510
Y.-L. Chou et al. / Bioorg. Med. Chem. Lett. 13 (2003) 507–511
Concurrent with our studies of the aniline and anthra-
nilic acid rings, we investigated substitution or replace-
ment of the benzothiophene ring (Table 4). Substitution
at the 3-position of the benzothiophene was required for
potency, with chloro or methyl substituents giving the
best result (1 and 47 vs 46). The larger 3-methoxy sub-
stituent reduced potency by ca. 20-fold (49 vs 4), while
the small polar 3-hydroxy substituent caused a potency
loss of more than 10,000-fold (50). The loss in activity
of the C-3 unsubstituted analogue (46) and the
3-hydroxy analogue (50) could both be due to con-
formational effects. The chloro and methyl substituents
should cause the benzothiophene to rotate out of the
plane of the adjacent amide bond. For compounds 46 and
50, the benzothiophene would be expected to be copla-
nar with the amide bond, due to lack of steric hin-
drance (46) or hydrogen bonding between the hydroxy
group and the adjacent carbonyl group (50). Changing
from benzothiophene to thiophene caused a 30- to 400-
fold loss of potency (1 vs 48 and 4 vs 51). Some
potency was regained when a methyl sulfone or bromo
substituent was added at the 4-position of the thiophene
(52 vs 51 and 54 vs 53). These results suggest that
benzothiophene gives a better fit in the S4 pocket than
does thiophene. However, a C-4 substituent on the
thiophene may improve S4 binding. Replacement of the
benzothiophene with other ring systems such as ben-
zene, naphthalene, pyrimidine, thiazole and pyridine
(55–59), all caused loss of f Xa activity to varying
degrees, although in most cases consideration was not
given to optimal substitution of these ring systems. The
most potency was retained by the isosteric 2-naphthyl
analogue (56, Ki,app=78 nM).
References and Notes
1. (a) Kochanny, M. J.; Adler, M.; Cheeseman, S.; Chou, Y.
L.; Davey, D. D.; Eagen, K. A.; Ewing, J.; Fitch, R.; Griedel,
B. D.; Karanjawala, R.; Lee, W.; Lentz, D.; Liang, A.; Mor-
rissey, M. M.; Phillips, G. B.; Post, J.; Sacchi, K. L.; Sakata, S.
T.; Shaw, K. J.; Snider, R. M.; Subramanyam, B.; Trinh, L.;
Vergona, R.; Walters, J.; Wang, Y. X.; White, K. A.; Whitlow,
M.; Wu, S. C.; Ye, B.; Zhao, Z. 221st National Meeting of the
American Chemical Society, San Diego, CA, Apr 1–5, 2001;
American Chemical Society: Washington, DC, 2001; MEDI-
16. (b) Kochanny, M. J.; Davey, D. D.; Eagen, K. A.; Griedel,
B. D.; Karanjawala, R.; Lentz, D.; Liang, A.; Morrissey, M.
M.; Phillips, G. B.; Sacchi, K. L.; Snider, R. M.; Trinh, L.
221st National Meeting of the American Chemical Society,
San Diego, CA, Apr 1–5, 2001; American Chemical Society:
Washington, DC, 2001; MEDI-120. (c) Chou, Y. L.; Eagen,
K. A.; Griedel, B. D.; Lentz, D.; Liang, A.; Morrissey, M. M.;
Shaw, K. J.; Wu, S. C.; Kochanny, M. J. 221st National
Meeting of the American Chemical Society, San Diego, CA,
Apr 1–5, 2001; American Chemical Society: Washington, DC,
2001; MEDI-123.
2. (a) Ahmad, S; Rawala-Sheikh, R.; Walsh, P. N. Semin.
Thromb. Hemost. 1992, 18, 311. (b) Mann, K. G.; Nesheim,
M. E.; Church, W. R.; Haley, P.; Krishnaswamy, S. Blood
1990, 76, 1.
3. (a) Harker, L. A.; Hanson, S. R.; Kelly, A. B. Thromb.
Haemostasis 1995, 74, 464. (b) Hara, T.; Yokoyama, A.;
Tanabe, K.; Ishihara, H.; Iwamoto, M. Thromb. Haemost.
