Discovery of potent, efficacious, and orally bioavailable inhibitors of blood coagulation factor Xa with neutral P1 moieties

Article abstract of DOI:10.1016/j.bmcl.2006.08.027

The bicyclic dihydropyrazolopyridinone scaffold allowed for incorporation of multiple P1 moieties with subnanomolar binding affinities for blood coagulation factor Xa. The compound 3-[6-(2′-dimethylaminomethyl-biphenyl-4-yl)-7-oxo-3-trifluoro-methyl-4,5,6,7-tetrahydro-pyrazolo[3,4-c]pyridine-l-yl]-benzamide 6d shows good fXa potency, selectivity, in vivo efficacy and oral bioavailability. Compound 6d was selected for further pre-clinical evaluations.

Full text of DOI:10.1016/j.bmcl.2006.08.027

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5584–5589
Discovery of potent, efficacious, and orally bioavailable inhibitors
of blood coagulation factor Xa with neutral P1 moieties
Donald J. P. Pinto,a,* Robert A. Galemmo, Jr.,e Mimi L. Quan,a Michael J. Orwat,a
Charles Clark,a Renhua Li,h Brian Wells,f Francis Woerner,f Richard S. Alexander,e
Karen A. Rossi,c Angela Smallwood,g Pancras C. Wong,b Joseph M. Luettgen,b
Alan R. Rendina,b Robert M. Knabb,b 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
eJohnson & Johnson, Pharmaceutical Research and Development, Exton, PA 19341, USA
fE.I. DuPont Central Research & Development, Wilmington, DE 19880, USA
gGlaxoSmithKline, King of Prussia, PA 19406, USA
hEli Lilly & Co. Indianapolis, IN, USA
Received 10 July 2006; revised 2 August 2006; accepted 3 August 2006
Available online 11 September 2006
Abstract—The bicyclic dihydropyrazolopyridinone scaffold allowed for incorporation of multiple P1 moieties with subnanomolar
binding affinities for blood coagulation factor Xa. The compound 3-[6-(20-dimethylaminomethyl-biphenyl-4-yl)-7-oxo-3-trifluoro-
methyl-4,5,6,7-tetrahydro-pyrazolo[3,4-c]pyridine-l-yl]-benzamide 6d shows good fXa potency, selectivity, in vivo efficacy and oral
bioavailability. Compound 6d was selected for further pre-clinical evaluations.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Thrombosis is initiated by the rupture of an artheroscle-
rotic plaque in arteries and subsequently leads to clot
formation and sets in motion disease states such as
thromboembolism, myocardial infarction, and stroke.
Despite the advances in identifying novel anticoagu-
lants, warfarin (Coumadinꢁ)1a–c remains the only
approved oral anticoagulant in the United States. Other
parenteral treatments are also available such as heparin,
low molecular weight heparins (LMWH), and fondapar-
inux.2,3,4a–c Blood coagulation factor Xa (fXa), a key
enzyme in the coagulation cascade, catalyzes the conver-
sion of prothrombin to thrombin, the final enzyme that
triggers clot formation.5a–g Pre-clinical animal models
have suggested that inhibiting fXa has the potential
for providing excellent antithrombotic efficacy with min-
imal risk of bleeding.5f,g
Recently, in a phase II study we have shown that a small
molecule fXa inhibitor razaxaban (BMS-561389 fXa
Ki = 0.19 nM, trypsin Ki > 10,000 nM) demonstrated
good antithrombotic efficacy and minimal bleeding in
humans.6a,b Razaxaban, a pyrazole-based aminobenzis-
oxazole fXa inhibitor with good potency, selectivity, and
oral bioavailability, was a follow-on candidate to an
earlier compound from the pyrazole class, that is,
DPC423.7 As part of a strategy to mask the arylaniline
P4 moiety, we recently described an optimization strat-
egy that led to the discovery of a highly potent and
orally bioavailable bicyclic pyrazole compound BMS-
740808 (Fig. 1).8 In this report, we wish to describe
further efforts to optimize the bicyclic dihydropyrazo-
lopyridinone series by replacing the aminobenzisoxazole
with other P1 moieties.
