1-[3-Aminobenzisoxazol-5′-yl]-3-trifluoromethyl-6-[2′-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1′]-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa

Article abstract of DOI:10.1016/j.bmcl.2006.02.069

Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5′-yl]-3-trifluoromethyl-6-[2′-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1′]-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa K_i = 30 pM) with a rapid onset of inhibition (2.7 × 10⁷ M^-1 s^-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.

Full text of DOI:10.1016/j.bmcl.2006.02.069

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4141–4147
1-[3-Aminobenzisoxazol-50-yl]-3-trifluoromethyl-6-[20-(3-(R)-
hydroxy-N-pyrrolidinyl)methyl-[1,10]-biphen-4-yl]-1,4,5,6-
tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a
highly potent, selective, efficacious, and orally bioavailable
inhibitor of blood coagulation factor Xa
Donald J. P. Pinto,a,* Michael J. Orwat,a Mimi L. Quan,a Qi Han,e
Robert A. Galemmo,, Jr.,f Eugene Amparo,a Brian Wells,g Christopher Ellis,j
Ming Y. He,k Richard S. Alexander,f Karen A. Rossi,c Angela Smallwood,i
Pancras C. Wong,b Joseph M. Luettgen,b Alan R. Rendina,b Robert M. Knabb,b
Lawrence Mersinger,g Charles Kettner,a Steven Bai,h Kan He,d
Ruth R. Wexlera and Patrick Y. S. Lama
aDiscovery Chemistry Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
bDiscovery Biology Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
cCADD Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
dPCO-MAP Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543, USA
eIncyte Corporation, Wilmington, DE 19880, USA
fJohnson & Johnson, Pharmaceutical Research and Development, Exton, PA 19341, USA
gE.I. DuPont Central Research and Development, Wilmington, DE 19880, USA
hCentocor, Malvern, PA 19355, USA
iGlaxoSmithKline, King of Prussia, PA 19406, USA
jProctor & Gamble Pharmaceuticals, Mason, OH 45040, USA
kPfizerGlobal R&D, LaJolla, CA 92121, USA
Received 4 January 2006; revised 15 February 2006; accepted 17 February 2006
Available online 30 May 2006
Abstract—Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold
optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel
bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido
moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-amin-
obenzisoxazol-50-yl]-3-trifluoromethyl-6-[20-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,10]-biphen-4-yl]-1,4,5,6-tetrahydropyrazol-
o-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki = 30 pM) with a rapid onset of inhibition (2.7 · 107 Mꢀ1 sꢀ1
)
in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor
of blood coagulation factor Xa.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Normal hemostasis is a balanced process of blood coag-
ulation and clot dissolution. In the course of a thrombo-
genic event, the hemostatic balance is disturbed, leading
to excess clot formation which, if not treated, leads to a
serious thrombogenic state. Currently, the only
Keywords: Razaxaban; BMS-740808; Coagulation; Warfarin;
Mutagenicity; Strategies; Clotting times; Factor Xa; Selectivity;
Permeability; Pharmacokinetics; Bioavailability.
