substituent with a longer substituent,
such as butyl analogue 2c (Table 1), results in a 2-fold
loss in potency, albeit still 3-fold more potent than the
0.15
2800
21
2a
H
Me
n-Bu
Me
0.16
0.013
0.06
900
300
300
15
16
11
SN429 (2b)
2c
2d N-Me amide
pyrroles
3a
3b
11.00
>2000
>1600
H
Br
0.80
0.29
900
300
56
32
a
Human purified enzymes were used. Ki values are averaged
from multiple determinations (n ) 2), and the standard deviations
b
are <30% of the mean. Ki’s were measured as in ref 14a,b. All
compounds gave satisfactory analytical data (C, H, N; (0.4% of
theoretical values).
F igu r e 3. X-ray structure of pyrazole 2b in bovine trypsin.
P 4 Mod ifica tion s. In the isoxazoline series,14 the
o-biphenylsulfonamide moiety and the corresponding
o-biphenylmethylsulfone were identified as optimal P4
substituents. The potent fXa binding activity shown for
these compounds is due to the highly constrained
orientation, in which the P1 benzamidine and the P4
biphenylsulfonamide substituents are in a 1,3-relation-
ship on the isoxazoline core. In these examples, the
terminal phenyl ring of the 2′-biphenylsulfonamide P4

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 571
Ta ble 3. In Vitro Activities Profile of P4 Variants in the
3-Trifluoromethylpyrazole Series
human
in vitro
FXaa
human
in vitro
thrombina
Ki, nM
human
in vitro
trypsina
Ki, nM
compd
I/II
X
Y
Ki, nM
2b
2k
CH
C-F
CH
N
CH
CH
CH
CH
N
CH
CH
CH
0.013
0.005
0.015
0.009
0.010
<0.005
0.008
<0.005
300
210
40
400
900
120
70
16
4.6
3.9
59
F igu r e 4. Binding model of pyrazole 17h in factor Xa.
14a
14b
14c
14d
14e
14f
I
I
I
I
II
II
half-lives ranging from 0.3 to 3.7 h. The pyrimidyl P4
analogues 2j and 14c show lower clearance than the
other compounds evaluated but still show moderate
duration of action due to their relatively low volume of
distribution. The analogue 14f has slightly higher
clearance than the other compounds in this set, but it
also has a higher volume of distribution and longer half-
life (3.8 h). When administered orally, these benz-
amidine analogues show poor oral bioavailability (<5%).
The poor permeability is likely a result of the highly
charged amidine functionality (pKa ∼ 10.7), which was
predicted based on the low Caco-2 permeability coef-
ficient Papp values obtained for these compounds.
The antithrombotic efficacy of a number of benz-
amidine analogues was evaluated in a rabbit arterio-
venous shunt thrombosis model (Table 4).26 These
compounds were administered by intravenous infusion,
and the antithrombotic effect was measured as the ID50
(dose which reduced clot weight by 50%). In this assay,
pyrazole analogues 2b, 2f, 2i, and 2j inhibit thrombus
formation in a dose dependent manner with ID50’s
ranging from 0.23 µmol/kg/h to 0.02 µmol/kg/h.
Ben zyla m in e P 1 An a logu es. To improve the oral
absorption of the pyrazole analogues, we adopted a
strategy of replacing the P1 benzamidine with a less
basic moiety (preferably with pKa < 10). One of the first
benzamidine replacements considered was the benzyl-
amine moiety (pKa of ∼8.8). We modeled the benzyl-
amine analogue 17h in the active site of fXa (Figure 4)
and determined that the conformation assumed by 17h
was very similar to that seen for benzamidine 2b. In
the case of the benzylamine analogue 17h , the single
amino functionality interacts with Asp-189, but is not
capable of a bidentate interaction such as that seen for
the benzamidine analogue 2b. In addition, since no
interaction is observed with Ser-190, some loss in fXa
activity was expected. Since the pyrazole series was
optimized to the 5-10 pM range, we were able to
sacrifice up to 2 orders of magnitude in potency in
exchange for compounds with improved oral absorption.
The in vitro data for the benzylamine analogues 17a -d
is reported in Table 5. A quick review of the data shows
similar fXa potency trends as seen in the benzamidine
series. The benzylamine analogue 17a (fXa Ki ) 2.5 nM)
is 190-fold less potent than its benzamidine counterpart
2a (fXa Ki ) 0.013 nM). The incorporation of a 3-F
substituent in the proximal phenyl ring resulted in 17b
with a modest improvement in potency (fXa Ki ) 1.6
N
20
C-F
CH
C-F
4.0
4.3
3.4
50
a,b
Refer to Table 1.
Ta ble 4. Dog Pharmacokinetics and Antithrombotic Efficacy of
Benzamidine Analogues
Caco-2c
rabbit A-V
Cl
Vdss
t1/2
F% Papp × 10-6 shuntd ID50
compd (L/kg/h)a (L/kg)a (h)a (po)b
cm/s
(µmol/kg/h)
2b
2f
2i
2j
14c
14f
0.67
0.47
0.76
0.36
0.33
1.10
0.29
0.47
0.21
0.20
0.25
3.80
0.82
4
0.30
NT
NT
NT
0.10
0.20
0.02
0.24
0.07
0.03
NT
3.72 NT
0.31 <1
3.32 <5
1.40 NT
3.80
0
NT
a
b
Dose of 1 mg/kg IV. Oral dose of 4 mg/kg. c Reference 27.
d
Reference 26.
unsubstituted pyrazole 2a . Since the methyl analogue
2b shows much better potency than the longer chain
alkyl substitutions, the 3-trifluoromethyl pyrazole was
prepared. Although it was anticipated that the trifluo-
romethyl substituent would exhibit a better hydrophobic
interaction with the fXa enzyme than the 3-methyl
substituent, the 3-trifluoromethylpyrazole o-biphenyl-
sulfonamide P4 analogue 14a (Table 3) and the corre-
sponding 3-methylpyrazole analogue 2b were found to
have similar potency. Comparable potencies were also
observed for the respective pyridyl analogues 14b and
2i. However, in the pyrimidyl series, a 4-fold improve-
ment was observed for the trifluoromethyl pyrimidyl
analogue 14c compared to the corresponding methyl
analogue 2j. A similar enhancement was also observed
for the 3-fluoro analogue 14d . The methylsulfonyl
analogue 14e was 2-fold more potent than the corre-
sponding sulfonamide 14a , and as anticipated, a further
increase in affinity was observed with the 3-fluoro
analogue 14f. Compounds 14d and 14f clearly suggest
that the 3-trifluoromethyl substituent, when taken
together with an optimized biaryl P4 moiety, results in
extremely high affinity molecules. These two compounds
are currently the most potent fXa inhibitors reported
in the literature to date.
In Vivo P r ofile of Ben za m id in e An a logu es. The
pharmacokinetic profiles for a select set of benzamidine
analogues were determined (Table 4). When dosed
intravenously in beagle dogs, the biaryl P4 analogues
2f and 2i have relatively low clearance and moderate

572 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Ta ble 5. Profile of Pyrazole Benzylamine P1 Analogues
Pinto et al.
human
in vitro
FXaa
human
in vitro
thrombina
Ki, nM
human
in vitro
trypsina
Ki, nM
rabbit A-V
shuntb ID50
(µmol/kg/h)
human
APTT,
µM
compd
R
X
R1
Ki, nM
HPBc
2b (amidine)
17a
17b
17c
17d
17f (amidine)
17e
17f
17g
17h
CH3
CH3
CH3
CH3
CH3
CF 3
CF3
CF3
CF3
CF3
CH
CH
C-F
CH
C-F
C-F
CH
C-F
CH
SO2NH2
SO2NH2
SO2NH2
SO2CH3
SO2CH3
SO2CH3
SO2NH2
SO2NH2
SO2CH3
SO2CH3
0.013 (0.03)d
2.70 (4.10)d
1.60 (2.20)d
0.89
300
21000
12000
21000
14000
50
14000
2000
5800
16
250
120
220
130
3.4
120
53
0.02
NT
NT
NT
0.90
NT
1.00
0.60
2.20
1.10 (0.15)e
0.44
2.30
0.92
1.30
1.10
70
63
70
NT
75
NT
83
93
77
89
0.48 (1.20)d
<0.005
0.91 (2.10)d
0.36 (1.00)d
0.38
6.40
0.91
2.20
4.86
100
60
C-F
0.15 (0.30)d
6000
a
b
Refer to Table 1. Reference 26. c HPB refers to human protein binding; all compounds gave satisfactory analytical data including C,
H, N within ( of theoretical values. Rabbit in vitro data. e IC50, which is the concentration to inhibit thrombus formation by 50% in
rabbits.