Keywords: Blood coagulation; Factor Xa; Thrombin, trypsin; Selec-
tivity; BMS-740808; Aminobenzisoxazole; P-methoxyphenyl and
3-phenylcarboxamido P1; Dog ‘N-in-one’ pharmacokinetics; Oral
bioavailability; Clearance; Vdss; Half-life; Rabbit AV Shunt efficacy.
*
The bicyclic dihydropyrazolopyridinone scaffold was
prepared as outlined in Scheme 1. The starting material
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.2006.08.027

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5584–5589
5585
OH
tuted compounds 8b–f (not shown in Scheme 1) were
also prepared. The aminobenzisoxazole pyrazole deriva-
tive 9 was prepared according to the methodology
previously reported.8
F3C
F3C
F
N
H
N
N
N
N
N
N
N
O
O
N
N
NH2
NH2
BMS-740808 (Fig. 1, fXa Ki = 0.03 nM) and several
analogs within the aminobenzisoxazole P1 series demon-
strated potent inhibitory activity against factor Xa.8 At-
tempts to incorporate additional diversity within our
factor Xa inhibitors prompted us to focus on identifying
non-aminobenzisoxazole P1 moieties that shared the
properties of razaxaban or BMS-740808. Table 1 lists
a set of substituted 2, 3, and 4-phenyl P1 bicyclic pyra-
zole compounds. Several of these compounds displayed
potent inhibition of factor Xa, and interestingly are con-
siderably more potent when compared to similarly
substituted analogs in the monocyclic pyrazole series.10
These data complemented that obtained for BMS-
740808, and thereby demonstrate the optimal nature
of this highly constrained and rigid bicyclic pyrazolopy-
ridinone scaffold, to maximize the inhibitor–fXa enzyme
binding interactions from the S1 to the S4 regions.
Although several of these inhibitors showed good fXa
binding affinity, the set of neutral P1 analogs described
herein are somewhat less potent when compared to sim-
ilar aminobenzisoxazole P1 analogs.8 In terms of the
SAR, when compared to the corresponding 2-amid-
ophenyl P1 analog 6b (fXa Ki = 2.70 nM), a 3-fold
improvement in binding affinity is seen for the 2-amin-
osulfonyl compound 6a (fXa Ki = 0.82 nM). However,
a 20-fold loss in fXa affinity is seen when compared to
O
N
O
N
Razaxaban
FXa Ki = 0.19nM
BMS-740808
FXa Ki = 0.03nM
Figure 1.
1 was readily prepared in excellent yield following a two-
step sequence from 4- iodoaniline and 5-bromovaleryl
chloride [10% NaOH, DCM followed by treatment with
potassium tert-butoxide]. Chlorination of 1 with phos-
phorus pentachloride in chloroform at reflux afforded
a,a-dichloro-piperidinone intermediate which on heat-
ing with excess morpholine provided the morpholine-en-
amine 2 in 70% yield. Treatment of enamine 2 with
trifluoroacetic anhydride in the presence of DMAP, fol-
lowed by acid hydrolysis, gave the trifluoroacetyl-piper-
idine-2,3-dione intermediate 3 in high yield (>90%).
Condensation of intermediate 3 with commercially
available 3-CN, 3-NO2, 4-Et, and 4-OMe phenylhydra-
zines afforded the requisite bicyclic pyrazole scaffolds
4c–f in good yield. The coupling of the 2-aminosulfo-
nyl-phenylhydrazine (prepared in situ by diazotization
of
2-amino-phenylsulfonamide
and
subsequent
reduction with tin chloride in HCl) afforded the 2-amino-
sulfonyl-phenyl bicyclic pyrazole intermediate 4a (R =
2-SO2NH2). In a similar manner, intermediate 4b
(R = 2-CONH2) was obtained by condensation of the
2-amidophenyl P1-phenylhydrazine (prepared in situ
from 2-aminobenzamide as described above) with the
tricarbonyl intermediate 3. Suzuki cross-coupling of
bicyclic pyrazole intermediates 4a–f with 2-formyl-phen-
ylboronic acid or 2-methylthiophenylboronic acid
afforded the corresponding 20-formyl-biaryl 5a–f or the
20-methylthio-biaryl intermediate 5g in good yields.
