Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3113
(9) Bonow, R. O.; Carabello, B.; de Leon, A. C. et al. ACC/AHA
Guidelines for the management of patients with valvular heart
disease. J. Heart Valve Dis. 1998, 7, 672-707.
(10) Nicolini, F. A.; Lee, P.; Rios, G.; Kottke-Marchant, K.; Topol, E.
J. Combination of platelet fibrinogen receptor antagonist and
direct thrombin inhibitor at low dosis markedly improves
thrombolysis. Circulation 1994, 89, 1802-1809.
(11) Sabatine, M. S.; Tu, T. M.; Jang, I.-K. Combination of a direct
thrombin inhibitor and a platelet glycoprotein IIb/IIIa blocking
peptide facilitates and maintaines reperfusion of platelet-rich
thrombus with alteplase. J. Thromb. Thrombolysis 2000, 10,
189-196.
(12) Wallenga, J. M.; Jeske, W. P.; Wallis, D. E.; Bakhos, M.; Lewis,
B. E.; Leya, F.; Fareed, J. Clinical experience with combined
treatment of thrombin inhibitors and GP IIb/IIIa inhibitors in
patients with HIT. Semin. Thromb. Hemostasis 1999, 25, Suppl.
1, 77-81.
(13) Lincoff, A. M.; Bittl, J. A.; Harrington, R. A. et al. Bivalirudin
and provisional glycoprotein IIb/IIIa blockade compared with
heparin and planned glycoprotein IIb/IIIa blockade during
percutaneous coronary intervention-REPLACE-2 randomized
trial. JAMA 2003, 289, 853-863.
(14) Morphy, R.; Kay, C.; Rankovic, Z. From magic bullets to designed
multiple ligands. Drug Discovery Today 2004, 9, 641-651.
(15) Church, F. C.; Phillips, J. E.; Woods, J. L. Chimeric antithrombin
peptide-Characterization of an Arg-Gly-Asp (RGD)-containing
and hirudin carboxyl terminus-containing synthetic peptide. J.
Biol. Chem. 1991, 266, 11975-11979.
Table 1. Biological Activity of 1,4-Benzoxazin-3(4H)-one
Derivatives: Inhibition of Serine Proteases Thrombin, Factor
Xa, and Trypsin, and Inhibition of Fibrinogen Binding to GP
IIb/IIIa Receptors
Ki FIIa Ki trypsin Ki FXa selectivity IC50 GP IIb/IIIa
(µM)
(µM)
(µM) FIIa/trypsin
(µM)
9
38.6
53.8
12.1
5.30
5.5
22.3
31.6
20.6
26.8
19.2
25.7
8.8
0.14
0.10
0.69
0.57
0.5
0.973
3.94
2a
10a
10b 21.3
8.4
6.84
12.1
10.5
17.7
5.5
7.30
10c
11
14
15
16
20
1a
21.6
49.4
16.3
20.6
23.3
3.7
3.66
0.4
0.206
5.19
0.3
9.6
21.1
54.1
54.6
0.5
59.05
3.94
46.2
16
2
4.2
24.63
1.64
14.9
26.5
>200
-
a Acetate salt.
0.21 µM) range. Compounds 1, 2, 9-11, 14-16, and 20
also inhibited thrombocyte aggregation induced by ADP
or collagen (results not presented), with compound 9
exerting the highest inhibition [IC50 (ADP) ) 63 µM;
IC50 (collagen) ) 85 µM].
(16) Owen, T. J.; Broersma, R. J., Jr; Krstenansky, J. L. Trifunctional
antithrombin and antiplatelet peptides. WO9429349, 1994.
(17) Wienen, W.; Nar, H.; Ries, U. J.; Priepke, H. W. M.; Hauel, N.
H.; Stassen, J. M. Pharmacological characterization of BIBR
830ZW: A novel, low molecular weight, synthetic combined
inhibitor of thrombin, Factor Xa and platelet aggregation.
Presented at the 18th Congress of the International Society of
Thrombosis and Haemostasis, July 6-12, 2001, Paris. Thromb.
