J. J. Cui et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2925–2930
2929
(OBA) ranking of lead candidates decreased as a func-
tion of the P1-arginine surrogate in the following order:
3Am6Bio (NC22, pKa=8.6)ꢅ3Am5Indaz (NC10,
pKa=3.6)>3Am6Indaz (NC11,13, pKa=3.8)>BzImid
(NC32,33, pKa=5.8)>Indole (NC37, ꢀnon-basic)
ꢅ5Indaz (NC1, ꢀnon-basic) >3Am7[N]6Bio (NC23,
pKa=4.7)>3OH5Indaz (NC41, pKa=6.3, OH form).
Although inhibitory potency was modest with our P1-3-
aminoindazole (3Am5Indaz, NC10) and 3-aminobenzi-
soxazole (3Am6Bio, NC22) prototypes, relative OBA
profiles in fasted or fed dogs dosed at 1 mg/kg were
quite intriguing. The impact of P1-regioisomers on inhib-
itory potency and OBA was pronounced in both series;
compare NC10 versus NC11 and NC21 versus NC22.
This empirical PK information, coupled with our prior
SAR5ꢂ7 and literature precedent,4,8,9 led us to pursue a
variety of novel second generation targets featuring
tethered P4-(substituted)aromatic groups judiciously
combined with the optimal P1-arginine surrogates iden-
tified above. This exercise resulted in the identification
of the highly potent and selective inhibitors NC17–20
and NC24–31, which demonstrated excellent PK prop-
erties in dogs comparable or superior to the reference
compounds L374,087 and L375,378 (Table 1). Incor-
poration of other leading bicyclic heteroaromatic P1-
arginine surrogates (BzImid, Indole, 3Am7[N]6Bio,
3OH5Indaz) afforded potent and selective inhibitors in
vitro, however they generally lacked useful in vivo
efficacy.
candidates NC17–20 and NC24–31 emerged that
demonstrated excellent PK properties. Numerous
favorable active-site interactions coupled with optimal
physical properties are deemed as critical factors for
conferring high potency, specificity, and useful PK
properties in this class of inhibitors.
Acknowledgements
We gratefully acknowledge L. Truong and P. R. Ber-
gum for in vitro pharmacological studies, and T. K.
Brunck, O. E. Levy and S. Y. Tamura for stimulating
discussions regarding antithrombotic inhibitor targets.
References and Notes
1. (a) Coleman, R. W.; Marder, V. J.; Salzman, E. W.; Hirsch,
J. In Hemostasis and Thrombosis, Basic Principles and Clinical
Practice, 3rd ed.; Colman, R. W., Hirsch, J., Marder, V. J.,
Salzman, E. W., Eds.; J. B. Lippincott: Philadelphia, 1994;
Chapters 1, 9, 57, and 80–86. (b) Vlasuk, G. P. Thromb. Hae-
mostas. 1993, 70, 212.
2. (a) Topol, E. Am. Heart J. 2001, 142, S22. (b) Vlasuk, G. P.
In New Therapeutic Agents in Thrombosis and Thrombolysis;
Sasahara, A. A, Loscalzo, J., Eds. Marcel Dekker: New York,
1997; Chapter 15.
3. (a) Recent reviews: (a) Hauptmann, J.; (special issue). Eur.
J. Clin. Pharm. 2002, 57, 751. (b) Sanderson, P. E. J. Annu.
Rep. Med. Chem. 2001, 36, 79.
Potency and selectivity in our series results from
numerous key binding interactions at each of the S1–S3
specificity pockets in the thrombin active site (Fig. 1).
As confirmed by an X-ray crystal structure of the
NC17–fIIa complex,10,11 inhibitor binding occurs in a
normal substrate-like mode, with the rigid P1-3-ami-
noindazole moiety participating in salt-bridge interac-
tions with Asp189 and water-mediated hydrogen-
bonding with Tyr228 at the S1 specificity pocket. Rela-
ted P1-analogues may partake in additional hydro-
phobic interactions with Val213 at S1. Other canonical
b-sheet H-bonds and van der Waals interaction at S2
and S3 are conserved. Although many structural varia-
tions are represented in our 44 targets, it appears that
overall drug potency and efficacy is governed by the
appropriate balancing of several factors, including
choice of P4-hydrophobe, P3-heterocycle and especially
the P1-arginine mimic. In general, P1-arginine surro-
gates with pKa’sꢀ3.6–8.6 confer the best OBA in this
series.