*
approved oral anticoagulant in the US is warfarin sodium
(Coumadinꢁ).1a,b,c Other parenteral treatments such
Corresponding author. Tel.: +1 609 818 5295; e-mail:
Donald.Pinto@bms.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.069

4142
D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
O
as heparin, low molecular weight heparins (LMWH),
and fondaparinux are also available for the prevention
and treatment of thromboembolic diseases.2,3,4a–c The
coagulation cascade provides many opportunities for
anticoagulant intervention. Factor Xa (fXa), a key en-
zyme in this cascade, is a trypsin-like serine protease
which catalyzes the conversion of prothrombin to
thrombin, the final enzyme that triggers clot forma-
tion.5a–g Several pre-clinical animal models have sug-
gested that targeting the fXa mechanism has the
potential of providing good antithrombotic efficacy with
minimal risk of bleeding.5f,g Recently, we described our
efforts which led to the identification of razaxaban
(BMS-561389), a potent factor Xa inhibitor with good
efficacy and minimal bleeding in animals and human-
s.6a,b,c Razaxaban, a pyrazole based fXa inhibitor, is
both potent and selective (fXa Ki = 0.19 nM, trypsin
Ki >10,000 nM) and has good oral bioavailability. The
improved selectivity profile of razaxaban, relative to ear-
lier candidates such as DPC423 (fXa Ki = 0.13 nM, tryp-
sin Ki = 60 nM),7 and DPC602 (fXa Ki = 0.91 nM,
trypsin Ki = 3500 nM)8 (Fig. 1), is the result of employ-
ing the aminobenzisoxazole (PI moiety) to take advan-
tage of the larger SI specificity pocket in factor Xa
(Ala 190 in factor Xa compared to Serl90 in trypsin).9
R
R
R
F3C
a,b,c,d
N
e,f,g
R
N
H2N
O
N
O
O
3
I
I
I
O
2
1
F3C
NH2
F
k
N
N
N
F
HO OH
h,i,j
O
B
I
CHO
NC
NC
4
R
F3C
R
R1
N
F3C
N
CHO
R2
N
N
O
N
N
F
N
O
l,m
NC
5
H2N
6 R = H
7 R = F
O
N
Scheme 1. General method of synthesis of the 3-trifluoromethylpy-
razolo-pyridinone factor Xa compounds. Reagents and conditions: (a)
5-Br-valeryl chloride, NaOH (10%) THF; (b) 2 equiv KOtBu; (c) PCl5
(3 equiv), CHCl3; (d) morpholine excess 140 ꢂC; (e) DMAP/TFAA/
DCM; (f) Et2O, 20% aq HCl; (g) HCl, H2O; (h) NaNO2, HCl, AcOH;
(i) SnCl2, H2O, HCl; (j) 3, MeOH; (k) (Ph3P)4Pd, K3PO4, dioxane; (l)
3-OH-pyrrolidine (2 equiv), NaCNBH3; (m) Ac-NHOH, K2CO3,
DMF.
phorus pentachloride, in refluxing chloroform, afforded
the aa-dichloro-piperidinone intermediate, which on
heating to reflux with excess morpholine provided the
morpholine-enamine 2 in about 70% yield. Treatment
of enamine 2 with trifluoroacetic anhydride in the pres-
ence of DMAP, followed by acid hydrolysis, afforded
the trifluoroacetyl-piperidine-2,3-dione intermediate 3
in high yield (>90%). Condensation of 3 with 3-cyano-
4-fluorophenylhydrazine afforded the required bicyclic
pyrazole scaffold intermediate 4 in moderate yield
(40–50%). Palladium-catalyzed Suzuki cross-coupling
of the iodo intermediate 4 with 2-formylphenylboronic
acid afforded the biarylaldehyde intermediate 5 (85%
yield). Reductive animation of 5 with various amines
provided the biarylaminomethyl P4 intermediates which
on further treatment with acetohydroxamic acid and
potassium carbonate in DMF9 afforded the desired
A
common feature in razaxaban, DPC423, and
DPC602 is the presence of the 5-pyrazolo-carboxamido
linker. As part of our optimization strategy, we sought
to modify the carboxamido portion of the molecule to
make it less susceptible to potential in vivo hydrolysis,
as we were keenly aware that metabolic hydrolysis of
this linker could potentially result in biarylamine frag-
ments which could be mutagenic.
The bicyclic pyrazolo-dihydropyridinone scaffold was
prepared according to the methodology outlined in
Scheme 1. The starting material 1 was readily prepared
in excellent yield (80–90%) following a two-step se-
quence from either 4-iodoaniline (R = H) or 2-fluoro-
4-iodoaniline (R = F) and 5-bromovaleryl chloride
(10% NaOH and DCM) followed by treatment with
potassium tert-butoxide. Chlorination of 1 with phos-
aminobenzisoxazole compounds
6
(R = H) or
7
(R = F) in moderate yields (40–50% yield for the two
steps). The bicyclic pyrazolo-azepinone scaffold analogs
were also constructed in a similar manner. The synthesis
of the pyrazolo-pyrimidinone compounds was accom-
plished according to the methodology described by
Ref. 10.