d
Ta ble 6. Dog Pharmacokinetic Profile of Benzylamine Analogues
Caco-2a
Cl
(L/kg/h)
Vdss
(L/kg)
t1/2
(h)
F%
(po)
Papp × 10-6
compd
X
R1
(cm/s)
R ) CH3
2b (amidine)
CH
CH
C-F
CH
SO2NH2
SO2NH2
SO2NH2
SO2CH3
SO2CH3
0.67
0.43
0.30
0.98
2.10
0.29
1.90
0.60
3.62
3.10
0.82
9.30
2.80
5.80
1.90
4.40
13
10
0.30 ( 0.07
0.20 ( 0.03
0.95 ( 0.02
1.20 ( 0.09
3.14 ( 0.10
17a
17b
17c
17d
C-F
73
R ) CF3
14f (amidine)
C-F
CH
C-F
CH
SO2CH3
SO2NH2
SO2NH2
SO2CH3
SO2CH3
1.10
0.75
0.10
0.44
0.24
3.80
4.36
0.34
5.82
0.90
3.80
7.88
4.70
9.50
7.50
<1
35
22
39
57
0.20 ( 0.01
bql
0.91 ( 0.11
3.38 ( 0.08
4.86 ( 0.33
17e
17f
17g
17h
C-F
a
b
IV dose ) 0.5 mg/kg. Oral dose ) 0.2 mg/kg. c Reference 27.
nM) and improved permeability in the Caco-2 assay
with a Papp value of 0.95 × 10-6 cm/s compared to 0.2 ×
10-6 cm/s (Table 6). Replacing the 2′-sulfonamide on the
terminal phenyl ring with the 2′-sulfonylmethyl sub-
stituent afforded 17c (fXa Ki ) 0.89 nM). This series
was further optimized by incorporating the 3-fluoro
substituent (17d ; fXa Ki ) 0.48 nM). Most notable was
the improvement in the Caco-2 permeability displayed
by analogues 17c and 17d , which have Papp values of
1.20 × 10-6 cm/s and 3.14 × 10-6 cm/s, respectively
(Table 6). The introduction of the trifluoromethyl pyr-
azole into the benzylamine P1 series provides com-
pounds with improved potency and an SAR which
parallels that observed for the methylpyrazole benzyl-
amine P1 series. The most potent compound was 17h
(Ki ) 0.15 nM) which contains the same P4 substituent
as 17d . More importantly the Caco-2 permeability for
17h (Papp ) 4.86 × 10-6 cm/sec) improves significantly.
All compounds produced good selectivity ratios for fXa
relative to thrombin and trypsin. Against relevant
human enzymes, 17h showed greater than 10000-fold
selectivity for inhibition of fXa over thrombin and 300-
fold selectivity over trypsin and kallikrein.29 In the
activated partial thromboplastin time (APTT) in vitro
clotting assay, the methylpyrazole benzylamine P1
analogues (Table 5) displayed clotting times within 2-
to 3-fold when compared to benzamidine 2b and are
slightly higher for the trifluoromethyl analogues. The
higher protein binding exhibited by the trifluoromethyl
pyrazole analogues (Table 5) over their 3-methyl pyr-
azole counterparts may account at least in part for these
changes. In the rabbit arterio-venous shunt thrombosis
model, the benzylamine analogues exhibit good anti-
thrombotic potency with ID50 values in the range of 0.6-

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 573
F igu r e 5. In vivo dog pharmacokinetics of DPC423 (17h ) and 17f.
Ta ble 7. Selectivity Profile for DPC423 (17h )
human enzymes
factor Xa
trypsin
thrombin
plasma kallikrein
activated protein C
factor IXa
factor VIIa
chymotrypsin
urokinase
Ki (nM)
0.15 ( 0.02
60 ( 6.5
6000
61a
1800a
F igu r e 6. Antithrombotic effect of DPC423 (17h ) in the rabbit
arterio-venous shunt thrombosis model.
2200 ( 0.2a
>15000a
>17000a
>19000a
>35000a
>45000a
44000a
2.2 µmol/kg/h (Table 5). With the high binding affinity
and good selectivity relative to thrombin and trypsin
coupled with the good Caco-2 permeability and anti-
thrombotic potency for these benzylamine analogues in
hand, we next addressed the pharmacokinetic properties
of the benzylamines.
plasmin
tPA
complement factor I IC50
a
Data from 17h TFA salt, others from HCl salt.
P h a r m a cok in etic P r ofile of Ben zyla m in e An a -
logu es. When methyl pyrazole sulfonamide 17a was
dosed via intravenous administration to beagle dogs, low
clearance and long half-life were observed (Table 6).
However, when dosed orally the compound demon-
strated relatively low oral availability, again consistent
with the low measured Caco-2 permeability. The 3-
fluoro analogue 17b also displayed low clearance and
poor oral absorption profile. Good oral bioavailability
(73%) was seen for 17d . Unfortunately, the high clear-
ance and short duration of action made this compound
unsuitable for further evaluation. Most of the 3-tri-
fluoromethylpyrazole benzylamine analogues show good
oral absorption and significantly longer duration of
action. The best profile was observed with 17h , which
had a lower clearance (0.24 L/kg/h) and a slightly larger
volume of distribution (Vdss) than the corresponding
sulfonamide 17f. These differences resulted in a longer
exposure (t1/2 of 7.5 h compared to 4.7 h) (Figure 5).
When dosed orally, 17h showed higher oral bioavail-
ability (F% ) 57%) compared to the corresponding
sulfonamide 17f (F% ) 22%) and the des-fluoro ana-
logue 17g (F% ) 39%; Table 6).
In rats, a higher total body clearance (5.1 ( 0.75 L/h/
kg) and Vdss (21 ( 1.9 L/kg) was observed for 17 h. The
terminal elimination half-life in rats was 4.6 ( 0.7 h
(mean ( SD; n ) 3). When dosed orally to rats, the
plasma concentrations are lower and more variable than
those encountered in dogs. The apparent oral bioavail-
ability of 17h in rats is 36%.
Compound 17h was shown to be 89% protein bound
as measured in human plasma by equilibrium dialysis.
In the rabbit arterio-venous shunt thrombosis model,
the compound inhibits thrombus formation with an ID50

574 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pinto et al.
(CDCl3) δ: 7.16 (d, J ) 1.5 Hz, 1H), 7.68 (t, 1H), 7.84 (m, 2H),
7.92 (dd, J ) 2 and 8 Hz, 1H), 8.08 (d, J ) 2.1 Hz, 1H).
) 1.1 µmol/kg/h and with a IC50 (concentration to inhibit
thrombus formation by 50%) value of 0.15 µM (Figure
6).30 The IC50 value obtained for 17h in the rabbit
thrombosis models reflects its potency in this model.31
Overall, compound 17h showed the best balance of good
permeability resulting in good oral bioavailability, low
clearance, and relatively long half-life. When combined
with its exceptionally good fXa potency and selectivity,
17h emerged as an ideal candidate for further develop-
ment as an antithrombotic agent (Table 7). DPC423, a
crystalline nonhygroscopic hydrochloride salt form of
17h , was selected for clinical development.
1-{3-[Am in o(im in o)m et h yl]p h en yl}-N-[2′-(a m in osu l-
fon yl) [1,1′-bip h en yl]-4-yl]-3-m eth yl-1H-p yr a zole-5-ca r -
boxa m id e, Tr iflu or oa cetic Acid Sa lt (2a ). The pyrazole 4b
(0.20 g, 0.92 mmol) was coupled according to the method
described by Quan et al.14b,c to 4′-amino[1,1′-biphenyl]-2-tert-
butylsulfonamide (0.28 g, 0.92 mmol) to afford 5 (0.36 g, 76%).
