Optimization of the beta-aminoester class of factor Xa inhibitors. Part 2: Identification of FXV673 as a potent and selective inhibitor with excellent In vivo anticoagulant activity.

Article abstract of DOI:10.1016/S0960-894X(02)00213-5

Further optimization of the beta-aminoester class of factor Xa (fXa) inhibitors is described culminating in the identification of 9c (FXV673), a potent and selective factor Xa inhibitor with excellent in vivo anticoagulant activity. An X-ray structure of FXV673 bound to human fXa is also presented. Based on its selectivity, potent in vivo activity and favorable pre-clinical safety profile, FXV673 was selected for further development and is currently undergoing clinical trials.

Full text of DOI:10.1016/S0960-894X(02)00213-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1671–1674
Optimization of the ꢀ-Aminoester Class of Factor Xa Inhibitors.
Part 2: Identification of FXV673 as a Potent and Selective
Inhibitor with Excellent In Vivo Anticoagulant Activity
Kevin R. Guertin,*,y Charles J. Gardner, Scott I. Klein, Allison L. Zulli, Mark Czekaj,
Yong Gong, Alfred P. Spada, Daniel L. Cheney, Sebastian Maignan,
Jean-Pierre Guilloteau, Karen D. Brown, Dennis J. Colussi, Valeria Chu,
Christopher L. Heran, Suzanne R. Morgan, Ross G. Bentley,
Christopher T. Dunwiddie, Robert J. Leadley and Henry W. Pauls*
Drug Innovation and Approval, Aventis Pharmaceuticals, Route 202-206, Bridgewater, NJ 08807, USA
Received 11 January 2002; accepted 28 February 2002
Abstract—Further optimization of the b-aminoester class of factor Xa (fXa) inhibitors is described culminating in the identification
of 9c (FXV673), a potent and selective factor Xa inhibitor with excellent in vivo anticoagulant activity. An X-ray structure of
FXV673 bound to human fXa is also presented. Based on its selectivity, potent in vivo activity and favorable pre-clinical safety
profile, FXV673 was selected for further development and is currently undergoing clinical trials. # 2002 Elsevier Science Ltd. All
rights reserved.
Factor Xa (fXa) is a pivotal serine protease situated at
the juncture of the intrinsic and extrinsic pathways of the
blood coagulation cascade. Its singular role in thrombin
activation and potentiating effects on clot formation
identify it as a target for therapeutic intervention.1 Thus,
the inhibition of fXa (in the physiologically relevant pro-
thrombinase complex) represents an attractive target for
the development of novel antithrombotic agents.2 In
response, numerous reports describing small molecule
factor Xa inhibitors have recently appeared in the litera-
ture.3 Compounds 1 and 2 are representative of the
b-aminoester class of factor Xa inhibitors developed at
Aventis.4
A fXa inhibitor may have considerable therapeutic
potential for the treatment of acute coronary syn-
dromes. In terms of clot penetration, a small, syntheti-
cally derived fXa inhibitor would provide a significant
advantage over biologically derived antithrombotics of
high molecular weight (e.g., Heparin).9 Furthermore,
there are theoretical arguments to support the potential
superiority of fXa inhibitors over direct fIIa inhibitors
such as decreased bleeding10 and elimination of rebound
thrombosis.11
Parenteral antithrombotic agents include intravenous
heparin,5 low molecular weight heparins (LMWH),5 IV
GPIIbIIIa antagonists6 and recently, direct thrombin
(fIIa) inhibitors such as IV argatroban.7 Each of the
preceding therapies have documented limitations.8
Possible indications for an intravenously-administered
fXa inhibitor include the treatment of deep vein throm-
bosis,12 unstable angina,13 adjunctive treatment with
thrombolytics (acute MI14), or in percutaneous trans-
luminal coronary angioplasty.15 Taken together, these
large patient populations present a major medical need
for improved antithrombotic therapy.
*Corresponding authors. Fax: +1-973-235-7122; e-mail: kevin.
guertin@roche.com (K. R. Guertin); fax: +1-908-231-3577; e-mail:
henry.pauls@aventis.com (H.W. Pauls).
yCurrent address: Roche Research Center, 340 Kingsland St., Nutley,
NJ 07110, USA.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00213-5

