Design, synthesis, and SAR of amino acid derivatives as factor Xa inhibitors1

Article abstract of DOI:10.1016/S0960-894X(01)00585-6

A series of potent and selective factor Xa inhibitors was synthesized using various readily available amino acids as central templates. The most potent compound displays IC₅₀ of 3 nM.

Full text of DOI:10.1016/S0960-894X(01)00585-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2947–2950
Design, Synthesis, and SAR of Amino Acid Derivatives as Factor
Xa Inhibitors1
Ting Su,* Yanhong Wu, Brandon Doughan, Zhaozhong J. Jia, John Woolfrey,
Brian Huang, Paul Wong, Gary Park, Uma Sinha, Robert M. Scarborough and
Bing-yan Zhu
COR Therapeutics, Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA
Received 28 June 2001; accepted 27 August 2001
Abstract—A series of potent and selective factor Xa inhibitors was synthesized using various readily available amino acids as cen-
tral templates. The most potent compound displays IC50 of 3 nM. # 2001 Elsevier Science Ltd. All rights reserved.
Thrombin, a plasma protease, plays a central role in
both venous and arterial thrombosis. It has been pos-
tulated that efficient regulation of thrombosis may be
achieved by regulation of thrombin. The current strate-
gies employed involve reduction of thrombin generation
as well as inhibition of the proteolytic activity of
thrombin. The prothrombinase complex is the sole site
of thrombin formation in the vasculature2 and factor
Xa (fXa) is the key component of the complex. Once
assembled into the prothrombinase complex with
cofactor Va and calcium ions on a phospholipid surface,
fXa converts prothrombin to thrombin. In recent years,
fXa has become a major target enzyme for new ther-
apeutic agents with potential for treatment of arterial
and venous thrombosis.3
benzamidine factor Xa inhibitors incorporating benza-
midine at P1 and this novel biphenylsulfonamide at P4
will be discussed.
Compounds 5–21 were readily prepared as shown in
Scheme 1. BOP coupling of 40-amino[1,10-biphenyl]-2-
tert-butylsulfonamide 35 with various N-Boc protected
amino acids, followed by BOC removal and subsequent
coupling to 3-cyanobenzoic acid afforded nitrile inter-
mediates 4. Two procedures for amidine formation were
used. One consisted of sequential reaction of the nitrile
We have previously reported the design and synthesis of
various dibasic fXa inhibitors using amino acids such as
3-OH-proline, m-amidinophenylalanine, and 2,3-dia-
mino propionic acid as templates (1 in Fig. 1).4 To dis-
cover novel inhibitors with improved bioavailability, we
focused our synthetic efforts on reducing the overall
basicity and enhancing the potency of this series of
compounds. Quan et al.5 has reported isoxazoline-con-
taining fXa inhibitors (2 in Fig. 1) in which the biphen-
ylsulfonamide moiety was designed to interact with the
S4 aryl-binding domain of the fXa active site. In this
report, the design, synthesis, and in vitro structure–
activity relationships of amino acid-based mono-
*Corresponding author. Fax: +1-650-244-9287; e-mail: tsu@corr.com
Figure 1. Design of amino acid-based inhibitors.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00585-6

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T. Su et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2947–2950
Table 1 (continued)
12
13
14
15
16
17
18
0.842
0.055
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.81
>10
1.77
>10
>10
2.15
4.39
0.888
0.0348
0.0348
0.0313
>10
0.003
Scheme 1. Synthesis of compounds 5–21. (a) NH2tBu, TEA, DCM;
(b) BuLi, {(CH3)2CHO}3B, 1 N HCl, THF; (c) p-bromoaniline,
Pd(Ph3P)4, Bu4NBr, NaOH, toluene; (d) Biphenyl 3, BOP, DIEA,
DMF, rt; (e) 50% TFA/DCM; (f) 3-cyanobenzoic acid, BOP, DIEA,
DMF; (g) H2S, TEA/Pyr; (h) MeI, acetone, reflux; (i) NH4OAc,
MeOH, reflux; (j) Neat TFA, rt or (g) NH2OH.HCl, TEA; (h) AcOH,
Ac2O; (i) 10% Pd/C, H2; (j) Neat TFA.