Reductive amination of intermediates 5a–c, 5d, and
5e,f with dimethylamine and sodium cyanoborohydride
in methanol provided the desired dimethylaminomethyl
biaryl P4 compounds 6a–c, 6e and 6j k.7a,8 The 3-car-
boxamidophenyl P1 compound 6d was obtained from
the 3-cyanophenyl compound 6c by treatment with
hydrogen peroxide in the presence of aqueous sodium
hydroxide. The reduction of the 3-nitrophenyl interme-
diate 6e with tin chloride in ethylacetate and concd
hydrochloric acid provided the 3-amino compound 6f.
Treatment of 6f with acetic anhydride and triethylamine
in dichloromethane gave the acetamide compound 6g.
Likewise, treatment of 6f with excess NaOCN in
dioxane at 100 ꢂC gave the urea compound 6h. The phe-
nylsulfonamide P1 compound 6i was obtained from the
3-aminophenyl compound 6f by treatment with metha-
nesulfonyl chloride in pyridine. The reductive amination
of the carboxaldyhyde intermediates 5a, 5c and 5f with
3-(R)-hydroxy-pyrrolidine and NaBH3CN in methanol
afforded the 3-(R)-hydroxy-pyrrolidinomethyl biaryl
P4 compounds 7a, 7c and 7f. The methylsulfonyl biaryl
P4 compound 8a was prepared by oxidation of the cor-
responding 20-methylthio-biaryl intermediate 5g with
MCPBA. In a similar fashion, other P1 phenyl-substi-
the aminobenzisoxazole P1 compound
9
(fXa
Ki = 0.04 nM, Table 1).8 Also noteworthy is the similar-
ity in the anticoagulant activity (as measured by the clot-
ting PT value) for compound 6a and the corresponding
aminobenzisoxazole P1 analog 9 (6a PT, EC2X
=
2.50 lM, 9 PT, EC2X = 2.70 lM). The result suggests
that in addition to protein binding differences, 6a may
have altered the overall coactivator complex.
The 3-amidophenyl compound 6d (fXa Ki = 0.18 nM)
and the 4-methoxyphenyl compound 6k (fXa
Ki = 0.35 nM) displayed sub-nanomolar fXa binding
affinity with moderate clotting activity. The 3-anilino
compound 6f and the 4-ethylphenyl compound 6j also
demonstrated low nanomolar fXa activity but were
weak in the clotting assay. Substituting the 3-anilino
moiety (compounds 6g–i) led to weaker inhibitors of
fXa. It is interesting to note, however, that the pyra-
zole-based compounds listed in Table 1 were several fold
more potent when compared to other fXa scaffolds bear-
ing neutral P1 moieties.11a–c Differences in the anticoag-
ulant activity were also seen for the 4-methoxyphenyl P1
compound 6k (PT EC2X = 6.4 lM) and the 4-ethyl com-
pound 6j (PT EC2X > 200 lM). As indicated above, it is
possible that plasma protein binding could account for
these high in vitro clotting values (PTs).