F3C
F3C
F
F
H
N
H
N
SO2NH2
SO2Me
N
N
N
N
O
O
H2N
NH2
The synthesis of the 3-methylpyrazolo-pyridinone com-
pounds is shown in Scheme 2. Coupling of 3-oxopenta-
noic acid 8 with the requisite aniline via oxalyl chloride
and DMF afforded the keto–amido intermediate 9 in
quantitative yield. This intermediate was further
reduced (LAH/THF), acylated (ethyloxalylchloride/di-
chloromethane), oxidized (Swern) and cyclized (sodium
methoxide in methanol) to afford the key lactam–dione
intermediate 11 in approximately 40% yield. Condensa-
tion of 11 with 4-fluoro-3-cyanophenylhydrazine fol-
lowed by the sequence of reactions described in
Scheme 1 afforded the desired 3-methylpyrazolo-tetra-
hydropyridinone compounds 12.
DPC602
FXa Ki = 0.91nM
Trypsin Ki = 3500nM
DPC423
FXa Ki= 0 .15nM
Trypsin Ki = 60nM
F3C
N
F
H
N
N
N
O
N
N
NH2
O
N
Razaxaban (DPC906)
FXa Ki = 0.19nM
Trypsin Ki > 10000nM
Figure 1. Pyrazole FXa inhibitors.

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
4143
O
I
O
H3C
F3C
F3C
a,b
OH
H3C
F
N
H
N
O
H
R
N
O
R
R
Strategy I
N
N
8
N
9
N
O
O
O
I
NH2
NH2
Bicyclic Pyrazoles
e,f H3C
HO
R
c,d
H3C
O
O
N
N
15
N
N
O
R
CO2Et
O
O
I
10
11
Strategy III
F3C
H3C
Strategy II
R
g,h,i,j,k
R
F3C
N
N
NR1R2
N
N
R
N
N
N
O
N
O
O
H2N
12
N
O
NH2
14
NH2
P4 indoline
replacements
O
O
N
N
Scheme 2. Synthesis of the 3-methylpyrazolo-pyridinone factor Xa
compounds. Reagents and condition: (a) (COCl)2, CHCl3; (b) DMAP,
4-iodo-aniline, CHCl3; (c) LAH, THF; (d) ethyl oxalyl chloride, TEA,
THF; (e) Swern; (f) NaOMe, MeOH reflux; (g) 3-CN-4-F-aniline,
NaNO2, HCl, AcOH; (h) SnCl2, H2O, HCl; (i) 3, MeOH, (Ph3P)4Pd,
K3PO4, dioxane; (j) 3-OH-pyrrolidine (2 equiv), NaCNBH3; (k) Ac-
NHOH, K2CO3, DMF.
Ketone
replacements
13
Figure 2. Pyrazole optimization strategies.
impacted the fXa potency. For example, compounds 6a
and 7a were more potent when compared to the azepinone
compounds 16a,b. Introduction of a double bond in the
azepinone ring showed no difference in potency (17a,b
compared to 16a,b). Despite the sub-nanomolar binding
affinity of these analogs, these compounds exhibited
weaker activity in the in vitro clotting assays than expect-
ed based on their potencies in the binding assays. To try to
address these weak clotting time values, we investigated
pyrazole bicyclic pyrimidinone compound 18.10
Although the potency of the pyrimidinone compound
18 in the binding assay was reduced relative to the pyraz-
olo-dihydropyridinone compound 6a, an improvement
was indeed observed in the clotting (PT) values. Addition-
al information on the pyrazolo-pyrimidinyl bicyclic scaf-
fold will be the subject of discussion in future
communications.10
With the discovery of razaxaban, our next focus was to
identify suitable candidates that lacked the potential
issue of an aniline P4. There is precedent in the literature
for mutagenicity with some biarylanilines.18 Moreover,
the biaryl fragment of DPC602 tested positive in the
Ames assay and was confirmed to be positive in the fol-
low-up assays for mutagenicity. On the other hand, it
was gratifying to determine that the aminomethyl imid-
azole-aniline P4 moiety of razaxaban tested negative in
the Ames assay. Several additional diverse aminobiaryls
and aminophenylimidazole found in our fXa inhibitors
were evaluated without a clear SAR (i.e., many were
not mutagenic but closely related compounds were).