19
20
0.0187
0.0967
Table 1. In vitro activity for compounds 5–21
21
1.78
>10
>10
with hydrogen sulfide, methyl iodide, and ammonium
acetate.6 The second method consisted of reaction of the
nitrile with hydroxyamine, acetic anhydride and hydro-
genation. Final removal of the t-butyl group afforded
free sulfonamides 5–21.
Compd
X
IC50, mM
Thrombin
>10
fXa
Trypsin
5
6
0.851
>10
We also prepared aminoindazole, aminobenzisoxazole,
and aminoisoquinoline analogues (22–25) using the
published procedures.7
0.676
0.635
0.337
0.0762
1.44
>10
>10
>10
>10
>10
>10
6.07
7.93
5.17
>10
>10
5.64
The enzyme inhibition constants for compounds 5–25
toward fXa and the structurally related serine proteases
thrombin and trypsin are summarized in Tables 1 and 2.8,9
7
8
The initial compound (5) prepared in this series contained
a glycyl residue, which displayed only modest fXa inhi-
bitory activity. Replacement of the glycyl residue with
isoleucyl or b-cyclohexylalanyl residue afforded 6 and 7,
which were comparable to 5 in fXa inhibitory activity.
Replacement of the glycyl residue with more hydro-
phobic phenylglycyl residue afforded 8 with 2.5-fold
enhancement in the potency. Its homologue phenyl ala-
nyl-containing analogue 9 exhibited 5-fold increase in
activity when comparing with 8. While the glutamic acid
analogue 10 had diminished inhibitory potency relative
to 5, its benzyl and cyclohexyl esters 11 and 12 had
9
10
11
0.644

T. Su et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2947–2950
Table 2. In vitro activity for compounds 22–25: P1 modifications
2949
Compd
Factor Xa IC50, mMa
>10
Thrombin IC50, mMa
>10
22
23
24
25
3.45
>10
2.77
>10
>10
>10
similar activity compared to 5. These data suggest that a
hydrophobic side chain is preferred and acidic side
chain is not preferred at the central position. Aspartic
acid cyclohexyl ester-containing analogue 13 was sig-
nificantly more potent (15-fold) than its homologue 12.
the S4 pocket bordered by Phe174, Trp215, and Tyr99.
Hydrogen bonding between this sulfonamide moiety
and the carboxylate side chain of Glu97 helps hold the
ligand in the active site. Additional hydrogen bonding
interactions can be observed between the backbone
carbonyl of Gly216 and the amide hydrogens in the
central position of the ligand. In our model, the benzene
ring of the d-phenylglycine (15) is accommodated in the
lipophilic ‘disulfide’ pocket as described by Jones et al.10
This interaction is not prefered with the enantiomer
derived from l-phenylglycine (8). In this case, the
To examine the stereochemical effect of the amino acid
residues employed, several d-amino acids were incorpo-
rated into the series. d-isoleucine-containing analogue
14 is 19-fold more potent than l-isoleucine-containing
analogue 6, d-phenylglycine-containing analogue 15 is
10-fold more potent than l-phenylglycine-containing
analogue 8 and d-phenylalanine-containing analogue 16
is 2.5-fold more potent than l-phenylalanine-containing
analogue 9. These data suggest an enzyme preference
for the R-configuration at the a-position of these amino
acids. Very recently, Jones et al.10 published a series of
potent phenylglycine containing benzamidine carbox-
amides fXa inhibitors based on a lead compound they
selected using their proprietary PRO-SELECT module.
In that module, they predicted that lipophilic d-amino
acids can pick up an additional interaction with ‘dis-
ulfide’ pocket adjacent to S1, comprising residues
Gln192, Cys191, Cys220 and Gly218. The lipophilic d-
amino acid derivatives were synthesized and found to be
the preferred central unit in their series. Molecular mod-
eling of our two phenylglycine enantiomers (8 and 15)
(Fig. 2) in the active site of fXa shows the benzamidine in
the S1 pocket with a bidentate interaction with Asp189
while the biphenylsulfonamide moiety projects towards
Figure 2. Proposed binding mode of compound 15 in the factor Xa
active site. The Connelly surface coloring represents proximal atoms
(red, blue, white, and teal identify oxygen, nitrogen, carbon and
hydrogen, respectively). The P1 and P4 binding pockets are labeled, as
are critical residues on the protein.