Incorporation of the 3-(R)-hydroxypyrrolidinomethyl
biaryl P4 moiety of BMS-7408088 led to compounds
shown in Table 2. Also included in this study were com-
pounds bearing the o-methylsulfonyl biaryl P4 moiety
found in DPC423 (e.g., compounds 8a–f) to determine

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D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5584–5589
O
F3C
N
a,b,c,d
N
e,f
N
N
O
N
O
O
O
I
I
O
I
2
1
3
F3C
N
F3C
N
N
N
g, h,i,j
CHO
k
N
O
O
HO OH
B
I
CHO
R
R
OH
k HO
4a R = 2-SO2NH2
4b R = 2-CONH2
4c R = 3-CN
4d R = 3-NO2
4e R = 4-Et
5a R = 2-SO2NH2
5b R = 2-CONH2
5c R = 3-CN
5d R = 3-NO2
5e R = 4-Et
B
SMe
F3C
N
SMe
N
N
4f R = 4-OMe
5f R = 4-OMe
O
5g
R
F3C
N
F3C
N
N
N
N
N
N
N
O
6c
m
O
l
CONH2
R
6d
6a R = 2-SO2NH2
6b R = 2-CONH2
6c R = 3-CN
6e R = 3-NO2
6j R = 4-Et
6k R = 4-OMe
F3C
N
N
o, p or q
or r
N
N
6f R = 3-NH2
6g R = 3-NHCOMe
6h R = 3-NHCONH2
6i R = 3-NHSO2Me
6e
O
R
OH
F3C
F3C
N
N
N
N
N
N
N
CHO
O
O
l or n
OH
R
R
HN
5a R = 2-SO2NH2
5c R = 3-CN
5f R = 4-OMe
7a R = 2-SO2NH2
7b R = 3-CONH2
7f R = 4-OMe
F3C
N
F3C
N
B(OH)2
SMe
N
N
N
N
SO2Me
H2NO2S
H2NO2S
k,n
+
O
O
I
8a
4a
Scheme 1. General method of synthesis of the 3-trifluoromethyl-dihdropyrazolopiperidinone factor Xa compounds. Reagents and conditions: (a) 5-
Br-valeryl chloride, NaOH (10%) DCM; (b) 2 equiv KO-t-Bu; (c) PCl5 (3 equiv),CHCl3; (d) morpholine excess 140 ꢂC; (e) DMAP/TFAA/DCM; (f)
Et2O, 20% aq HCl; (g) subs-aniline, NaNO2, HCl, AcOH; (h) SnCl2, H2O, HCl, ethylacetate; (i) MeOH reflux; (j) TFA/DCM; (k) (Ph3P)4Pd, K3PO4,
dioxane; (l) amine (2 equiv), NaCNBH3; (m) H2O2, NaOH (10%); (n) MCPBA/DCM; (o) [H]Pd/C 10%, MeOH; (p) Ac2O, TEA, DCM; (q) NaOCN,
AcOH heat; (r) MsCl, pyridine, DCM.
if they provided superior profiles previously observed
with BMS-740808 and DPC423. Comparable fXa bind-
ing affinities were observed for the o-methylsulfonyl
compound 8a (fXa Ki = 0.74 nM) and the dimethylami-
nomethyl biaryl compound 6a (fXa Ki = 0.82 nM).
However, unlike compound 9 (fXa Ki = 0.04 nM),8 the
3-(R)-hydroxy-pyrrolidinomethyl biaryl P4 compounds
7a (fXa Ki = 1.2 nM) and 8a (fXa Ki = 0.74 nM) were
less potent. Similarly, the compound bearing the 3-(R)-
hydroxypyrrolidinomethyl biaryl P4 moiety 7c (fXa
Ki = 0.76 nM) was less potent than the compound bear-
ing the dimethylaminomethyl biaryl P4 moiety 8c (fXa
Ki = 0.18 nM), and the corresponding dimethylaminom-
ethyl biaryl P4 compound 6k (fXa Ki = 0.34 nM). The 3-
amidophenyl P1 compound 7b (fXa Ki = 0.72 nM) was
also less potent when compared to the corresponding
3-amidophenyl compound 6d (fXa Ki = 0.18 nM) (Table
1). Di-substituted phenyl P1 compounds (8d–f) did not

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5584–5589
5587
Table 1. In vitro activity for various bicyclic P1 moieties
F3C
and 7a–b are shown in Table 3. In general, the com-
pounds showed good Caco-2 permeability (Papp) values,
and correlated well with the high oral bioavailability
(F%) seen in dogs. The pharmacokinetic profile (dogs)
for these compounds shows a moderate clearance pro-
F3C
N
N
N
N
N
N
N
4
N
O
O
2
file, moderate to high volume of distribution (Vdss
)
3
R
NH2
and long terminal half-lives (t1/2). In the rabbit AV
Shunt thrombosis model, compounds 6d, 7a, and 7b
inhibited thrombus formation in a dose-dependent man-
ner, but at significantly higher IC50 values when com-
pared to razaxaban and or BMS-740808.6–8
Compounds 6d and 7a compared favorably to razax-
aban and BMS-740808. However, the analogs were less
potent in the rabbit arteriovenous shunt (AV Shunt)
thrombosis model.