With this set of data in hand, it became clear that
advancement of additional molecules from the monocy-
clic pyrazole series would require tedious investigations
into the hydrolysis of the carboxamido linker and test
each aminophenyl P4 in the Ames assay. To circumvent
this, three strategies (Fig. 2) were employed in an effort
to prevent cleavage of the carboxamido linker to the
corresponding aminobiaryls.
The effect of modifications on the biaryl moiety is illus-
trated in Table 2. In general, most of the compounds
show potent inhibition of fXa. The SAR clearly shows
a preference for a diamino-substitution on the biaryl
moiety. For example, compound 6a (fXa Ki = 0.04 nM)
and compound 6b (fXa Ki = 0.11 nM) compared to
compound 6d (fXa Ki = 0.34 nM). The potency was en-
hanced when one of the ethyl groups of 6b was replaced
with a methyl group (6c, fXa Ki < 0.10 nM). Pyrrolidine
6e was potent against fXa (Ki = 0.15 nM), however, a
significant enhancement in potency was seen by
the introduction of a 3(R)-hydroxyl moiety on the
pyrrolidine ring (e.g., 6f fXa Ki = 0.03 nM). The 3(S)-
pyrrolidine isomer 6g (fXa Ki = 0.12 nM) was about
fourfold less potent. Between the two compounds, 6f
showed the best balance of potency in the binding assay
and in the in vitro clotting assay. The morpholine and
piperazine containing compounds 6i–k were potent
fXa inhibitors, but were weaker in the clotting assay.
In general, the compounds in this series were highly
selective relative to trypsin (Ki > 10,000), but showed re-
duced selectivity for thrombin. Compound 6f showed
the highest affinity for thrombin (Ki = 35 nM). As previ-
ously described, the pyrazolo bicyclic compounds lack-
ing the 20-fluorobiaryl substituent are more potent in
Herein, we wish to report on the progress made with strat-
egy I. The other approaches (strategies II and III) will be
discussed in subsequent publications.10 In strategy I, the
potential in vivo cleavage of the carboxamido linker is
prevented by its cyclization to the pyrazole scaffold. Table
1 depicts some of the pyrazolo bicyclic scaffolds which
were investigated. In general, these scaffolds maintained
the subnanomolar inhibitory activity against factor Xa,
previously observed with razaxaban and the monocyclic
pyrazole prototype 15.19 In fact, 6a was the most potent
compound with a fivefold enhancement in potency
relative to razaxaban. While these results were gratifying,
some minor SAR differences did emerge with this study.
For example, in the series lacking the tied-back ring, the
ortho-fluoro P4 substituent added potency, however, the
ortho-fluoro P4 substituted in the biaryl compound
7a was comparatively less potent to the corresponding
unsubstituted analog 6a. The scaffold size clearly

4144
D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
Table 1. In vitro activity for various bicyclic pyrazole scaffolds
F3C
N
R
NMe2
N
N
O
NH2
O
N
Compound
Scaffold
R
H
fXa Ki (nM)
PT EC2x (lM)
F3C
N
N
6a
0.04
2.7
N
N
O
O
F3C
7a
F
H
F
H
F
H
F
F
0.13
0.81
0.85
0.60
0.60
0.25
0.26
0.19
5.6
4.0
8.5
6.9
15
N
N
F3C
N
16a
N
N
O
O
O
O
O
F3C
N
N
N
N
16b
N
N
F3C
17a
N
N
F3C
17b
N
N
H
N
F3C
1810
1.4
4.2
2.1
N
N
F3C
1519
NH
N
N
O
F3C
Razaxaban6a
NH
N
N
O
Kis obtained from purified human enzymes and are averaged from multiple determinations (n = 2). PT values are measured according to Ref. 6.
the binding and clotting assays than those with the sub-
stituent (i.e., 6a vs 7a and 6f vs 7c). The replacement of
the 3-CF3 with a 3-CH3 substituent on the pyrazole scaf-
fold was also investigated (Table 2). The compounds
were less potent in the binding assay, but still showed
sub-nanomolar inhibition and improved potency in the
clotting assay.
the oral bioavailability seen with these compounds.