LRMS (ESI+): 500 (M + H)+. 1H NMR (CDCl3) δ: 1.04 (s, 9H),
3.88 (s, 1H), 7.05 (s, 1H), 7.18-7.37 (m, 3H), 7.59-7.77 (m,
4H), 7.83 (dd, J ) 2.5 and 8 Hz, 1H), 7.80 (m, 2H), 8.02 (m,
2H). Compound 5 was dissolved in dry methanol (10 mL) and
cooled to 0 °C. Into this solution was bubbled dry HCl gas for
15 min. The reaction mixture was sealed and stirred at room
temperature for 18 h. Removal of solvents in vacuo afforded
the imidate intermediate which was immediately treated with
a saturated solution of ammonia in methanol (10 mL). The
reaction mixture was stirred at room temperature for 12 h,
concentrated, and the crude benzamidine purified by reverse
phase HPLC to afford 0.15 g of pyrazole benzamidine 2a (45%)
as the TFA salt. LRMS (ESI+): 461 (M + H). 1H NMR (DMSO-
d6) δ: 7.23-7.38 (m, 5H), 7.56-7.78 (m, 5H), 7.80-7.86 (dt, J
) 1.4 and 10 Hz, 2H), 7.93 (d, J ) 1.9 Hz, 1H), 8.01 (dd, J )
1.9 and 12 Hz, 2H), 9.06 (bs, 2H), 9.45 (bs, 2H), 10.70 (s, 1H).
HRMS (C23H20N6O3S): calcd 461.2678; found, 461.2587. HPLC
purity >95%. Anal. calcd for C23H20N6O3S‚1.3TFA‚0.2H2O: C,
50.21, H, 3.57, N, 13.72; found, C, 50.11, H, 3.65, N, 13.88.
Eth yl-1-(3-cya n op h en yl)-3-m eth yl-p yr a zol-5-yl-ca r box-
yla te (6). Prepared in 75% yield following the methodology of
Ashton.18 LRMS (CI/NH3): 256 (M + H). 1H NMR (CDCl3) δ:
1.31 (t, 3H), 2.36 (s, 3H), 4.3 (q, 2H), 6.86 (s, 1H), 7.58 (t, 1H),
7.70 (dd, 1H), 7.76 (t, 1H).
1-(3-Cya n op h en yl)-3-m et h yl-1H -p yr a zol-5-ylca r b ox-
ylic Acid (6a ). Ethyl-1-(3-cyanophenyl)-3-methyl-pyrazol-5-
yl-carboxylate (0.55 g, 2.1 mmol) was dissolved in THF (20
mL) and to this was added LiOH (0.5 M, 5,6 mL). The reaction
mixture was stirred at room temperature for 18 h, quenched
with water (50 mL), and extracted with ethyl acetate (2 × 50
mL). The aqueous layer was acidified, and the organics were
extracted with ethyl acetate (2 × 50 mL), dried (magnesium
sulfate), and evaporated to afford pure acid (0.44 g, 90%).
LRMS (CI/NH3): 228 (M + H). 1H NMR (DMSO-d6) δ: 2.27
(s, 3H), 6.89 (s, 1H), 7.09 (t, 1H), 7.82 (dd, 1H), 7.91 (d, 1H),
8.02 (t, 1H).
1-(3-Cyan oph en yl)-N-[2′-(ter t-bu tylam in osu lfon yl) [1,1′-
biph en yl]-4-yl]-3-m eth yl-1H-pyr azole-5-car boxam ide, Tr i-
flu or oa cetic Acid Sa lt (8). To a dichloromethane solution
(20 mL) of 1-(3-cyanophenyl)-3-methyl-pyrazol-5-yl carboxylic
acid (0.2 g, 0.88 mmol) was added oxalyl chloride (0.12 mL,
1.32 mmol). The reaction mixture was stirred at room tem-
perature for 2 h. To this solution was then added 4′-amino-
[1,1′-biphenyl]-2-tert-butylsulfonamide (0.27 g, 0.88 mmol) and
triethylamine (0.50 mL), and the reaction mixture was stirred
at room temperature for 24 h. The reaction was quenched with
water (50 mL), extracted with ethyl acetate (2 × 50 mL),
washed with brine (50 mL), and dried (magnesium sulfate).
Evaporation in vacuo afforded an oil which was chromato-
graphed on silica gel (CH2Cl2:MeOH, 9:1) to afford the title
compound (0.39 g, 99%). LRMS (ESI-): 458 (M - H). 1H NMR
(CDCl3) δ: 1.03 (s, 9H), 2.42 (s, 3H), 3.64 (s, 1H), 6.76 (s, 1H),
7.30 (d, 1H), 7.50 (m, 3H), 7.58 (m, 2H), 7.68 (d, J ) 7.8 Hz,
3H), 7.76 (d, J ) 8.2 Hz, 1H), 7.80 (d, J ) 7.9 Hz, 1H), 8.05 (s,
1H), 8.16 (d, J ) 8.0 Hz, 1H).
1-{3-[Am in o(im in o)m et h yl]p h en yl}-N-[2′-(a m in osu l-
fon yl)[1,1′-biph en yl]-4-yl]-3-m eth yl-1H-pyr azole-5-car box-
a m id e (2b). Compound 8 (0.39 g, 0.85 mmol) was dissolved
in anhydrous MeOH (20 mL) saturated with HCl. The reaction
mixture was stirred at room temperature for 24 h and
concentrated and the residue redissolved in MeOH (20 mL).
Ammonium carbonate was added (1.0 g, 10.00 mmol), and the
reaction mixture was stirred at room temperature for 18 h.
Concentration followed by purification of the residue via
reverse phase HPLC afforded pyrazole 2b (0.15 g, 36%). LRMS
(ESI+): 475 (M + H). 1H NMR (DMSO-d6) δ: 2.50 (s, 3H), 7.03
Con clu sion s
Optimization of the core template of our fXa inhibitors
led to a highly potent pyrazole analogue 2b. Further
optimization resulted in benzamidine analogues such
as 14d and 14f, which are the most potent fXa inhibitors
reported in the literature to date. While the benz-
amidine analogues demonstrated good factor Xa potency
and selectivity, poor oral bioavailability and relatively
short duration of action precluded further development
of these compounds. Incorporation of a less basic ben-
zylamine P1 with a pKa of ∼8.8, followed by careful SAR
manipulations resulted in the identification of analogue
17h , a highly potent, selective, and orally bioavailable
inhibitor of fXa. This compound is a potent antithrom-
botic agent which inhibits thrombus formation in a dose
dependent manner. The nonhygroscopic hydrochloride
salt form DPC423 was selected for clinical development.
Exp er im en ta l Section
Preparations of the final products and intermediates are
included as Supporting Information. All reactions were run
under an atmosphere of dry nitrogen. All solvents were used
without purification as acquired from commercial sources.
NMR spectra were obtained with a Varian VXR-300a spec-
trometer. Microanalyses were performed by Quantitative
Technologies Inc. and were within 0.4% of the calculated
values. Mass spectra were obtained on a HP 5988A MS/HP
particle beam interface. Flash chromatography was done using
EM Science silica gel 60. HPLC purifications were performed
on a Ranin Dynamax SD200 instrument using a C18 reverse
phase column with acetonitrile/water (containing 0.05% TFA)
as a mobile phase. HPLC purity in most cases was >95%.
Various P4 starting materials such as 4′-amino[1,1′-biphenyl]-
2-tert-butylsulfonamide, 4′-amino-3′-fluoro[1,1′-biphenyl]-2-
tert-butylsulfonamide, 3-fluoro-2′-(methylsulfonyl)[1,1′-biphenyl]-
4-amine, 2-(6-amino-3-pyridinyl)benzene-tert-butylsulfonamide,
2-(6-amino-3-pyrimidinyl)benzene-tert-butylsulfonamide sub-
stituted aminobiaryls, and aminopyridylphenylsulfonamides
were obtained as per procedures described by Quan.14a-c
Eth yl-1-(3-cya n op h en yl)-3-m eth yl-1H-p yr a zole-5-ca r -
boxyla te (4a ) a n d 1-(3-Cya n op h en yl)-3-m eth yl-1H-p yr -
a zole-5-ca r boxylic Acid (4b). A solution of 3-cyanophenyl-
hydrazine hydrochloride (3.38 g, 19.98 mmol) and methyl-4-
(dimethylamino)-2-oxo-3-butenoate (3.14 g, 19.98 mmol) in
acetic acid (75 mL) was refluxed for 18 h. The solution was
concentrated and the residue chromatographed on silica gel
(hexane/ethyl acetate, 8:2) to afford the desired pyrazole
1
regioisomer 4a (0.81 g, 17%). LRMS (ESI+): 242 (M + H). H
NMR (CDCl3) δ: 3.77 (s, 3H), 4.20 (q, 2H), 7.16 (d, J ) 1.5
Hz, 1H), 7.68 (t, 1H), 7.84 (m, 2H), 7.92 (dd, J ) 2 and 8 Hz,
1H), 8.08 (d, J ) 2.1 Hz, 1H). The above solid was saponified
with LiOH (0.010 g, 0.2 mmol) in a THF/water solution (4:1,
25 mL). The reaction mixture was acidified with HCl (1 N),
and the organics were extracted with ethyl acetate (2 × 50
mL), dried(magnesium sulfate), and evaporated to a colorless
1
solid 4b (0.70 g, 98%). LRMS (ESI+): 214 (M + H). H NMR

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 575
(s, 1H), 7.27 (s, 2H), 7.32 (d, J ) 8.5 Hz, 1H), 7.37 (d, J ) 8.3
Hz, 2H), 7.62 (m, 2H), 7.70 (d, J ) 7.8 Hz, 2H), 7.75 (d, J )
8.2 Hz, 1H), 7.83 (t, 1H), 7.97 (s, 1H), 8.03 (d, 1H), 9.09 (s,
2H), 9.44 (s, 2H), 10.66 (s, 1H). HRMS (C24H23N6O3S): calcd
475.1552; found, 475.1537. HPLC purity >95%. Anal. calcd
for C24H22N6O3S‚1.0TFA: C, 53.06, H, 3.94, N, 14.28; found,
C, 53.07, H, 3.89, N, 14.10.