Haemostasis 2001, Suppl.: Abstr. P756.
(18) (a) Docking was performed with AutoDock Tools version 3.0.5.
(b) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Har,
T. W. E.; Belew; R. K.; Olson, A. J. Automated docking using a
Lamarckian genetic algorithm and an empirical binding free
energy function. J. Comput. Chem. 1998, 19, 1639-1662.
(19) (a) A P3 carboxylic group is present in some thrombin inhibitors,
e.g. melagatran19b and dabigatran,19c in order to increase oral
bioavailability of these compounds with a basic P1 benzamidine
moiety. (b) Evans, H. C.; Perry, C. M.; Faulds; D. Ximelagatran/
melagatran - A review of its use in the prevention of venous
thromboembolism in orthopaedic surgery. Drugs 2004, 64, 649-
678. (c) Eriksson; B. I., Dahl, O. E. Prevention of venous
thromboembolism following orthopaedic surgery. Clinical po-
tential of direct thrombin inhibitors. Drugs 2004, 64, 577-595.
(20) HyperChem (Release 5.01 for Windows, Hypercube, Inc. 1996).
(21) 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.
(22) Shridhar, D. R.; Gandhi, S. S.; Rao, S. K. A Facile synthesis of
2-alkyl(aryl)-6- and -7-nitro-3-oxo-3,4-dihydro-2H-1,4-benzox-
azines Synthesis 1982, 986-987.
The docking experiments showed that the benzami-
dine and benzoxazinone moieties of the target com-
pounds fit well into the thrombin S1 and S2 pockets,
respectively, whereas the interactions with the S3
binding pocket are not optimal. To increase both the
anticoagulant and antiaggregatory potencies of this type
of compounds, further optimization of the P3 part of
molecules is in progress. This is expected to result in
compounds with improved thrombin inhibitory and
fibrinogen receptor antagonist potencies.
In conclusion, we describe a novel class of potential
antithrombotic compounds, capable of acting both as
thrombin inhibitors and fibrinogen receptor antagonists.
Such composite antithrombotic agents, which still re-
quire potency and selectivity optimization, constitute a
promising approach in the search for new antithrom-
botics which could replace the currently used combined
antithrombotic therapy.
Supporting Information Available: Experimental pro-
cedures and spectroscopic data of intermediates and final
compounds are available free of charge via the Internet at
(23) Rutar, A.; Kikelj, D. Selective alkylation of 3-oxo-3,4-dihydro-
2H-1,4-benzoxazine-2-carboxylates. Synth. Commun. 1998, 28,
2737-2749.
(24) Chiou, S.; Shine, H. J. A simplified procedure for preparing 3,5-
di-substituted-1,2,4-oxadiazoles by reaction of amidoximes with
acyl chlorides in pyridine solution. J. Heterocycl. Chem. 1989,
26, 125-128.
(25) Bolton, R. E.; Coote, S. J.; Finch, H.; Lowdon, A.; Pegg, N.;
Vinader, M. V. 3-Substituted-1,2,4-oxadiazolin-5-one; a useful
amidine protecting group. Tetrahedron Lett. 1995, 36, 4471-
4474.
(26) (a) Cheng, Y.-C.; Prusoff, W. H. Relationship between the
inhibition constant (KI) and the concentration of the inhibitor
which causes 50% inhibition (I50) of an enzymatic reaction.
Biochem. Pharmacol. 1973, 22, 3099-3108. (b) Lottenberg R.;
Hall J. A.; Blinder M.; Binder E. P.; Jackson C. R. The action of
thrombin on peptide para-nitroanilide substrates - substrate
selectivity and examination of hydrolysis under different reaction
conditions. Biochim. Biophys. Acta 1983, 742, 539-557.
(27) Addicks, E.; Mazitchek, R.; Giannis, A. Synthesis and biological
investigation of novel tricyclic benzodiazepinedione-based RGD
analogues. ChemBioChem 2002, 3, 1078-1088.
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