The pharmacokinetic profile for compounds 6a,d, and
e was comparable to that of razaxaban with moderate
half life, whereas compounds 6f,g,h and 7c showed low
clearance, low Vdss, moderate half life and high oral bio-
availability. This is quite possibly due to the hydroxyl
substituent on the cyclic amino moiety (the rationale
for the improved pharmacokinetics due to the presence
of the hydroxyl substituent on the P4 moiety is not
understood). Although the 3-methylpyrazolo com-
pounds 12a–d were relatively potent in vitro, their phar-
macokinetic profiles were moderate to poor. Overall
compounds 6a, f, 7c, and 12c had the best in vitro poten-
cy and dog pharmacokinetic profiles.
The permeability Caco-2 assay, pharmacokinetics
(determined in dogs), and in vivo antithrombotic effica-
cy (rabbit arterio-venous shunt thrombosis model) are
shown in Table 3. The measured oral bioavailability
(F %) was good in most cases. The pharmacokinetics
and Caco-2 permeability values could in part explain

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
4145
Table 2. In vitro activity—biaryl P4 analogs
thrombus formation with IC50 values of 510, 600,
1000, and 600 nM, respectively. The high IC50 values
were in good agreement with the high clotting (PT)
values. It is also possible that this could be attributed
to the low plasma free fraction for these compounds
(>98% plasma protein bound for 7a–c).
F3C
R
R
R1
R1
N
N
N
N
N
N
O
O
NH2
NH2
O
O
The X-ray structure of compound 6f (Fig. 3) bound in
the active site of fXa shows a tight inhibitor enzyme
complex17 and follows the general trend seen with razax-
aban. In the SI pocket, the aminobenzisoxazole PI
N
N
6 R = H
7 R = F
12a-c R = H
12d R = F
Compound R1
fXa
Ha
PT IC2x
˚
Ki (nM) Ki (nM) (lM)
group is in close proximity to Aspl89 (2.8 A). The 3-trif-
luoromethylpyrazole substituent engages the enzyme at
the outer ridge of the SI region and is in close contact
with the disulfide bridge. The pyrazole N-2 ring atom
interacts with the NH backbone of Gln192 (3.3 A).
The carboxamido carbonyl oxygen of 6f strongly inter-
˚
acts with the NH of Gly216 (3.0 A) and positions the
biaryl moiety in the S4 region in an optimal fashion.
The terminal phenyl ring of the biphenyl forms an edge
to face interaction with Try215, and is neatly p stacked
between the Try99 and Phe174 residues. The 3-(R)-
6a
6b
NMe2
NEt2
0.04
0.11
43
97
2.7
3.0
3.2
3.7
3.2
3.6
4.8
4.9
6c
6d
N(Me)Et
NHMe
Pyrrolidine
<0.1
57
˚
0.34
0.15
150
58
6e
6f
6g
3(R)-OH-pyrrolidine 0.03
35
59
3(S)-OH-pyrrolidine
4-OH-piperidine
Morpholine
0.12
0.22
0.35
0.64
0.71
0.13
0.24
6h
6i
71
130
280
230
75
41
6j
6k
4-NH-piperazine
4-Me-piperazine
NMe2
14
31
7a
7b
5.6
Pyrrolidine
—
6.1
8.4
2.1
1.4
2.8
4.9
2.1
7c
12a
3(R)-OH-pyrrolidine 0.16
72
NMe2
Pyrrolidine
0.55
0.31
380
120
130
250
540
12b
12c
3(R)-OH-pyrrolidine 0.27
3(R)-OH-pyrrolidine 0.34
12d
Razaxaban6a
—
0.19
Kis obtained from purified human enzymes and are averaged from
multiple determinations (n = 2). PT values are measured according to
Ref. 6.
In the rabbit arteriovenous shunt (AVShunt) thrombo-
sis model,5f the 3(R)-hydroxypyrrolidinyl compound 6f
was found to be the most potent compound from this
series (IC50 = 135 nM). The dimethylaminomethyl bia-
ryl compound 6a was about twofold less potent
(IC50 = 223 nM). The pyrazole compound 6f was
97.4% protein bound (equilibrium dialysis assay).16 In
the same model, compounds 6h and 7a–c inhibited
Figure 3. Overlap X-ray structures of BMS-740808 (6f, orange)20 and
razaxaban (white)6a in the active site of factor Xa.