1-(3-Cya n op h en yl)p yr r ole (15a ). 2,5-Dimethoxytetrahy-
drofuran (0.44mol, 59.5 mL) was added to an acetic acid (200
mL) solution of 3-aminobenzonitrile (47.45 g, 0.40 mol). The
solution was stirred at reflux overnight, allowed to cool to room
temperature, and diluted with ethyl acetate (250 mL). The
combined ethyl acetate layers were washed with brine (3 ×
200 mL) and saturated sodium carbonate (200 mL), dried over
magnesium sulfate, and filtered through a plug of silica gel.
Evaporation in vacuo afforded the title compound (62.82 g,
93%). LRMS (H2O-CI): 169 (M + H). 1H NMR (CDCl3) δ: 6.41
(t, J ) 2.2 Hz, 2H), 7.09 (t, J ) 2.2 Hz, 2H), 7.51-7.55 (m,
2H), 7.60-7.66 (m, 2H).
Gaseous anhydrous HCl was bubbled through the solution for
0.5 h and allowed to stir overnight at room temperature. The
reaction was concentrated in vacuo, and the residue was
redissolved in anhydrous methanol (100 mL). Ammonium
carbonate (0.43 g, 4.45 mmol) was added and the reaction
mixture stirred overnight at room temperature. Evaporation
in vacuo afforded a solid mass which was purified via reverse
phase HPLC to afford the title compound as a colorless solid.
1
LRMS: (ESI+): 460.3 (M + H). H NMR (DMSO-d6) δ: 6.39
(t, 1H), 7.15-7.17 (m, 1H), 7.20 (s, 2H), 7.25-7.31 (m, 4H),
7.48-7.57 (m, 2H), 7.60-7.64 (m, 4H), 7.74-7.78 (m, 1H), 7.84
(bs, 1H), 7.98 (dd, J ) 7.7, 1.4 Hz), 9.14 (bs, 2H), 9.38 (bs,
2H). HPLC purity >95%. HRMS (C24H22N5O3S): calcd 460.1443;
found, 460.1447.
1-(3-Cya n op h en yl)-2-for m yl-4-br om op yr r ole. 1-(3-Cy-
anophenyl) pyrrole-2-carboxaldehyde (6.06 g, 30.89 mmol) was
combined with N-bromosuccinimide (6.60 g, 37.06 mmol) in
anhydrous CCl4 (150 mL) and stirred at room temperature
overnight. The residue was treated with ethyl acetate (50 mL),
filtered through a silica gel, and concentrated in vacuo. The
residue was then recrystallized from ethyl acetate to afford
the title compound as a light brown solid (4.49 g, 53%). LRMS
1-(3-Cya n op h en yl)p yr r ole-2-ca r b oxa ld eh yd e (15b ).
Phosphorus oxy-chloride (191.8 mmol, 17.90 mL) was added
over 0.5 h to N,N-dimethylformamide (191.8 mmol, 14.1 mL)
at 0 °C. The reaction mixture was cooled to 0 °C and diluted
with 1,2-dichloroethane (100 mL). A solution of 1-(3-cyano-
phenyl)pyrrole (29.33 g, 174.4 mmol) in 1,2-dichloroethane (250
mL) was added slowly via an addition funnel, and the mixture
was heated to reflux for 0.5 h. The solution was cooled to room
temperature, and to this mixture were added sodium acetate
(86.55 g, 1.05 mol) and water (250 mL). The solution was
heated to reflux for 0.5 h, diluted with ethyl acetate (250 mL),
washed with brine (25 mL) and saturated aqueous sodium
carbonate (250 mL), dried over magnesium sulfate, filtered
through a plug of silica gel, and evaporated in vacuo to afford
a brown solid. Recrystallization with ethyl acetate afforded
the title compound as tan solid (28.4 g, 83%). LRMS (NH3-
1
(CI/NH3): 292 (M + NH4). H NMR (CDCl3) δ: 7.06 (dd, J )
1.8, 0.9 Hz, 1H), 7.14 (d, J ) 2.0 Hz, 1H), 7.57-7.60 (m, 2H),
7.62-7.63 (m, 1H), 7.72-7.75 (m, 1H), 9.52 (d, J ) 0.8 Hz,
1H). HPLC purity >95%.
1-{3-[Am in o(im in o)m et h yl]p h en yl}-N-[2′-(a m in osu l-
fon yl)[1,1′-biph en yl]-4-yl]-4-br om o-N-m eth yl-1H-pyr r ole-2-
ca r boxa m id e, Tr iflu or oa cetic Acid Sa lt (3b). Following
the procedures described above, 1-(3-cyanophenyl)-2-formyl-
4-bromopyrrole was converted into the title compound. LRMS
1
(ESI+): 538.2 (M + H). H NMR (DMSO-d6) δ: 7.21 (s, 2H),
7.24-7.30 (m, 4H), 7.51 (s, 2H), 7.54-7.57 (m, 1H), 7.62-7.66
(m, 4H), 7.77-7.80 (m, 1H), 7.86 (bs, 1H), 7.98 (d, J ) 7.3 Hz,
1H), 9.01 (bs, 2H), 9.36 (bs, 2H); HPLC purity >95%. HRMS
(C24H21BrN5O3S): calcd 538.054; found, 538.055.
1
CI): 214 (M + NH4). H NMR (CDCl3) δ: 6.44-6.47 (m, 1H),
7.07 (d, J ) 1.1 Hz, 1H), 7.17 (dd, J ) 3.7 and 1.5 Hz, 1H),
7.54-7.71 (m, 4H), 9.58 9s, 1H).
P r ep a r a tion of 1-(3-Cya n op h en yl)-3-tr iflu or om eth yl-
p yr a zole-5-ca r b oxylic Acid (12). A solution of 1,1,1-tri-
fluoro-2,4-pentanedione (1.35 mL, 11.2 mmol), 3-bromo-
phenylhydrazine hydrochloride (3 g, 13.4 mmol) in glacial
acetic acid (20 mL), and 2-methoxyethanol (10 mL) was heated
to reflux for 2 h. The solvents were removed in vacuo, and the
residue was dissolved in ethyl acetate (100 mL). The ethyl
acetate solution was washed with 1 N HCl (10 mL), saturated
NaHCO3 (50 mL), and brine (50 mL) and dried (magnesium
sulfate). Purification via silica gel flash chromatography
[hexanes/ethyl acetate (8:1)] afforded the pyrazole intermedi-
ates 9a and 9b as a 8:2 mixture of the two isomers (3.42 g,
100%), the desired 5-methylpyrazole isomer pre-dominating.
The mixture was combined with 1-methyl pyrrolidinone (7 mL)
and copper cyanide (1.3 g, 14.5 mmol) and heated to reflux
for 18 h. The reaction mixture was cooled to room temperature,
diluted with ethyl acetate (100 mL), and filtered. The filtrate
was washed with water (100 mL) and brine (50 mL) and dried
(magnesium sulfate). The crude mixture was purification via
silica gel flash chromatography (hexane ethyl acetate, 9:1) to
afford a mixture of cyanophenyl intermediates 10a and 10b
(1.15 g, 40%). To the mixture of 10a and 10b (0.65 g, 2.59
mmol) in carbon tetrachloride (20 mL) was added N-bromo-
succinimide (0.48 g, 2.7 mmol) and benzoylperoxide (20 mg).