O
N
9
Compound
R
fXa Ki
(nM)
PT EC2X
(lM)
9
—
0.04
0.82
2.70
12.0
0.18
20.0
2.00
88.0
54.0
47.0
2.60
0.35
2.70
2.50
—
6a
6b
6c
6d
6e
6f
6g
6h
6i
2-SO2NH2
2-CONH2
3-CN
—
3-CONH2
3-NO2
3-NH2
2.63
—
15
The X-ray structure of the 3-carboxamido-phenyl com-
pound 6d18 bound to factor Xa (2.1 A resolution) is
˚
3-NHCOMe
3-NHCONH2
3-NHSO2Me
4-Et
—
—
shown in Figure 2. Overall the compound binds to
fXa in a similar manner to BMS-740808.8 A strong
interaction between the P1 amidophenyl NH proton
—
228.3
6.40
6j
6k
´
4-OMe
˚
with Asp 189 (2.92A) of Xa is prominent in the S1 re-
Kis obtained from purified human enzymes and are averaged from
multiple determinations (n = 2). PT values are measured according to
Ref. 6.
gion of the enzyme. However, the X-ray structure does
not show any specific S1 interaction with the P1 amid-
ophenyl carbonyl moiety. The pyrazole N-2 nitrogen
interacts with Gln192 backbone. The pyrazolopyridi-
none carbonyl forms a strong interaction with Gly216
and the dimethylaminomethyl biaryl P4 is neatly stacked
between the S4 enzyme residues Tyr99, Phe174, and
Trp215 thus making for a tight enzyme–inhibitor
complex.
Table 2. Comparative in vitro fXa binding affinity of pyrazolopyrid-
inone compounds bearing novel P1 moieties
OH
F3C
F3C
N
N
N
N
N
SO2Me
N
4
N
4
Figure 3 shows an overlap model of the 4-methoxyphe-
nyl P1 compound 6k and the 2-aminosulfonylphenyl
compound 8a. The oxygen atom of the 4-methoxy P1
moiety appears to form a lipophilic-p interaction with
the S1 Tyr228 residue at the bottom of the S1 pocket
of fXa. The loss of the fXa binding affinity seen with
the 4-ethyl compound 6j highlights the importance of
the oxygen atom in compound 6k. In the fXa model,
the 2-aminosulfonyl functionality (compound 6a) ap-
pears to orient toward the catalytic triad, and interacts
with Ser195 and the carbonyl oxygen atom of the inhib-
itor itself. Interestingly, this binding mode differs from
the binding mode previously seen with the ortho-benzyl-
amine P1 compound DPC602,9 wherein the amino moi-
ety of the P1 benzylamine interacts with Asp 189. The
rest of the P4–S4 interactions are identical. Based on
the overlap model of 6a and 6k, it was clear that com-
bining the ortho and para phenyl P1 substituents on
one molecule should be beneficial in terms of potency.
This was confirmed by the high fXa potencies seen with
compounds 8d–f. Unfortunately, these analogs were
poor in the clotting assay, presumably due to an increase
in protein binding.