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T. Su et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2947–2950
central templates. These readily available amino acids
gave us rapid access to the critical structure–activity
information within this class of compounds. Hydro-
phobic interactions between the side chain of the amino
acid residue and fXa and the orientation of the side
chain played important role for enhancing the potency
of this series of inhibitors. Further exploration of amino
acid templates aimed at improving pharmacokinetic prop-
erties will be the subject of additional communications
from our laboratories.
Figure 3. Overlap of the lowest energy docked conformations of
compound 18 and compound 19 (green) in the factor Xa active site.
The side chains of residues Asp189 and Glu97 are shown for perspec-
tive, and residues associated with compound 19 are colored green.
Docked conformations of compound 18 were consistently more stable
than the most favorable binding conformations of compound 19.
References and Notes
1. For preliminary account of this work, see: Su, T.; Wu, Y.;
Doughan, B.; Jia, Z. J.; Woolfrey, J.; Huang, B.; Wong, P.;
Sinha, U.; Park, G.; Scarborough, R. M.; Zhu, B.-Y. Abstracts
of Papers, Part 2, 221st National Meeting of the American
Chemical Society, San Diego, CA, April 1–5, 2001; American
Chemical Society: Washington, DC, 2001; MEDI 061.
2. Mann, K. G.; Nesheim, M. E.; Church, W. R.; Haley, P. E.;
Krishnaswamy, S. Blood 1990, 76, 1.
3. (a) Zhu, B.-Y.; Scarborough, R. M. Annu. Rep. Med. Chem.
2000, 35, 83. (b) Zhu, B.-Y.; Scarborough, R. M. Curr. Opin.
Cardiovasc. Pulm. Renal Invest. Drugs 1999, 1, 63. (c) Fevig,
J. M.; Wexler, R. R. Annu. Rep. Med. Chem. 1999, 34, 81. (d)
Scarborough, R. M. J. Enzyme Inhibit 1998, 14, 15. (e) Ripka,
W.; Brunck, T.; Stansens, P.; LaRoche, Y.; Lauwereys, M.;
Lambeir, A. M.; Lasters, I.; DeMaeyer, M.; Vlasuk, G.; Levy,
O.; Miller, T.; Webb, T.; Pearson, T. D. Med. Chem. 1995, 30,
88. (f) Claeson, G. Blood Coagulat. Fibrinolysis 1994, 5, 411.
4. Wu, Y.; Yang, W.; Doughan, B.; Tran, K.; Sinha, U.;
Scarborough, R. M.; Su, T. Abstracts of Papers, Part 2, 219th
National Meeting of the American Chemical Society, San
Francisco, CA, March 26–30, 2000; American Chemical
Society: Washington, DC, 2000; MEDI 190.
5. (a) Quan, M. L.; Liauw, A. Y.; Ellis, C. D.; Pruitt, J. R.;
Carini, D. J.; Bostrom, L. L.; Huang, P. P.; Harrison, K.;
Knabb, R. M.; Thoolen, M. J.; Wong, P. C.; Wexler, R. R. J.
Med. Chem. 1999, 42, 2752. (b) Quan, M. L.; Ellis, C. D.;
Liauw, A. Y.; Alexander, R.; Knabb, R. M.; Lam, G. N.;
Wong, P. C.; Wexler, R. R. J. Med. Chem. 1999, 42, 2760.
6. Su, T.; Naughton, M. A.; Smyth, M. S.; Rose, J. W.; Arf-
sten, A. E.; McCowan, J. R.; Jakubowski, J. A.; Wyss, V. L.;
Ruterbories, K. J.; Sall, D. J.; Scarborough, R. M. J. Med.