The reaction mixture was refluxed for 6 h, cooled, filtered, and
concentrated to yield the crude bromide. The bromide was
dissolved in a mixture of dioxane/water (1:1, 20 mL), and
calcium cabonate (0.46 g, 4.6 mmol) was added. The solution
was heated on a steam bath for 6 h, cooled, filtered, and
concentrated in vacuo. Purification via silica gel flash chro-
matography (hexanes/ethyl acetate, 1:1) afforded the desired
alchohol 11a (0.31 g, 44%). LRMS (CI-NH3): 268.1 (M + H),
285 (M + NH4). 1H NMR (CDCl3) δ: 8.07 (s,1H), 8.01 (dd, J )
2.2,8.05 Hz, 1H), 7.77 (d, J ) 7.7 Hz, 1H), 7.68 (t, J ) 8.05
Hz, 1H), 6.76 (s,1H), 4.72 (d, J ) 5.85 Hz, 2H), 2.02 (t, J )
5.86 Hz, 1H). To 11a (0.18 g, 0.67 mmol) in acetonitrile (5 mL)
was added sodium periodate (0.3 g, 1.4 mmol), water (5 mL),
and a crystal of ruthenium(III)chloride hydrate. The reaction
1-(3-Cya n oph en yl)-N-[2′-(ter t-bu tyla m in osu lfon yl)[1,1′-
bip h en yl]-4-yl]-N-m eth yl-1H-p yr r ole-2-ca r boxa m id e, (R
) H, 16a ). A solution of 1-(3-cyanophenyl)pyrrole-2-carbox-
aldehyde (5.14 g, 26.20 mmol) and acetone/water (1:1, 300 mL)
was cooled to 0 °C. To this solution was added potassium
permanganate (12.42 g, 78.60 mmol) over 0.5 h, and the
reaction mixture was allowed to warm to room temperature.
The mixture was quenched with sodium bisulfite (10.90 g,
104.8 mmol), and the solution was made acidic with HCl (10%).
The solution was filtered through a plug of Celite, extracted
with ethyl acetate (3 × 100 mL), washed with brine (200 mL),
dried over magnesium sulfate, and evaporated in vacuo to
afford the corresponding carboxylic acid derivative 15c (4.11
g, 74%) as a colorless solid. LRMS (ESI-): 211.2 (M - H). The
1-(3-cyanophenyl)pyrrole-2-carboxylic acid (2.77 g, 13.05 mmol)
obtained above was dissolved in anhydrous DMF (50 mL), and
to this mixture were added triethylamine (1.98 mL, 19.58
mmol), benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluoro-phosphate (8.66 g, 19.58 mmol), and 4′-amino[1,1′-
biphenyl]-2-tert-butylsulfonamide (6.03 g, 19.84 mmol). The
reaction mixture was heated at 50 °C for 18 h, cooled to room
temperature, and quenched with water (200 mL). The organics
were extracted with ethyl acetate (2 × 100 mL), washed with
brine (100 mL), dried over magnesium sulfate, and concen-
trated in vacuo. Purification of the residue via flash chroma-
tography (hexane/ethyl acetate, 3:2) afforded the title com-
pound (1.9 g, 29%). LRMS (NH3-CI): 516 (M + NH4). 1H NMR
(CDCl3) δ: 1.01 (s, 9H), 3.67 (bs, 1H), 6.38 (dd, J ) 3.7 and
2.9 Hz, 1H), 6.96-6.98 (m, 2H), 7.28 (dd, J ) 7.7 and 1.5 Hz,
1H), 7.43-7.49 (m, 2H), 7.51-7.66 (m, 8H), 7.94 (bs, 1H), 8.15
(dd, J ) 7.8, 1.3 Hz, 2H).
1-{3-[Am in o(im in o)m et h yl]p h en yl}-N-[2′-(a m in osu l-
fon yl)[1,1′-biph en yl]-4-yl]-N-m eth yl-1H-p yr r ole-2-ca r box-
a m id e, Tr iflu or oa cetic Acid Sa lt (3a ). To a cold (0 °C)
anhydrous methyl acetate (60 mL) solution of 16a (R ) H, 0.37
g, 0.74 mmol) was added methanol (0.30 mL, 7.4 mmol).

576 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pinto et al.
was stirred for 18 h at room temperature, filtered, and
concentrated in vacuo. The aqueous solution was extracted
with ethyl acetate (2 × 50 mL) and evaporated to afford the
desired carboxylic acid 13 (0.17 g, (89.9%)). LRMS (ESI-):
was collected and recrystallized from MeOH/EtOAc to afford
0.34 g (82.9%) of a colorless crystalline solid. mp: 288 °C. H
1
NMR (DMSO-d6) δ: 10.83 (s, 1H), 8.44 (s, 2H), 8.11 (dd, J )
1.1, 7.7 Hz, 1H), 7.81 (m, 6H), 7.62 (m, 2H), 7.44 (dd, J ) 1.1,
7.3 Hz, 1H), 7.41 (dd, J ) 1.8, 11.4, 1H), 7.25 (dd, J ) 1.5, 8.1
Hz, 1H), 4.13 (s, 2H), 2.94 (s, 3H). HRMS (C25H21F4N4O3S):
calcd 533.1192; found, 533.1282; HPLC purity > 95%; Anal.
calcd for C25H21F4N4O3S1‚1.0HCl; C, 52.77, H, 3.72, N, 9.85,
Cl, 6.23; found, C, 52.86, H, 3.45, N, 9.74, Cl, 6.30.
1
280.2 (M - H). H NMR (CDCl3 + DMSO-d6) δ: 7.82 (d, J )
1.47 Hz), 7.78 (dd, J ) 8.0, 1.47 Hz, 1H), 7.63 (t, J ) 7.3, 8.42,
1H), 7.29 (s, 1H).
1-{3-[Am in o(im in o)m et h yl]p h en yl}-N-[2′-(a m in osu l-
fon yl)[1,1′-biph en yl]-4-yl]-3-(tr iflu or om eth yl)-1H-pyr azole-
5-ca r boxa m id e, Tr iflu or oa cetic Acid Sa lt (14a ). To the
pyrazole carboxylic acid 12 (0.35 g, 1.2 mmol) dissolved in
methylene chloride (50 mL) was added oxalyl chloride (0.15
mL, 1.7 mmol) and 2 drops of DMF. The reaction was stirred
for 2 h at room temperature and concentrated in vacuo. The
residue was redissolved in methylene chloride (10 mL). 4′-
Amino[1,1′-biphenyl]-2-tert-butylsulfonamide (0.38 g, 1.25 mmol)
and N,N-dimethylaminopyridine (0.38 g, 3.1 mmol) were
added, and the reaction was stirred at room temperature for
24 h. The reaction was concentrated and the residue quenched
with HCl (3 N, 50 mL). The organics were extracted with ethyl
acetate (2 × 100 mL), washed with saturated NaHCO3 (50 mL)
and brine (50 mL), and dried (magnesium sulfate). The crude
product was purified via silica gel flash chromatography
(hexanes/ethyl acetate, 1:1) to afford the desired coupled
Ack n ow led gm en t . The authors thank Tracy
Bozarth, Andrew Leamy, Thomas Reilly, Martin Thoolen,
Carol Watson, Earl Crain, David Christ, Danielle
Timby, Cecila Chi, Pieter Stouten, Gregory Nemeth,
Gerry Everloff, and J anan J ona for their efforts result-
ing in the discovery of DPC423. The authors also
acknowledge the help of J oanne Smallheer, Thomas
Maduskuie, Mona Patel, and Steven Bai in the prepara-
tion of this manuscript.
