Nonbenzamidine tetrazole derivatives as factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00951-4

Factor Xa (fXa) is an important serine protease that holds the central position linking the intrinsic and extrinsic activation mechanisms in the blood coagulation cascade. Therefore, inhibition of fXa has potential therapeutic applications in the treatmen

Full text of DOI:10.1016/S0960-894X(02)00951-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 369–373
Nonbenzamidine Tetrazole Derivatives as Factor Xa Inhibitors
Mimi L. Quan,* Christopher D. Ellis, Ming Y. He, Ann Y. Liauw, Francis J. Woerner,
Richard S. Alexander, Robert M. Knabb, Patrick Y. S. Lam, Joseph M. Luettgen,
Pancras C. Wong, Matthew R. Wright and Ruth R. Wexler
Bristol-Myers Squibb Co., PO Box 80500, Wilmington, DE 19880-0500, USA
Revised 15 October 2002; accepted 7 November 2002
Abstract—Factor Xa (fXa) is an important serine protease that holds the central position linking the intrinsic and extrinsic acti-
vation mechanisms in the blood coagulation cascade. Therefore, inhibition of fXa has potential therapeutic applications in the
treatments of both arterial and venous thrombosis. Herein we describe a series of tetrazole fXa inhibitors containing benzamidine
mimics as the P1 substrate, of which the aminobenzisoxazole moiety was found to be the most potent benzamidine mimic. SR374
(12) inhibits fXa with a Ki value of 0.35 nM and is very selective for fXa over thrombin and trypsin.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cycle have been shown to be potent fXa inhibitors.4
Optimization of the pyrazole series has led to a series of
extremely potent fXa inhibitors which culminated in
clinical development candidate DPC 423.5,6 Herein we
would like to report on a series of tetrazole fXa inhibi-
tors in which the benzamidine moiety has been replaced
with a less basic P1 residue.
Factor Xa (fXa) is the site of convergence of the intrin-
sic and the extrinsic coagulation pathways. Factor Xa in
combination with factor (Va) and calcium forms the
prothrombinase complex that converts prothrombin to
thrombin via proteolysis. This activation is a highly
amplified process.1 The concentration of fXa is lower
than the concentration of thrombin at the thrombotic
site, and hence a smaller dose of a fXa inhibitor may
prevent thrombosis effectively. Furthermore, since fXa
inhibitors affect coagulation specifically, this mechanism
may decrease the risk of abnormal bleeding. Preclinical
studies has indicated less bleeding risk with fXa inhibi-
tors than thrombin inhibitors.2 Therefore, orally active
fXa inhibitors should represent an important advance in
the management of both chronic arterial and venous
thrombosis.
Results and Discussion
We have reported on a variety of benzamidine mimics
which we employed in an isoxazoline and a pyrazole
series that afforded potent and selective fXa inhibitors
with improved pharmacokinetic profile.5À8 We now
wish to report on a series of tetrazole based fXa inhibi-
tors which employ the same set of benzamidine mimics
that we have previous described (Table 1). In this case,
aminobenzisoxazole 2 was found to be the most potent
inhibitor, which was only 26-fold less active than the
corresponding benzamidine 1. Chloroaniline 3 was
approximately 150-fold less potent, while p-methoxy 4,
m-benzylamine 5, and methoxyaniline 6 were >200-fold
less active. As we have previously shown,5À8 these ben-
zamidine mimics have excellent selectivity over throm-
bin and trypsin, with the possible exception of
benzylamine 5, which while 1000-fold selective over
thrombin, is only 5-fold selective over trypsin with a Ki
value of 65 nM.
A large number of small molecule fXa inhibitors have
been disclosed in recent years.3 We have reported on a
series of benzamidine fXa inhibitors containing a vicin-
ally-substituted heterocyclic core.4 Pyrazole, triazole,
and tetrazole compounds with a m-benzamidine P1
moiety linked to a nitrogen in the 5-membered hetero-
*Corresponding author. Tel.: +1-320-467-5897; fax: +1-320-467-
6881; e-mail: mimi.quan@bms.com
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00951-4