Chem. 1997, 40, 4308.
hydrogen binding to the backbone carbonyl is greatly
inhibited by the change in conformation necessary to
place the phenyl group in the lipophilic ‘disulfide’
pocket.11
To determine whether the conformational restriction
within the central unit would modulate activity, the
glycyl residue was replaced by several cyclic amino
acids. The modifications, which included the incorpora-
tion of a proline, afforded analogue 17 with loss of
activity. However, incorporation of the tetra-
hydroisoquinoline-3-carboxyl group generated this ser-
ies’ most potent compound 18, with 283-fold enhanced
activity in the fXa inhibitory assay relative to the cor-
responding glycine-containing analogue 5. Interestingly,
the d-isomer of this bulky amino acid 19 is less potent
than the corresponding l-isomer 18. Molecular model-
ing of these two enantiomers suggests that favorable P1
region binding factors are somewhat disrupted by
inverting the stereocenter from d- (18) to l- (19) (Fig. 3).
Introduction of an H-bonding hydroxy group to the tet-
rahydroisoquionline ring resulted in analogues 20 and 21
with significantly reduced activity for both isomers.
As shown in Table 1, all compounds displayed good
selectivity for fXa over thrombin and trypsin. High
selectivity against a panel of serine proteases including
tissue plasminogen activator (tPA), activated protein C
(APC) and plasmin, was also observed for these com-
pounds (data not shown). For example, compound 15
has IC50 values of 0.0348 mM for Xa, >180 mM for
tPA, 6.56 mM for APC and 18.1 mM for plasmin. 18 has
IC50 values of 0.003 mM for Xa, >180 mM for tPA, 73
mM for APC and 77.9 mM for plasmin. The pharmaco-
kinetic properties of the most potent analogues 15 and
18 were evaluated in Sprague–Dawley rats. The oral
bioavailability of 15 and 18 was found to be less than
5%. In order to increase the bioavailability of these
monobenzamidine leads, amidine groups in 8, 15, and
18 were replaced by aminoindazole (22 and 23), amino-
benzisoxazole (24) and aminoisoquinoline (25). Similar
approaches have been used by other laboratories12
where potent compounds have been reported. However,
the replacement within our series resulted in a more
than 100 to 1000-fold drop in fXa inhibitory potency.
7. Lam, P. Y.; Clark, C. G.; Dominguez, C.; Fevig, J. M.;
Han, Q.; Li, R.; Pinto, D. J.-P.; Pruitt, J. R.; Quan, M. L. WO
patent 9857951; Chem. Abstr. 1999, 130, 66494.
8. Sinha, U.; Ku, P.; Malinowski, J.; Zhu, B.-Y.; Scarborough,
R. M.; Marlowe, C. K.; Wong, P. W.; Lin, P. H.; Hollenbach,
S. J. Eur. J. Pharmacol. 2000, 395, 51.
9. Betz, A.; Wong, P. W.; Sinha, U. Biochemistry 1999, 38,
14582.
10. Jones, S. D.; Liebeschuetz, J. W.; Morgan, P. J.; Murray,
C. W.; Rimmer, A. D.; Roscoe, J. M. E.; Waszkowycz, B.; Welsh,
P. M.; Wylie, W. A.; Young, S. C.; Martin, H.; Mahler, J.; Brady,
L.; Wilkinson, K. Bioorg. Med. Chem. Lett. 2001, 11, 733.
11. Computational results were accomplished using Sybyl
(Tripos, Inc.) associated modules incorporating MMFF94
force field and charges, as well as the SA_DOCKING module
of InsightII (Accelrys) along with the cff91 force field and
charges.
12. Choi-Sledeski, Y. M.; Becker, M. R.; Green, D. M.;
Davis, R.; Ewing, W. R.; Mason, H. J.; Ly, C.; Spada, A.;
Liang, G.; Cheney, D.; Barton, J.; Chu, V.; Brown, K.;
Colussi, D.; Bentley, R.; Leadley, R.; Dunwiddie, C.; Pauls,
H. W. Bioorg. Med. Chem. Lett. 1999, 9, 2539.
In summary, we have synthesized a series of potent and
selective fXa inhibitors using various amino acids as