Su p p or tin g In for m a tion Ava ila ble: Experimental data
for compounds 2c-2n , 14b-14f, 17b-17f, and all relevant
biological protocols. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
product 0.41 g (58%). LRMS (ESI+): 590 (M + Na). H NMR
(CDCl3 + DMSO-d6) δ: 9.88 (s, 1H), 8.18 (dd, J ) 7.69, 1.47
Hz, 1H), 7.87 (d, J ) 1.83 Hz, 1H), 7.79 (m,4H), 7.64 (m, 3H),
7.50 (m, 3H), 7.30 (d, J ) 7.3 Hz, 1H), 3.67 (s, 1H), 1.02 (s,
9H). Following the Pinner amidine reaction described previ-
ously, the desired product 14a was obtained (46%). LRMS
(ESI+): 529.03 (M + H). 1H NMR (DMSO-d6) δ: 10.85 (s, 1H),
9.47 (s, 1.5H), 9.20 (s, 1.5H), 8.05 (s, 1H), 8.04 (dd, J ) 7.69,
1.84 Hz, 1H), 7.96 (m, 2H), 7.82 (d, J ) 7.69 Hz, 1H), 7.75 (s,
1H), 7.68 (d, J ) 8.79 Hz, 2H), 7.62 (m, 2H), 7.39 (d, J ) 8.43
Hz, 2H), 7.32 (sm, 3H). HRMS (C24H20F3N6O3S): calcd 529.1270;
Refer en ces
(1) This work was partially presented: (a) Wexler, R. R. The design
and synthesis of orally bioavailable noncovalent factor Xa
inhibitors. 27th National Medicinal Chemistry Symposium,
Kansas City, MO, J une 13-17, 2000. (b) Pinto, D. J . P.; Orwat,
M. J .; Wang, S.; Amparo, E.; Pruitt, J . R.; Rossi, K. A.;
Alexander, R. S.; Fevig, J . M.; Cacciola, J .; Lam, P. Y. S.; Knabb,
R. M.; Wong, P. C.; Wexler, R. R. The discovery of a novel
pyrazole SN429, a highly potent inhibitor of coagulation factor
Xa. Abstracts of Papers, 217th National Meeting of the American
Chemical Society, Anaheim, CA, March 21-25, 1999; American
Chemical Society: Washington, DC, 1999; MEDI-006.
(2) (a) Hirsh, J .; Fuster, V. Guide to anticoagulant therapy. Part 2:
Oral anticoagulants. Circulation 1994, 89, 1469-1480. (b) Hirsh,
J . Oral anticoagulant drugs. New Engl. J . Med. 1991, 324, 1865-
1875.
(3) Lefkovits, J .; Topol, E. J . Direct thrombin inhibitors in cardio-
vascular medicine. Circulation 1994, 90, 1522-1568.
(4) (a) Rihal, C. S.; Flather, M.; Hirsh, J .; Yusuf, S. Advances in
antithrombotic drug therapy for coronary artery disease. Eur.
Heart J . 1995, 16, 10-21. (b) Fareed, J .; Callas, D. D.; Hoppen-
steadt, D.; J eske, W.; Walenga, J . M. Recent development in
antithrombotic agents. Exp. Opin. Invest. Drugs. 1995, 4 (50),
389-412. (c) Hirsh, J . Use of Warfarin (Coumadin). Heart Dis.
Stroke 1993, 2, 209-216. (d) Dalen, J . E.; Hirsh, J . Antithrom-
botic therapy. Introduction. Chest 1992, 102, 303S-304S.
(5) (a) Stein, P. D.; Grandison, D.; Hua, T. A. Therapeutic level of
anticoagulation with warfarin in patients with mechanical
prosthetic heart valves; review of literature and recommenda-
tions based on internal normalized ratio. Postgrad. Med. J . 1994,
70 (suppl 1), S72-S83. (b) Hirsh, J .; Poller, L. The international
normalized ratio. A guide to understanding and correcting its
problems. Arch. Int. Med. 1994, 154, 282-288.
(6) (a) Berry, C. N.; Girardot, C.; Lecoffre, C.; Lunven, C. Effects of
synthetic thrombin inhibitor argatroban on fibrin or clot-
incorporated thrombin: comparison with heparin and recombi-
nant hirudin. Thromb. Haemostat. 1994, 72, 381-386. (b) Kelly,
A. G.; Marzee, W. M.; Krupski, W.; Bass, A.; Cadroy, Y.; Hanson.
S. R.; Harker, L. A. Hirudin interruption of heparin-resistant
arterial thrombus formation in baboons. Blood 1991, 77, 1006-
1012.
(7) (a) Tapparelli, C.; Metternich, R.; Ehrhardt, C.; Cook, N. S.
Synthetic low-Molecular weight thrombin inhibitors: molecular
design and pharmacological profile. Trends Pharmacol. Sci.
1993, 14, 366-376. (b) Maffrand, J . P. Direct thrombin inhibi-
tors. Nov. Rev. Fr. Hematol. 1992, 34, 405-419.
found, 529.1267. HPLC purity >95%. Anal. calcd for C24H19
-
F3N6O3S‚1.2TFA‚1.0H2O; C, 46.40; H, 3.27; N, 12.30; found,
C, 46.11; H,3.06; N, 12.05.
1-[3-(Am in om eth yl)p h en yl]-N-[2′-(a m in osu lfon yl)[1,1′-
b ip h en yl]-4-yl]-3-m et h yl-1H -p yr a zole-5-ca r b oxa m id e,
tr iflu or oa cetic Acid Sa lt (17a ). 1-(3-Cyanophenyl)-5-
[(2′-aminosulfonyl-[1,1′]-biphen-4-yl)aminocarbonyl]-3-meth-
ylpyrazole 13a (0.19 g, 0.41 mmol) was dissolved in ethanol
(20 mL). To this solution was added TFA (0.5 mL) and
palladium on carbon (10%, 10 mg), and the mixture was
hydrogenated on a Parr apparatus at 40 psi for 18 h. The
mixture was filtered, concentrated, and purified by reverse
phase HPLC to afford the title compound 17a (17 mg, 9%).
1
LRMS (ESI+): 462 (M + H). H NMR (DMSO-d6) δ: 10.66 (s,
1H), 8.22 (bd, 2H), 8.03 (dd, J ) 1.47, 6.22 Hz, 1H), 7.70 (d, J
) 8.79 Hz, 2H), 7.67 (m, 2H), 7.64 (m, 5H), 7.37 (d, J ) 8.43
Hz, 2H), 7.32 (m, 2H), 6.93 (s, 1H), 4.13 (d, J ) 4.03 Hz, 2H),
2.33 (s, 3H). HRMS (C24H24N5O3S): calcd 462.1599; found,
462.1589. HPLC purity >95%. Anal. calcd for C24H23N5O3S‚
1.0TFA‚1.5H2O; C, 51.82, H, 4.52, N, 11.62; found, C, 51.80,
H, 4.35, N, 11.29.
1-[3-(Am in om eth yl)p h en yl]-N-[3-flu or o-2′-(m eth ylsu l-
fon yl)[1,1′-biph en yl]-4-yl]-3-(tr iflu or om eth yl)-1H-pyr azole-
5-ca r boxa m id e, Tr iflu or oa cetic Acid Sa lt (17h ). Prepared
following the procedure for 17a . LRMS (ESI+) 532 (M + H).
1H NMR (DMSO-d6) δ: 10.75 (s, 1H), 8.23 (m, 3H), 8.11 (dd,
J ) 7.69 and 1.46 Hz, 1H), 7.96 (dd, J ) 6.96, 1.47 Hz, 1H),
7.81 (m, 8H), 7.26 (dd, J ) 1.47 and 8.06 Hz, 1H), 4.16 (q, J )
5.49 Hz, 2H), 2.94 (s, 3H). HRMS (C25H21F4N4O3S): calcd
533.1192; found, 533.1279. HPLC purity >95%. Anal. calcd
for C25H20F4N4O3S‚1.1TFA; C, 49.65, H, 3.23, N, 8.52, found,
C, 49.73, H, 2.98, N, 8.40.
1-[3-(Am in om eth yl)p h en yl]-N-[3-flu or o-2′-(m eth ylsu l-
fon yl)[1,1′-biph en yl]-4-yl]-3-(tr iflu or om eth yl)-1H-pyr azole-
5-ca r boxa m id e, Hyd r och lor id e Sa lt (DP C423). Compound
17h (0.47 g, 0.727 mmol) was dissolved in absolute methanol
(30 mL). The mixture was cooled to 0 °C, and dry HCl gas
was bubbled for 15 min. To this solution was added diethyl
ether (400 mL) upon which a white solid precipitated. The solid
(8) (a) Rosenberg, J . S.; Beeler, D. L.; Rosenberg, R. D. Activation
of human prothrombin by highly purified human factor V and
FXa in presence of human antithrombin. J . Biol. Chem. 1995,
250, 1607-1617. (b) Davre, E. W.; Fujikawa, K.; Kisiel, W. The
coagulation cascade: Initiation, maintenance and regulation.
Biochemistry 1991, 30, 10363-10370. (c) Rosenberg, R. D.;
Damus, P. S. Purification and mechanism of action of human
antithrombin-heparin cofactor. J . Biol. Chem. 1973, 248, 6498-
6505.
(9) Mann, K. G.; Nesheim, M. E.; Church, W. R.; Haley, P.;
Krishnaswamy, S. Surface-dependent enzyme complexes. Blood
1990, 76, 1-16.