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M. L. Quan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 369–373
Table 1. Benzamidine mimics: comparison of fXa potency and
selectivity
Table 3. Optimization employing aminobenzisoxazole as the P1
substrate
Compd
P1
fXa Ki
(nM)
Thrombin Ki
(nM)
Trypsin Ki
(nM)
Compd
X
F
RFXa
Thrombin
Ki (nM)
Trypsin
Ki (nM)
Ki (nM)
1
2
300
6.4
0.037
0.98
11
>21,000
>1600
>1600
2.9
1500
>1600
2
H
F
F
0.98
0.35
0.39
1500
3
4
5.4
8.0
1600
>1600
>1600
SR374 (12)
600
400
>5200
>1600
10,000
13
5
6
13
19
>21,000
69
fXa, thrombin, and trypsin Ki values were obtained using human
purified enzymes. See ref 16 for assay conditions.
4000
>1600
fXa, thrombin, and trypsin Ki values were obtained using human
purified enzymes. See ref 16 for assay conditions.
Attempts to further optimize the chloroaniline and
aminobenzisoxazole series are shown in Tables 2 and 3.
Ortho-halogen substitution on the inner phenyl ring
afforded slight improvement in fXa affinity (1.5–4.5
fold). Replacement of the terminal phenylsulfonamide
with a pyrrolidinylamide group decreased the fXa affi-
nity by 2.5-fold. The chloroanilines showed good selec-
tivity for fXa over thrombin and trypsin as shown in
Table 2. The modest decrease in potency with the pyro-
lidinylamide modification was also observed when amino-
benzisoxazole was employed as the P1 moiety (11 vs 2). On
the other hand, ortho-fluoro substitution increased fXa
affinity by 3-fold (12). As we have previously reported for
both the isoxazoline and the pyrazole series, replacement
of the sulfonamide group with a methysulfone moiety did
not effect fXa affinity (13). The fluoro-substituted analo-
gues 12 and 13 are only 10-fold less potent than benzami-
dine SN357 and they both show excellent selectivity for
fXa relative to thrombin and trypsin.
Table 2. Optimization employing chloroaniline as the P1 substrate
Compd
RX
FXa
N
Thrombin Trypsin
Ki (nM)
Ki (nM)
Ki (nM)
7
8.0
7900
>1600
3
8
CH
5.4
3.3
1600
1600
>1600
>1600
In order to elucidate the binding mode of this series of
compounds, a crystal structure of SW712 (10) com-
plexed to human thrombin was obtained (Fig. 1).9 In
the thrombin crystal structure, a 64-degree dihedral
angle was observed between the chloroaniline ring and
the tetrazole ring, leading to a 3.1 Angstrom contact
between the backbone nitrogen of Glu192 and the N2 of
the tetrazole ring. A similar interaction between the
pyrazole and the backbone nitrogen of residue Glu192
was observed in the crystal structure of the DPC 423
fXa complex.12 The presence of a hydrogen bond
acceptor in this position appears to be important for
activity. The oxygen of the amide of the linker group
and the backbone nitrogen of Gly218 are within hydro-
gen bonding range with a distance of 3.0 Angstroms.
CCl
9
CF
CF
1.2
3.0
SW712 (10)
1200
>1600
fXa, thrombin, and trypsin Ki values were obtained using human
purified enzymes. See ref 16 for assay conditions.

M. L. Quan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 369–373
371
Figure 1. Structure of SW712 complexed to human thrombin.
Figure 2. Structure of SW712 (atomtype) from the human thrombin-
SW712 complex superimposed on the coordinates of fXa (red).
The pyrolidine group is found in the P4 pocket with a
4.2 Angstrom distance to Trp215. The nitrogen of the
chloroaniline interacts with side-chain oxygens of
Asp189 with contacts of 2.9 and 3.0 Angstroms. The
chlorine of the chloroaniline interacts with Ala190 with a
contact of 3.8 Angstroms. The increased steric bulk
associated with the substitution of Ser190A in trypsin (Ala
is found in fXa) would explain the selectivity of this series
of compounds over trypsin. Specifically, the oxygen of the
serine side-chain would sterically clash with the chlorine
of the inhibitor. Given that sequence identity between fXa
and thrombin, we superimposed the coordinates of
SW712 from the thrombin complex on the structure of
fXa.13 Analogous interactions could be found in the
model of SW712 superimposed into the fXa active site as
were observed in the thrombin crystal structure (Fig. 2).
The pharmacokinetic profile of SR374 (12) was studied
in dogs. Compared with benzamidine 1, the clearance
was reduced by two-fold and the half-life was increased
by five-fold (Table 4). More significantly, SR374
showed 74% bioavailability in dog. The antithrombotic
efficacy of SR374 was studied in the rabbit A-V shunt
model.14 Following intravenous administration, the
ID50 was found to be 5.8 mmol/kg.
Table 4. Dog PK profile and antithrombotic efficacy of SR374 com-
pared to 1
Compd
fXa
Ki (nM)
Dose
(mg/kg)
Clearance t1=2
(L/h/kg) (h) (mmol/kg/h)
ID50
1
SR374 (12)
0.037
0.36
1.0 (iv)
0.5 (iv); 0.2 (po)
0.62
0.30
0.65
3.1
0.13
5.8
Synthesis
PK Data were obtained dog N-in-1 studies in dogs. SR374 has a
bioavailability of 74%. ID50’s were obtained in the rabbit A-V shunt
model.14
The preparation of the chloroaniline derivatives is
exemplified by the synthesis of SW712 (10) shown in
Scheme 1. Synthesis of SW712.