4. Recent FIIa inhibitors: (a) Linusson, A.; Gottfries, J.; Ols-
son, T.; Oernskov, E.; Folestad, S.; Norden, B.; Wold, S. J.
Med. Chem. 2001, 44, 3424. (b) Steinmetzer, T.; Schweinitz,
A.; Kunzel, S.; Wikstrom, P.; Hauptmann, J.; Sturzebecher, J.
J. Enz. Inhib. 2001, 16, 241. (c) Coburn, C. A.; Rush, D. M.;
Williams, P. D.; Homnick, C.; Lyle, E. A.; Lewis, C. D.;
Lucas, B. J.; DiMuzio-Mower, J. M.; Juliano, M.; Kreuger,
J. A.; Vastag, K.; Chen, I. W.; Vacca, J. P. Bioorg. Med.
Chem. Lett. 2000, 10, 1069.
5. (a) Minami, N. K.; Reiner, J. E.; Semple, J. E. Bioorg. Med.
Chem. Lett. 1999, 9, 2625. (b) Reiner, J. E.; Lim-Wilby, M. S.;
Brunck, T. K.; Uong, T. H.; Goldman, E. A.; Abelman, M. A.;
Nutt, R. F.; Semple, J. E.; Tamura, S. Y. Bioorg. Med. Chem.
Lett. 1999, 9, 895. (c) Owens, T. D.; Semple, J. E. Bioorg.
Med. Chem. Lett. 1998, 8, 3683. (d) Semple, J. E.; Rowley,
D. C.; Owens, T. D.; Minami, N. K.; Uong, T. H.; Brunck,
T. K. Bioorg. Med. Chem. Lett. 1998, 8, 3525.
6. (a) Semple, J. E.; Levy, O. E.; Minami, N. K.; Owens, T. D.;
Siev, D. V. Bioorg. Med. Chem. Lett. 2000, 10, 2305. (b)
Tamura, S. Y.; Levy, O. E.; Reiner, J. E.; Uong, T. H.; Gold-
man, E. A.; Brunck, T. K.; Semple, J. E. Bioorg. Med. Chem.
Lett. 2000, 10, 745. (c) Ho, J. Z.; Levy, O. E.; Gibson, T. S.;
Nguyen, K.; Semple, J. E. Bioorg. Med. Chem. Lett. 1999, 9,
3459.
7. (a) Ho, J. Z.; Gibson, T. S.; Semple, J. E. Bioorg. Med.
Chem. Lett. 2002, 12, 743. (b) Reiner, J. E.; Siev, D. V.; Ara-
ldi, G. L.; Cui, J. J.; Ho, J. Z.; Reddy, K. M.; Mamedova, L.;
Vu, P. H.; Lee, K. S.; Minami, N. K.; Gibson, T. S.; Ander-
son, S. M.; Bradbury, A. E.; Nolan, T. G.; Dixon, S. A.; Ma,
M. G.; Semple, J. E. Bioorg. Med. Chem. Lett. 2002, 12, 1203.
8. Sanderson, P. J.; Dorsey, B. D.; Naylor-Olsen, A. M.;
Gardell, S. J.; Lynch, J. J.; Shafer, J. A.; Vacca, J. P. Bioorg.
Med. Chem. Lett. 1998, 8, 817.
In conclusion, our investigations on novel thrombin
inhibitor scaffolds incorporating P3-heterocyclic dipep-
tide surrogates along with perusal of reference inhibitors
led to the design, synthesis, and elucidation of potent,
selective thrombin inhibitors NC1–NC44 that feature a
range of weakly basic bicyclic P1-arginine mimics. After
identifying the P1-3-aminobenzisoxazole (3Am6Bio)
and 3-aminoindazole (3Am5Indaz) pharmacophores
from cassette oral dosing studies in dogs, we optimized
potency/PK parameters by judicious combination of
these arginine surrogates with several optimal P4-(sub-
stituted)aromatic groups. Potent and selective inhibitor
9. Sanderson, P. E. J.; Lyle, T. A.; Dorsey, B. D.; Gardell,
S. J.; Shafer, J. A.; Vacca, J. P. J. Med. Chem. 1998, 41,
4466.