Discovery of a Selective Inhibitor of Factor Xa
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4 577
(10) (a) Fevig, J . M.; Wexler, R. R. (Doherty, A. M., Ed.) Anticoagu-
lants: Thrombin and Factor Xa inhibitors. Annu. Rep. Med.
Chem.1999, 34, 81-100. (b) Sanderson, P. E. J . Small, non-
covalent serine protease inhibitors. Med. Res. Rev. 1999, 19,
179-197. (c) Hauptmann, J .; Sturzebecher, J . Synthetic inhibi-
tors of thrombin and factor Xa: from bench to bedside. Thromb.
Res. 1999, 93, 203-241. (d) Fareed, J .; Callas, D.; Hoppensteadt,
D. A.; Lewis, B. E.; Bick, R. L.; Walenga, J . M. Antithrombin
agents as anticoagulants and antithrombotics: implications in
drug development. Semin. Hematol. 1999, 36 (1, Suppl. 1), 42-
56. (e) Kimball, S. D. Oral thrombin inhibitors: Challenges and
progress. Handb. Exp. Pharmacol. 1999, 132 (Antithrombotics),
367-396. (f) Vacca J . P. (Bristol, J . A., Ed.) Annu. Rep. Med.
Chem. 1998, 33, 81-90.
(11) (a) Zhu, B.-Y.; Scarborough, R. M. Recent advances in inhibitors
of factor Xa in the prothrombinase complex. Curr. Opin. Car-
diovasc., Pulm. Renal Invest. Drugs 1999, 1 (1), 63-87. (b) Al-
Obeidi, F.; Ostrem, J . A. Factor Xa inhibitors by classical and
combinatorial chemistry. Drug Discovery Today 1998, 3 (5), 223-
231. (c) Scarborough R. M. Coagulation factor Xa: The pro-
thrombinase complex as an emerging therapeutic target for
small molecule inhibitors. J . Enzymol. Inhib. 1998, 14, 15-25.
(d) Hara, J .; Yokoyama, A.; Tanabe, K.; Ishihara, H.; Iwamoto,
M. DX-9065a an orally active specific inhibitor of factor Xa
inhibits thrombosis without affecting bleeding time in rats.
Thromb. Haemost. 1995, 74, 635-639. (e) Schaffer, J . A.;
Davidson, J . T.; Vlasuk, G. P.; Siegl, P. K. Antithrombotic
efficacy of recombinant tick anticoagulant peptide: a potent
inhibitor of coagulation factor Xa in a primate model of arterial
thrombosis. Circulation 1991, 84, 1741-1748.
(17) Ashton, W. T.; Doss, G. A. A. Regioselective route to 3-alkyl-1-
aryl-1H-pyrazole-5-carboxylates: Synthetic studies and struc-
tural assignments. J . Heterocycl. Chem. 1993, 30, 307-311.
(18) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977,
48, 4171-4174.
(19) Molecular models were compared using the Insight II program
developed by Molecular Simulations Inc. molecular modeling
program (version 97.2). For similar models, refer to ref 14.
Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.; Park, C.
H.; Bode, W.; Blankenship, D. T.; Cardin, A. D.; Kisiel, Structure
of human Des (1-45) factor Xa at 2.2 Ang. resolution W. J . Mol.
Biol. 1993, 232 (3), 947-966.
(20) (a) Krishnaswamy, S.; Vlasuk, G. P.; Bergum, P. W. Assembly
of the prothrombinase complex enhances the inhibition of bovine
factor Xa by tick anticoagulant peptide. Biochemistry 1994, 33,
7897-7907. (b) J ordan, S. P.; Waxman, L.; Smith, D. E.; Vlasuk,
G. P. Tick anticoagulant peptide: Kinetic analysis of the
recombinant inhibitor with blood coagulation factor Xa. Bio-
chemistry 1990, 29, 11095-111000. (c) Waxman, L.; Smith, D.
E.; Arcuri, K. E.; Vlasuk, G. P. Tick anticoagulant peptide (TAP)
is a novel inhibitor of blood coagulation factor Xa. Science 1990,
248, 593-596.
(21) (a) Brandstetter, H.; Ku¨hne, A.; Bode, W.; Huber, R.; Von der
Saal, W.; Wirthensohn, K.; Engh, R. A. J . Biol. Chem. 1996, 47,
29988-29992. (b) Stubbs, M. T.; Structural Aspects of Factor
Xa Inhibition. Curr. Pharm. Des. 1996, 2, 543-552. (c) Stubbs,
M. T.; Huber R.; Bode, W. Crystal structures of factor Xa
inhibitors in complex with trypsin: structural grounds for
inhibition of factor Xa and selectivity against thrombin. FEBS
Lett. 1995, 375, 103-107.
(12) (a) Lynch, J . J .; Sitko, G. R.; Lehman, E. D.; Vlasuk, G. P.
Primary prevention of coronary arterial thrombosis with the
factor Xa inhibitor rTAP in a canine electrolytic injury model.
Thromb. Haemost. 1995, 74, 640-645. (b) Ramjit, D. R.; Sta-
bilito, D. R.; Lehman, I. I.; Lynch, J . J .; Vlasuk, G. P. Conjunctive
enhancement of enzymatic thrombolysis and prevention of
thrombotic reocclusion with the selective factor Xa inhibitor, Tick
anticoagulant peptide: Comparison to hirudin and heparin in
a canine model of acute coronary artery thrombosis. Circulation
1992, 85, 805-815.
(22) (a) Brandstetter, H.; Turk, D.; Hoeffken, H. W.; Grosse, D.;
Sturzebecher, J .; Martin, P. D.; Edwards, B. F.; Bode, W. Refined
2.3 Å X-ray crystal structure of bovine thrombin complexes
formed with the benzamidine and arginine-based thrombin
inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for
improving antithrombotics. J . Mol. Biol. 1992, 226, 1085-1099.
(b) Turk, D.; Sturzebecher, J .; Bode, W. Geometry of binding of
the N-alpha-tosylated piperidides of m-amidino, p-amidino and
p-guanidino phenylalanine to thrombin and trypsin. X-ray
crystal structures of their trypsin complexes and modeling of
their thrombin complexes. FEBS Lett. 1991, 287, 133-138. (c)
Banner, D. W.; Hadvary, P. Crystallographic analysis at 3.0-A
resolution of the binding to human thrombin of four active site-
directed inhibitors. J . Biol. Chem. 1991, 266, 20085-20093. (d)
Bode, W.; Turk, D.; Sturzebecher J . Geometry of binding of the
benzamidine and arginine-based inhibitors N-alpha-(2-naphthyl-
sulphonyl-glycyl)-DL-p-amidinophenylalanyl-piperidine (NA-
PAP) and (2R,4R)-4-methyl-1-[N-alpha-(3-methyl-1,2,3,4-tet-
rahydro-8-quinolinesulphonyl)-L-arginyl]-2-piperidine carboxylic
acid (MQPA) tohuman alpha-thrombin. X-ray crystallographic
determination of the NAPAP-trypsin complex and modeling of
NAPAP-thrombin and MQPA-thrombin. Eur. J . Biochem. 1990,
193, 175-182. (e) Matsuzaki, T.; Sasaki, C.; Okumura, C.;
Umeyama, H. J . X-ray analysis of a thrombin inhibitor-trypsin
complex. Biochem. (Tokyo) 1989, 105, 949-952.
(23) Bovine trypsin was purchased from Worthington (cat. #3707)
and used without further purification. Crystals were grown by
vapor diffusion using 20 µL hanging drops containing 10-30 mg/
mL â trypsin, 35 mM tris pH 7.5, 2.5 mM benzamidine, 100 mM
ammonium sulfate, and 6-12% W/V PEG 8000. The drops were
equilibrated at 5 °C over 50 mM tris pH 7.5, 200 mM ammonium
sulfate, and 12-24% W/V PEG 8000. Crystals appeared after
one week. Benzamidine was removed by letting the crystals soak
overnight in a stabilizing solution containing 50 mM tris pH 7.5,
200 mM ammonium sulfate, and 20% W/V PEG 8000. The
crystals were transferred to a solution containing 20 mM sodium
phosphate pH 7.5, 20% PEG 8000, and 0.1% glutaraldehyde for
30 min to cross-link. These crystals were then transferred to a
solution containing the inhibitor. The inhibitor solution was
prepared by first dissolving 1 mg of inhibitor in 5 µL of DMSO.