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M. L. Quan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 369–373
Scheme 2. Synthesis of SR374.
Scheme 1. 4-Chloro-3-nitroaniline was reacted with
ethyl oxalate and Et3N in CH2Cl2 to give compound 15,
which was converted to tetrazole 16 by heating to reflux
with PPh3 in CCl4, followed by reaction with NaN3.
3-Fluoro-4-nitrobenzoic acid was converted to the
corresponding acyl chloride and then coupled with pyr-
rolidine to give compound 18. Reduction of 18 afforded
aniline 19, which was reacted with tetrazole 16 under
Weinreb conditions to produce amide 20. Reduction of
the nitro group of 20 with SnCl2 gave the chloroaniline
SW712 (10), which was purified by HPLC to afford the
final compound as the TFA salt.
of 3.1 h and oral bioavailability of 74%. SR374 also
showed antithrombotic efficacy with an ID50 of 5.8
mmol/kg/h.
Acknowledgements
We wish to thank Earl Crain, Angela Smallwood, and
Carol Watson for technical assistance.
References and Notes
The preparation of the aminobenzisoxazole derivatives
is exemplified by the synthesis of SR374 (12) as shown
in Scheme 2. 2-Fluoro-5-nitrobenzonitrile was reduced
to aniline 22 with SnCl2. The tetrazole 24 was formed
using the same methods as described above for the
chloroaniline series employing aniline 22 as starting
material. Weinreb coupling of tetrazole 12 and biaryl
derivative 2515 provided amide 26. The aminobenisox-
azole ring was then formed by reaction of 26 with ace-
tone oxime and potassium t-butoxide, followed by
heating to reflux with HCl and ethanol. Removal of the
t-butyl group with TFA gave the final product as the
TFA salt.
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Conclusions
We have designed and synthesized a series of tetrazole
compounds vicinally-substituted with P1 and P4 resi-
dues. The benzamidine group in the picomolar inhibitor
1 was replaced with a variety of less basic and nonbasic
moieties. Chloroaniline and aminobenzisoxazole were
found to be the most potent benzamidine replacements
with low nanomolar to subnanomolar fXa affinity.
SR374 (12) has a fXa Ki of 0.35 nM and was found to
be more selective compared to benzamidine 1. The
pharmacokinetic profile of aminobenzisoxazole SR374
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9. Crystals of human thrombin–hirugen complex were pre-
pared by the method described by Skrzypczak-Jankun et al.10

M. L. Quan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 369–373
373
Soaks of the enzyme-inhibitor complex were prepared by the
method described in Galemmo et al.11 Data were collected on
an R Axis II image plate mounted on a Rigaku RU200
rotating anode; crystals diffracted to 2.5 A. Data were refined
using CNX (Molecular Structure Inc.) with a final R-value of
0.209. Unambigous electron density was observed for the
inhibitor.
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values were obtained from human purified enzymes. All assays
are run in micro-titer plates using a total volume of 250 mL in
0.1 M Sodium Phosphate Buffer containing 0.2 M NaCl and
0.5% polyethylene glycol 6000 at pH 7.0. The compounds are
run at 10 mM, 3.16 mM, 1.0 mM, 0.316 mM, 0.1, 0.0316, 0.01,
and 0.00316 mM. Plates are read for 30 min at 405 nm. Rates are
determined for the controls (no inhibitor) and for the inhibitors.
%Enzyme activity is determined from these rates and used in
the following formula to determine Ki: Ki=1000ÃInhibitor
Concentration/(((Km+S)ÀSÃACT)/(ACTÃKm)À1); where S=
Substrate Concentration; ACT=% Enzyme Activity for
inhibitor. All compounds were tested in duplicate studies and
were compared with the same internal standards. These assays
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