O
O
2
2
3
3
R
R
A
B
Compound
Type
R
fXa Ki PT EC2X
(nM)
(lM)
7a
7b
A
A
A
B
B
B
B
B
B
2-SO2NH2
3-CONH2
4-OMe
1.20
0.72
0.76
0.74
2.00
0.18
0.18
4.30
3.50
8.20
6.20
9.90
31.9
31.9
—
7c
8a
2-SO2NH2
2-CONH2
4-OMe
8b
8c
8d
8e
2-CONH2, 4-OMe
2-SO2NH2, 4-OMe 0.12
8f
BMS-740808
2-NH2, 4-OMe
0.20
0.03
13.2
3.60
Kis obtained from purified human enzymes and are averaged from
multiple determinations (n = 2). PT values are measured according to
Ref. 6.
show any enhancements in binding affinities when com-
pared to the mono-substituted 4-methoxyphenyl P1
compound 8c. The 3-amidophenyl P1 compounds 6d
and 7b, and the 2-aminosulfonylphenyl P1 compounds
6a and 7a provided the best combination of in vitro
potency and clotting activity.
Key kinetic parameters for compound 6d from detailed
mechanism studies are summarized in Table 4. As
expected for an active site inhibitor, 6d is competitive
toward a tripeptide chromogenic substrate, but is a
mixed-type inhibitor (with ca. 3-fold lower affinity for
the ES complex) versus prothrombin, the physiological
substrate, for which the binding interactions are
The Caco-2 permeability (Papp), dog pharmacokinetics
(PK), and efficacy in the rabbit arteriovenous shunt
(AV Shunt) thrombosis models for compounds 6a, 6d

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D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5584–5589
Table 3. Permeability, pharmacokinetic, and in vivo anti-thrombotic profiles
Caco-2a
Papp · 10ꢁ6 cm
Clb (L/kg/h)
t1/2
F%b
(po)
Rabbitc AV
Shunt IC50 (nM)
b
Vdss (L/kg)
b
Compound
(po) (h)
6a
6d
5.8
5.5
1.1
2.9
5.56
1.7
0.50
0.43
0.29
0.43
1.1
8.1
4.0
3.5
2.6
3.4
1.6
11.6
13
52
93
93
72
84
82
—
500
645
>780
340
135
7a
7b
9.4
7.2
5.3
5.1
Razaxaban6a
BMS-740808
0.35
a,b,cCaco-2, dog PK, and rabbit AV Shunt were measured according to Ref. 6.
Table 4. Detailed factor Xa kinetic parameters for 6d
Human factor Xa parameter
6d Parameter value
Gly 218
25 ꢂC Ki (tripeptide substrate)
37 ꢂC Ki (tripeptide substrate)
37 ꢂC Ki (prothrombin)
37 ꢂC Ki (saturating prothrombin)
25 ꢂC association rate constant
25 ꢂC dissociation rate constant
37 ꢂC association rate constant
37 ꢂC dissociation rate constant
0.14 nM
0.41 nM
1.3 nM
3.7 nM
2.9 · 107
Asp 189
M
ꢁ1 sꢁ1
Glu 97
4.1 · 10ꢁ3 sꢁ1
Ty r 99
2.1 · 107
M
ꢁ1 sꢁ1
8.4 · 10ꢁ3 sꢁ1
His 57
Kis obtained from purified human enzymes and are averaged from
multiple determinations (n = 2). Prothrombinase inhibition, associa-
tion, anddissociation rate constantsweremeasuredaccordingto Ref. 12.
Ser 195
were linear up to 5 lM 6d, suggesting that binding oc-
curs either by a simple one step mechanism or by a
two-step mechanism with very weak initial complex for-
mation (initial Ki ꢀ 5 lM). Differences in the dissocia-
tion rate constants calculated from the relationship,
Ki =kdissoc/kassoc, account for the higher Kis at 37 ꢂC.
Similar results were obtained with razaxaban and will
be reported elsewhere.12
Figure 2. Factor Xa-bound X-ray structure of carboxamide 6d.