1995, 74, 635.
4. (a) Sanderson, P. E. J. Annu. Rep. Med. Chem. 2001, 36, 79.
(b) Rai, R.; Sprengeler, P. A.; Elrod, K. C.; Young, W. B.
Curr. Med. Chem. 2001, 8, 101. (c) Zhu, B. Y.; Scarborough,
B. M. Annu. Rep. Med. Chem. 2000, 35, 83. (d) Vacca, J. P.
Curr. Opin. Chem. Biol. 2000, 4, 394. (e) Ewing, W. R.; Pauls,
H. W.; Spada, A. P. Drugs Future 1999, 24, 771.
5. Compound 1 was originally purchased from Maybridge
Chemical Company. A recent publication from Axys Pharma-
ceuticals has appeared detailing their initial SAR investigations
of this compound: Shrader, W. D.; Young, W. B.; Sprengeler,
P. A.; Sangalang, J. C.; Elrod, K.; Carr, G. Bioorg. Med. Chem.
Lett. 2001, 11, 1801. Our independent results described herein
are consistent with the findings of the Axys group.
6. A detailed description of the identification and character-
ization of compound 1 is the subject of another publication:
Liang, A. M.; Light, D. R.; Kochanny, M.; Rumennik, G.;
Trinh, L.; Lentz, D.; Post, J.; Morser, J.; Snider, M. Biochem.
Pharmacol. In press.
7. Enzyme assay procedures.11 The activities of human f Xa,
human thrombin and bovine trypsin were determined kineti-
cally as the initial rate of cleavage of a peptide p-nitroanilide
by the enzyme. The assay was performed at room temperature
in flat-bottom microtiter plates in a final volume of 200 mL.
The reaction mixture consisted of 50 mM Tris–HCl, 150 mM
NaCl, 2.5 mM CaCl2, and 0.1% polyethylene glycol 6000, pH
7.5, with enzyme and substrate at the following concen-
trations: (1) f Xa assay: 0.04–1 nM f Xa and 164 mM S-2222;
(2) thrombin assay: 16 nM thrombin and 300 mM S-2302; and
(3) trypsin assay: 16 nM bovine trypsin and 127 mM S-2266.
Standard techniques with at least four substrate dilutions were
used to determine the Km for a given enzyme and substrate.
The substrate concentration listed is equal to the Km. Controls
without the test inhibitors or with a reference compound were
also run in each assay plate. Enzyme was incubated with test
compounds for 10 min; the reaction was then started by the
addition of the substrate. Reaction rates were determined by
measuring the rate of the absorbance change at 405 nm in a
ThermoMax microplate reader (Molecular Devices Corp.,
Sunnyvale, CA, USA).
Compounds 1, 4, and 20 were tested for anticoagulant
activity using the in vitro prothrombin time (PT) assay.
Despite their f Xa inhibitory activity, none of the com-
pounds prolonged PT 2-fold at concentrations up to 500
mM. This result was attributed to the poor solubility of
these compounds, along with their high lipophilicity,
which likely results in high plasma protein binding.
Related results have been reported for a similar series of
lipophilic f Xa inhibitors and for a series of lipophilic
thrombin inhibitors.8c,10 In these reports, potency of
compounds in PT (f Xa inhibitors) or in vitro activated
partial thromboplastin time (APTT, thrombin inhibi-
tors) assays did not correlate solely with activity against
f Xa or thrombin, but was additionally a function of
lipophilicity.
We have explored structure–activity relationships
around the novel non-amidine f Xa inhibitor 1 by sys-
tematic modifications of each of the aryl rings. Small
hydrophobic substituents were found to be optimal at
C-3 on the benzothiophene ring. On the central ring,
halogen or methyl substitution at C-5 is critical for high
f Xa potency, and a second substituent may be intro-
duced at the 3-position. The only significant increase in
potency was obtained by adding a chloro or bromo
substituent to the 4-position of the aniline ring. These
substitutions resulted in subnanomolar non-amidine
f Xa inhibitors. Further optimization of this template
will be the subject of future publications.