This was followed by a 40-fold dilution of the inhibitor/DMSO
solution into the first stabilizing solution. Data were collected
one week after inhibitor addition. A crystal of the trypsin-
inhibitor complex was mounted and sealed in a glass capillary.
An R-AXIS image plate detector was used for X-ray data
acquisition. A Rigaku RU-200 rotating anode X-ray generator
operating at 50 kV/100 mA equipped with a graphite monochro-
mator was used for data collection. The trypsin data were
collected at 40 °Celsius using an Enraf Nonius cooling device.
Data frames of 2° rotation about the spindle axis, φ, were
collected, with exposure times of 30 min/frame, for total angular
rotation ranges about φ of 90°. Data were processed using the
Raxis data processing software (Molecular Structure Corp.).
Crystals grew in space group P212121 with the following unit
cell parameters: a ) 54.8 Å, b ) 59.6 Å, c ) 66.8 Å. Data greater
than one σ were used in refinement and were 92% complete.
The XPLOR (Bru¨nger, A. T.; Kuriyan, J .; Karplus, J . Crystal-
lographic R factor refinement by molecular dynamics. Science,
(13) a) Phillips, G.; Davey, D. D.; Eagen, K. A.; Koovakkat, S. K.;
Liang, A.; Ng, H. P.; Pinkerton, M.; Trinh, L.; Whitlow, M.;
Beatty, A. M.; Morrissey, M. M. Design, synthesis and activity
of 2,6-diphenoxypyridine-derived factor Xa inhibitors. J . Med.
Chem. 1999, 42, 1749-1756. (b) Galemmo, R. A., J r.; Maduskuie,
T. P.; Dominguez, C.; Rossi, K. A.; Knabb, R. M.; Wexler, R. R.;
Stouten, P. F. W. The de novo design and synthesis of cyclic urea
inhibitors of factor Xa: initial SAR studies, Bioorg. Med. Chem.
Lett. 1998, 8, 2705-2710. (c) Maduskuie, T. P. J r.; McNamara,
K. J .; Ru, Y.; Knabb, R. M.; Stouten, P. F. W. Rational design
and synthesis of novel potent Bis-phenylamidine carboxylate
factor Xa inhibitors. J . Med. Chem. 1998, 41, 53-62. (d) Quan,
M. L.; Pruitt, J . R.; Ellis, C. D.; Liauw, A. Y.; Galemmo, R. A.;
Stouten, P. F. W.; Wityak, J .; Knabb, R. M.; Thoolen, M. J .;
Wong, P. C.; Wexler, R. R. Design and synthesis of isoxazoline
derivatives as factor Xa inhibitors. Bioorg. Med. Chem. Lett.
1997, 7, 2813-2818. (e) Sato, K.; Kawasaki, T.; Yaniuchi, Y.;
Hismichi, N.; Koshio, H.; Matsumoto, Y. YM-60828, a novel
factor Xa inhibitor: Separation of its antithrombotic effects from
its prolongation of bleeding time. Eur. J . Pharmacol. 1997, 339,
141-146. (f) Nagahara, T.; Yukoyama, Y.; Inamura, K.; Kataku-
ra, S.; Yamaguchi, H.; Hara, T.; Iwamoto, M. Design, synthesis
and biological activities of orally active coagulation factor Xa
inhibitors. Eur. J . Med. Chem. 1995, 30, 139. (g) Nagahara, T.;
Yukoyama, Y.; Inamura, K.; Hara, T.; Iwamoto, M. Dibasic
(amidinoaryl)propanoic acid derivatives as novel blood coagula-
tion factor Xa inhibitors. J . Med. Chem. 1994, 37, 1200-1207.
(h) Sturzebecher, J .; Markwardt, F.; Walsmann, P. Synthetic
inhibition of serine proteinases XXIII. Inhibition of factor Xa
by diamidines. Thromb. Res. 1980, 17, 545-548. (i) Sturze-
becher, J .; Markwardt, F.; Walsmann, P. Synthetic inhibitors
of serine proteinases XIV. Inhibition of factor Xa by derivatives
of benzamidines. Thromb. Res. 1976, 9, 637-646.
(14) (a) Quan, M. J .; Liauw, A. Y.; Ellis, C. D.; Pruitt, J . R.; Bostrom,
L. L.; Carini, D. J .; Huang, P. P.; Harrison, K.; Knabb, R. M.;
Thoolen, M. J .; Wong, P. C.; Wexler, R. R. Design and synthesis
of isoxazoline derivatives as factor Xa inhibitors. 1. J . Med.
Chem. 1999, 42, 2752-2759. (b) Quan, M. J .; Ellis, C. D.; Liauw,
A. Y.; Alexander R.; Knabb, R. M.; Lam G. N.; Wong, P. C.;
Wexler, R. R. Design and synthesis of isoxazoline derivatives
as factor Xa inhibitors. 2. J . Med. Chem. 1999, 42, 2760-2773.
(15) Pruitt, J . R.; Pinto, D. J .; Quan, M. L.; Estrella, M. J .; Bostrom,
L. L.; Knabb, R. M.; Wong, P. C.; Wexler, R. R. Isoxazolines and
isoxazoles as factor Xa inhibitors. Bioorg. Med. Chem. Lett. 2000,
10 (8), 685-689.
(16) J ones, R. G.; Whitehead, C. W. Vic-Dicarboxylic acid derivatives
of pyrazole, isoxazole and pyrimidine. J . Org. Chem. 1955, 20,
1342-1347.

578 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 4
Pinto et al.
1987, 235, 472-475) program was used for crystallographic
refinement. Simulated annealing (at a maximum temperature
of 3000 °C) was followed by B-factor refinement. The refined
coordinates of trypsin (Krieger, M.; Kay, L. M.; Stroud, R. M.
Structure and specific binding of trypsin: comparison of inhib-
ited derivatives and a model for substrate binding. J . Mol. Biol.
1974, 83, 209-230) were used to calculate the initial phases for
the enzyme-inhibitor structure. The inhibitor was built with
the program QUANTA (Molecular Simulations Inc.). No major
adjustments to the protein model were needed during the course
of the refinements. The final crystallographic R-factor was
19.2%.
(28) Gibaldi, M.; Perrier, D. Pharamcokinetics, 2nd ed.; Marcel
Dekker: New York, 1982.
(29) Kallikrein, although essential for clotting of plasma in vitro, the
in vivo significance of this hemostatic deficiency is not considered
to be important, as patients with this abnomality do not require
replacement therapy following hemostatic challenge from trauma
or surgery. Mann, K. G.; Lorand, L. Introduction: Blood
coagulation. Methods Enzymol. 1993, 222, 1-10.
(30) It also appears that measuring anti-fXa activity is a sensitive
method for assessing fXa inhibitors ex vivo. The APTT and PT
values for factor Xa inhibitors are not sensitive enough to
monitor the antithrombotic effect of factor Xa inhibitors. Wong,
P. C.; Crain, E. J .; Knabb, R. M.; Meade, R. P.; Quan, M. L.;
Watson, C. A.; Wexler, R. R.; right, M. R.; Slee, A. M. Nonpeptide
factor Xa inhibitors II. Antithrombotic evaluation in a rabbit
model of electrically induced carotid artery thrombosis. J .
Pharmacol. Exp. Ther. 2000, 295, 212-218.
(24) Kiely, S.; J ohn, J . S. The synthesis of Methyl 1-Aryl-2-pyrrole-
carboxylates. J . Heterocycl. Chem. 1987, 24, 1137-1139.
(25) Kettner, C.; Mersinger, L.; Knabb, R. The selective inhibition
of thrombin by peptides of boroarginine. J . Biol. Chem. 1990,
265 (30), 18289-18297.
(26) Wong, P. C.; Quan, M. L.; Crain, E. J .; Watson, C. A.; Wexler,
R. R.; Knabb, R. M. Nonpeptide Factor Xa Inhibitors I. Studies
with SF303 and SK549, a new class of potent antithrombotics.
J . Pharmacol. Exp. Ther. 2000, 292, 351-357.
(27) Hilgers, A. R.; Conradi, R. A.; Burton, S. Caco-2 cell monolayers
as a model for drug transport across the intestinal mucosa.
Pharm. Res. 1990, 7, 902-910.
(31) Wong, P. C.; Crain, E. J .; Watson, C. A.; Pinto, D. J .; Wexler, R.
R.; Wright, M. R.; Knabb, R. M. Antithrombotic effects of DPC
423, a potent and orally active nonpeptide factor Xa inhibitor,
in rabbit models of thrombosis. Circulation 2000, 102 (18, Supp.
II), 130.
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