Asp 189
Table 5 shows the enzyme selectivity profile of 6d com-
pared to razaxaban. The compound is highly selective
over a range of enzymes evaluated.
Gln 192
Gly 216
Glu 97
Trp 215
In summary, we have further demonstrated the effective-
ness of the pyrazolopiperidinone scaffold to accommo-
date numerous neutral P1 moieties which bind to
factor Xa in a highly optimized manner. The 3-[6-(20-
dimethylaminomethyl-biphenyl-4-yl)-7-oxo-3-trifluoro-
methyl-4,5,6,7-tetrahydro-pyrazolo [3,4-c]pyridine-l-yl]-
benzamide 6d provides the best balance of fXa potency,
enzyme selectivity, oral bioavailability, and the potency
in the AV Shunt antithrombotic model. Compound 6d
was selected for further pre-clinical evaluations. Various
Tyr 99
Ser 195
His 57
Tyr 228
Figure 3. Overlap fXa model of the 4-methoxyphenyl P1 compound 6k
and 2-aminosulfonyl P1 compound 8a.
predominantly at exosites.13 Similar substrate-depen-
dent inhibition mechanisms were also observed with
the Daiichi fXa inhibitor, DX-9065a.14 As a result of
this inhibition mechanism, 6d is a potent, low nanomo-
lar inhibitor in the presence or absence of saturating lev-
els of the physiological substrate. The second-order rate
constant for association determined by stopped-flow
spectrofluorimetry is rapid and near the diffusion con-
trolled limit at both 25 ꢂC and 37 ꢂC. Similar rapid onset
of inhibition has been reported for the Pfizer Xa inhibi-
tor PD0313052,15 and optimized thrombin inhibitors.16
Plots of the observed rate constants versus inhibitor
Table 5. Enzyme selectivity profile of 6d
Human enzyme
Ki (lM)
Razaxaban6a
6d
Factor Xa
Thrombin
0.00018
0.78
>1.8
0.00019
0.540
>2.3
Chymotrypsin, trypsin,
APC, factor IXa, factor VIIa,
plasmin, urokinase, tPA
Kis obtained from purified human enzymes and are averaged from
multiple determinations.17

D. J. P. Pinto et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5584–5589
5589
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2001, 44, 566; (b) Wong, P. C.; Quan, M. L.; Crain, E. J.;
Watson, C. A.; Wexler, R. R.; Knabb, R. M. J. Pharma-
col. Exp. Ther. 2000, 292, 351.
other optimization strategies using this pyrazole scaffold
will be reported in due course.
Acknowledgments
8. Pinto, D. J. P.Orwat, M. J.; Quan, M. L.; Fevig, J.; Han,
Q.; Galemmo, R. A., Jr.; Amparo, E.; Wells, B.;
Alexander, R. S.; Rossi, K. A.; Smallwood, A.; Wong,
P. C.Luettgen, J. M.; Rendina, A.; Knabb, R. M.;
Mersinger, L.; Kettner, C.; Bai, S.; He, K.; Wexler, R.
R.; Lam, P. Y. S. Bioorg. Med. Chem. Lett. 2006, 16, 4141.
9. Pruitt, J. R.; Pinto, D. J. P.; Galemmo, R. A.; Alexander,
R. S.; Rossi, K. A.; Wells, B. L.; Drummond, S.; Bostrom,
L. L.; Burdick, D.; Bruckner, R.; Chen, H.; Smallwood,
A.; Wong, P. C.; Wright, M. R.; Bai, S.; Luettgen, J. M.;
Knabb, R. M.; Lam, P. Y. S.; Wexler, R. R. J. Med.
Chem. 2003, 46, 5298.
The authors thank Dr. Steven Sheriff for depositing the
fXa/6d complex into the PDB. The authors thank Mrs.
Joanne Smallheer, Dr. James Corte, and Dr. Paul Gilli-
gan for their help in proof reading the manuscript, and
Earl Crain and Mrs. Carol Watson for providing the
in vivo data.
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deposited in the PDB with Accession No. 2G00.