Table 3. Permeability, pharmacokinetic, and in vivo antithrombotic profiles
Caco-2a Papp · 10ꢀ6 cm
Clb (L/kg/h)
F %d (po)
Rabbitc AVShunt IC50 (nM)
d
Vdss (L/kg)
d
t1/2 (po) (h)
Compound
6a
6d
4.7
—
0.98
1.66
1.01
0.35
0.27
0.21
1.05
0.79
0.35
3.2
4.5
9.7
5.0
1.6
1.12
1.4
8.8
4.8
2.8
6.4
3.3
0.75
1.1
3.4
3.8
7.4
5.3
5.1
4.5
6.4
6.4
4.9
5.9
1.6
1.7
1.7
1.1
5.3
50
67
37
82
52
102
83
85
81
13
10
44
62
84
223
—
6e
6f (BMS-740808)
—
—
1.7
—
135
—
6g
6h
0.4
8.9
bql
13.0
6.8
—
510
600
1000
600
—
7a
7b
7c
12a
12b
12c
1.9
—
d
6.2
—
0.42
0.94
1.1
12d
Razaxaban6a
—
5.56
340
a,b,c Caco-2, dog PK and rabbit AVShunt were measured according to Refs. 7a and b.
d ID50 determined to be 0.6 lg/kg/h.

4146
D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
hydroxypyrrolidinomethyl moiety is within bonding dis-
tance with the electron-rich residues of the S4 region.
Overall, these compounds fit into the fXa enzyme active
site in a highly complementary manner.
Acknowledgment
The authors thank Dr. Steven Sheriff for depositing the
BMS-740808/fXa complex into the PDB.
To fully appreciate the binding affinity seen with 6f a
detailed kinetic study was conducted.11 As expected
for an active site-directed inhibitor, 6f was competitive
with a tripeptide chromogenic substrate, but exhibited
mixed-type inhibition with the physiological substrate,
prothrombin, where the binding interactions are pre-
dominantly at exosites.12 Such substrate-dependent
modes of inhibition were also observed with the Daiichi
fXa inhibitor, DX-9065a.13 There was no evidence for
any significant binding of 6f to the zymogen, fX. One
consequence of this inhibition mechanism is that 6f is
a potent, low nanomolars, inhibitor in the presence or
absence of saturating levels of the physiological sub-
strate. The second order rate constant for association
determined by stopped-flow spectrofluorimetry is rapid
and near the diffusion controlled limit at both 25 ꢂC
and 37 ꢂC (2.7 · 107 and 1.8 · 107 Mꢀ1 sꢀ1, respective-
ly). Similar rapid onset of inhibition has been reported
for optimized thrombin inhibitors.14 The plots of the ob-
served rate constants versus inhibitor were linear up to
10 lM for 6f, suggesting that binding occurs either by
a simple one-step mechanism or by a two-step mecha-
nism with very weak initial complex formation (initial
Ki ꢁ 10 lM). Differences in the dissociation rate
References and notes
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P. Y. S.; Pinto, D. J.; Wexler, R. R.; Knabb, R. M. J.
Pharmacol. Exp. Ther. 2002, 303, 993; (g) Wong, P. C.;
Pinto, D. J.; Knabb, R. M. Cardiovasc. Drug Rev. 2002,
20(2), 137; The race to an orally active fXa inhibitor.
Recent advances (h) Quan, M. L.; Smallheer, J. Curr.
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constants calculated from the relationship, Ki = kdissoc
/
6. (a) Quan, M. L.; Lam, P. Y. S.; Han, Q.; Pinto, D. J.; He,
M.; Li, R.; Ellis, C. D.; Clark, C. G.; Teleha, C. A.; Sun, J.
H.; Alexander, R. S.; Bai, S. A.; Luettgen, J. M.; Knabb,
R. M.; Wong, P. C.; Wexler, R. R. J. Med. Chem. 2005,
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Watson C, Staus A. ISTH XXth Congress, Sydney
Australia, August 6–12, 2005.