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K. R. Guertin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1671–1674
In the preceding publication,4b methyl was established
as the preferred C3 sidechain of the b-aminoester class
of fXa inhibitors. P4 optimization indicated that aro-
matic rings substituted with H-bonding and/or posi-
tively charged functions were favored, for example 2.
Herein, we report further optimization of the biaryl P4
moiety culminating in the identification of a potent and
selective factor Xa inhibitor for further development.
Table 1. SAR of P-4 modified inhibitors
Compd
Ar
Ki (nM)
fXa
5.3
flla
Trypsin
69
1
3250
2800
Chemistry
The synthesis of the P4 modified inhibitors is outlined in
Scheme 1.16 The b-aminoester intermediate 34b is cou-
pled to the isomeric pyridylphenyl carboxylic acids
which are either commercially available, or prepared by
Suzuki chemistry as previously described.17 Conversion
of the resulting nitrile intermediates 4a–c to their corre-
sponding benzamidines is carried out under standard
conditions delivering the target b-aminoester fXa inhi-
bitors 5a–c, in good yield.
5a
5b
5c
7a
0.89
8.0
0.5
1.2
51
368
76
4000
>4000
>4000
36
The phenyl-N-methylpyridinium and phenylpyridyl-
N-oxide based inhibitors require N-methylation and
N-oxidation steps, respectively, prior to final conver-
sion to their corresponding benzamidines 7a–c, 9a–c.
Inhibitors bearing the imidazoylphenyl P4 group were
prepared by coupling of aminoester intermediate 3 with
the requisite imidazolyphenyl-carboxylic acids;17 ammo-
nolysis provides the amidines 10a–c.
7b
7c
0.8
2.7
>4000
>4000
52
168
9a
9b
0.58
2.0
>4000
>4000
36
130
Discussion
Given the desirability of incorporating H-bonding and/
or positively charged centers at the distal ring of the P4
group, compounds were prepared wherein these features
were incorporated into the aromatic rings per se. The
structure–activity relationships surrounding the inhibitors
incorporating heteroaromatic rings into the P4 ligand are
outlined in Table 1. Compounds were assayed against
human fXa, thrombin and trypsin as previously descri-
bed.18 The improvement in in vitro potency observed for
5a may be attributed to the hydrogen bonding potential
9c
0.4
6.0
>4000
>4000
>4000
>4000
301
>2900
490
10a
10b
10c
46
3.0
170
Scheme 1. Reagents and conditions: (a) ArCOOH, TBTU, diisopropylamine, DMF, rt; (b) MeI, CH2Cl2, reflux; (c) (i) H2S,pyridine, Et3N, rt; (ii)
MeI, acetone, reflux; (iii) NH4OAc, MeOH, reflux; (d) (i) HCl, MeOH, rt; (ii) NH3, MeOH, reflux; (e) m-CPBA,CH2Cl2, rt.

K. R. Guertin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1671–1674
1673
Table 2. In vitro comparison of selected b-aminoester fXa inhibitors
Compd
Ratio: Ki enz./Ki fXa
2X APTT (mM)
flla
Tryp. APC Plasmin tPA
Human
Rat
1
9c
610
13
>103
>104
87
>103
>103
>104
3.9
0.41
5.1
0.80
>104 750
against the related serine proteases fIIa, APC, plasmin
and tPA and acceptable selectivity (ꢀ750-fold) against
trypsin. Clearly, the overall selectivity profile is vastly
superior to earlier b-aminoester based prototypes such as
inhibitor 1 (Table 2). Related work, published sepa-
rately,18 indicated that FXV673 is a reversible, competitive
inhibitor of factor Xa.
Figure 1. Time-to-occlusion versus dose of FXV673 in the rat FeCl2-
induced carotid artery thrombosis model. Dose is iv bolus+60min of
infusion.
FXV673 inhibited coagulation of human plasma in
vitro, doubling activated partial thromboplastin
time (APTT) at a concentration of 0.41 mM (Table 2).
This illustrates that FXV673 is an effective inhibitor of
fXa in the physiologically relevant human prothrom-
binase complex; similar results were observed in rat
plasma.
of the pyridine ring, presumably via a productive inter-
action with the bound water of the electron rich cation
hole. The cation hole, not found in thrombin, trypsin,
APC or plasmin, represents an opportunity for enhan-
cing both fXa inhibitory potency and selectivity against
these related serine proteases. Structurally related inhi-
bitors, incorporating electron deficient phenyl-N-methyl-
pyridinium (e.g., 7a–c) and phenylpyridyl-N-oxide (e.g.,
9a–c) P4 groups also showed improved potency relative to
the parent biphenyl derivative 1 (Table 1). Compounds
based on more basic imidazole rings (10a–c) offered no
advantage over the pyridyl inhibitors.
The potent fXa inhibitor FXV673 was evaluated in a rat
model of ferrous chloride-induced arterial thrombosis. A
dose-dependent increase in time to occlusion (Fig. 1) was
observed with a maximal effective dose at about 50 mg/
kg bolus and 5 mg/kg/min iv maintenance infusion. This
dose corresponded to a 40% reduction in thrombus
mass relative to control. At twice the dose, the direct
thrombin inhibitor argatroban was not significantly
different from vehicle. FXV673 produced qualitatively
In particular, the sub-nanomolar N-oxide based inhibitor
9c, designated FXV673, showed exquisite selectivity
Figure 2. X-ray of FXV673 in Factor Xa. Red spheres represent bound water.