7. (a) Pinto, D. J. P.; Orwat, M. J.; Wang, S.; Fevig, J. M.;
Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.;
Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.;
Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.;
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2001, 44, 566; (b) Wong, P. C.; Quan, M. L.; Crain, E. J.;
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R. S.; Rossi, K. A.; Wells, B. L.; Drummond, S.; Bostrom,
L. L.; Burdick, D.; Bruckner, R.; Chen, H.; Smallwood,
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A.; Smallwood, A. M.; Rossi, K. A.; Wright, M. R.; Bai, S.
A.; He, K.; Luettgen, J. M.; Wong, P. C.; Knabb, R. M.;
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kassoc, account primarily for the higher Ki at 37 ꢂC
(0.00008 lM) and for the curved time courses observed
at 25 ꢂC (data not shown), which are often seen with
slow, tight binding inhibitors (t1/2 off = 13 min at 25 ꢂC
and 8 min at 37 ꢂC).15 Similar results were obtained with
razaxaban and will be reported elsewhere.11 Table 4
shows the enzyme selectivity profile of 6f compared to
that of razaxaban. With the exception of thrombin,
the compound is highly selective over a range of
enzymes evaluated.
In conclusion, structural modification of the pyrazole
scaffold resulted in the identification of a pyrazolo-
pyridinone scaffold with excellent potency, selectivity,
and favorable pharmacokinetics. Stability (pH) studies
on 6f showed no degradation of the molecule. Also, met-
abolic studies showed no cleavage of the carboxamido
moiety. Overall, compound 6f demonstrated excellent
potency, desirable enzyme kinetic properties, and selec-
tivity in vitro, good permeability, favorable pharmaco-
kinetic and antithrombotic efficacy profile. The
crystalline HCl salt of 6f was selected for further pre-
clinical evaluation as BMS-740808.
Table 4. Enzyme selectivity profile of 6f (BMS-740808)
10. Fevig, J. M.; Cacciola, J.; Buriak, J., Jr.; Rossi, K. A.;
Knabb, R. M.; Luettgen, J. M.; Wong, P. C.; Bai, S. A.;
Wexler, R. R.; Lam, P. Y. S. Bioorg. Med. Chem. Lett.
2006, 16, 3755.
11. Experimental details for the mechanism studies are
described in a manuscript in preparation on razaxaban.
12. Krishnaswamy, S. J. Thromb. Haemost. 2004, 3, 54.
13. Rezaie, A. R. J. Thromb. Haemost. 2003, 89, 112.
Human enzyme (Ki)
BMS-740808,
6f, (lM)
Razaxaban
(lM)6a
Factor Xa
Thrombin
0.00003
0.035
0.00019
0.540
Trypsin, plasma kallikrein, APC,
factor IXa, factor VIIa, plasmin,
Chymotrypsin, tPA
>2.8
>2.3

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4141–4147
4147
14. Elg, M.; Gustafsson, D.; Deinum, J. J. Thromb. Haemost.
1997, 78, 1286.
15. Data were analyzed by standard methods established for
slow binding inhibitors Kettner, C.; Mersinger, L.; Knabb,
R. J. Biol Chem. 1990, 265, 18289.
18. (a) Ioannides, C.; Lewis, D. F. V.; Trinick, J.; Neville, S.;
Sertkaya, N. N.; Kajbaf, M.; Gorrod, J. W. Carcinogen-
esis 1989, 10, 1403; (b) Chung, K. T.; Chen, S.-C.; Wong,
T. Y.; Li, Y.-S.; Wei, C.-L.; Chou, M. W. Toxicol. Sci.
2000, 56, 351.
16. Pacific, G. M.; Viani Clin. Pharmacokinet 1992, 23, 449.
17. The X-ray structure of 6f bound to human fXa was
19. Quan, M. L.; Han, Qi.; Fevig, J.; Lam, P. Y. S.; Bai, S.;
Knabb, R. M.; Luettgen, J. M.; Wong, P. C.; Wexler, R.
R. Bioorg. Med. Chem. Lett. 2006, 16, 1795–1798.
20. Coordinates and structure amplitudes for 6f have been
deposited in the PDB with accession no. 2FZZ.
˚
obtained at 2.2 A resolution with a crystallographic R-
value of 0.260 (R-free of 0.296) with a 1r cutoff according
to the methodology described in Ref. 7.