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K. R. Guertin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1671–1674
similar results in an acute canine model of arterial and
venous electrolytic injury.
3. For comprehensive reviews, see preceding article.
4. (a) Klein, S. I.; Czekaj, M.; Gardner, C. J.; Guertin, K. R.;
Cheney, D. L.; Spada, A. P.; Bolton, S. A.; Brown, K.;
Colussi, D.; Heran, C. L.; Morgan, S. R.; Leadley, R. J.;
Dunwiddie, C. T.; Perrone, M. H.; Chu, V. J. Med. Chem.
1998, 41, 437. (b) Czekaj, M.; Klein, S. I.; Guertin, K. R.;
Gardner, C.; Zulli, A. L.; Pauls, H. W.; Spada, A. P.; Cheney,
D. L.; Brown, K. D.; Colussi, D. J.; Chu, V.; Leadley, R. J.;
Dunwiddie, C. T. Bioorg. Med. Chem. Lett. 2002, 12, 1667.
5. For a comprehensive review, see: Samama, M. M.; Bara,
L.; Gouin-Thibault, I. Drugs 1996, 52, 8.
6. Jordan, R. E.; Nakada, M. T.; Weisman, H. E. Biopharm.
Ind. Perspect. 1999, 35.
7. Hursting, M. J.; Alford, K. L.; Becker, J. C.; Brooks, R. L.;
Joffrion, J. L.; Knappenberger, G. D.; Kogan, P. W.; Kogan,
T. P.; McKinney, A. A.; Schwartz, R. P. Semin. Thromb.
Hemost. 1997, 23, 503.
8. (a) Leadley, R. J. Curr. Top. Med. Chem. 2001, 1, 151. (b)
Freedman, M. D. J. Clin. Pharmacol. 1992, 32, 196. (c)
Walenga, J. M.; Bick, R. L. Med. Clin. North Am. 1998, 82,
635. (d) Kleiman, N. S. Am. Heart J. 1999, 138, S10.
9. Weitz, J. L.; Hudoba, M.; Massel, D.; Maraganore, J.;
Hirsh, J. Biocim. Biophys. Acta 1994, 1227, 113.
10. Schmaier, A. H. Handb. Exp. Pharmacol. 2000, 140, 277.
11. Gold, H. K.; Torres, F. W.; Garabedian, H. D.; Werner,
W.; Jang, I.-K.; Khan, A.; Hagstrom, J. N.; Yasuda, T.;
Leinbach, R. C.; Newell, J. B.; Bovill, E. G.; Stump, D. C.;
Collen, D. J. Am. Coll. Cardiol. 1993, 21, 1039.
An X-ray structure of FXV673 bound to human fXa is
illustrated in Figure 2. The data reveals an extended
inhibitor conformation with the expected bridging
interaction between the benzamidine group and D189 as
well as a direct H-bond from the amidine to the car-
bonyl of G219. The phenylpyridyl-N-oxide P4 group is
inserted deep into the S4 site and there are extensive Van
der Waals contacts made between three aromatic resi-
dues (Y99, F174 and W215) from fXa and the distal
pyridine-N-oxide ring. The oxide oxygen makes an H-
bond to bound water. In addition, there is a hydrogen
bond between the inhibitor P4 amide carbonyl oxygen
and the NH of G219. In contrast to our earlier fXa
docking studies on inhibitor 1,4a the C1-carbomethoxy
substituent of FXV673 is remote from the catalytic triad
region, fitting snugly into a cleft defined by the disulfide
bridge (C191 and C220), G219, Q192 and R147. The C1
carbomethoxy carbonyl oxygen is involved in a hydrogen
bond to the Q192 backbone NH (not shown).
In conclusion, the SAR surrounding the b-aminoester
series of fXa inhibitors has been expanded, culminating
in the identification of 9c (FXV673) as a potent, compe-
titive and reversible inhibitor (Ki=0.4 nM) with a high
level of selectivity against related serine proteases. In vivo
experiments have demonstrated that this compound is
highly efficacious in animal models of arterial thrombosis.
Based on these data and a favorable preclinical safety
profile, FXV673 has been selected for further development
and is currently undergoing clinical trials.
12. Haas, S. Hemostasis 1996, 26, 39.
13. Falk, E. Circulation 1985, 71, 699.
14. Cannon, C. P. J. Thromb. Thrombolysis 1995, 2, 205.
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16. Satisfactory spectral data were obtained for all com-
pounds. Final products were purified by C18 reverse-phase
HPLC (eluant: 0.1% TFA H2O/CH3CN gradient).
17. Gong, Y.; Pauls, H. W. SynLett. 2000, 6, 829. Gong, Y.;
Pauls, H. W.; Spada, A.; Czekaj, M.; Liang, G.; Chu, V.;
Colussi, D.; Brown, K.; Goa, J. Bioorg. Med. Chem. Lett.
2000, 10, 217.
18. Chu, V.; Brown, K.; Colussi, D.; Gao, J.; Bostwick, J.;
Kasiewski, C.; Bentley, R.; Morgan, S.; Guertin, K.; Pauls,
H. W.; Gong, Y.; Zulli, A.; Perrone, M. H.; Dunwiddie, C. T.;
Leadley, R. J. Thromb. Res. 2001, 103, 309.
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