Isoxazolines and Isoxazoles as Factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00097-4

3,4,5-Trisubstituted isoxazolines (2) and isoxazoles (3) were prepared and evaluated for their in vitro and in vivo antithrombotic efficacy. They were compared to 3,5,5-trisubstituted isoxazolines (1) for Factor Xa selectivity and potency. They were also compared in an arterio-venous (A-V) shunt model of thrombosis. (C) 2000 DuPont Pharmaceuticals Company. Published by Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00097-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 685±689
Isoxazolines and Isoxazoles as Factor Xa Inhibitorsy
James R. Pruitt,* Donald J. Pinto, Melissa J. Estrella, Lori L. Bostrom,
Robert M. Knabb, Pancras C. Wong, Matthew R. Wright and Ruth R. Wexler
The DuPont Pharmaceuticals Company, PO Box 80500, Wilmington, Delaware 19880-0500, USA
Received 25 October 1999; accepted 15 December 1999
AbstractÐ3,4,5-Trisubstituted isoxazolines (2) and isoxazoles (3) were prepared and evaluated for their in vitro and in vivo
antithrombotic ecacy. They were compared to 3,5,5-trisubstituted isoxazolines (1) for Factor Xa selectivity and potency. They
were also compared in an arterio-venous (A-V) shunt model of thrombosis. # 2000 DuPont Pharmaceuticals Company. Published
by Elsevier Science Ltd. All rights reserved.
Factor Xa (fXa) catalyzes the production of thrombin
from prothrombin and sits at the junction of the intrin-
sic and extrinsic pathways of the coagulation cascade.1
It has recently been suggested that fXa inhibitors may
be more e€ective as antithrombotic agents than direct
inhibitors of thrombin and may have less bleeding risk,
leading to a better safety-ecacy ratio.2
and 9) appear to be roughly equivalent in potency. All
the compounds are selective inhibitors of fXa relative to
thrombin and trypsin. We believe that H-bond accep-
tors at the 5-position of the regioisomeric isoxazolines
(1 and 2) enhance potency.
The isoxazole in Table 2 may have di€erent require-
ments for substituent R because of its ¯at structure.
Charged amino (10) and bulky tri¯uoromethyl (11)
groups are not well tolerated. Methoxymethyl (12) and
hydroxymethyl derivatives (13) show much greater
potency for fXa. Remarkably, the unsubstituted iso-
xazole ring (SA862) provides the greatest improvement
in potency. We believe that with an isoxazoline core, the
R group hydrogen bonds to the enzyme and orients the
amide bond. With the isoxazole core, this in¯uence on
the amide bond may be unnecessary and the R group
may be unable to obtain an optimal hydrogen bond to
the enzyme.
We have previously published on isoxazoline bisamidine
inhibitors of fXa.3 Further optimization of this series
resulted in the isoxazoline-5-carboxamides (1).4 These
compounds are very potent inhibitors of fXa, selective
against other serine proteases and have been shown to
inhibit thrombus formation as e€ectively as thrombin
inhibitors. It was our desire to discover what aspects of
the isoxazoline core were necessary for activity. A recent
report from our laboratories has shown pyrrolidine and
isoxazolidine benzamidines to be potent inhibitors of
fXa.5 In this paper we describe our successful e€orts to
improve activity by changing the point of attachment of
the amide moiety and subsequently aromatizing the
isoxazoline ring, eliminating all chiral centers.
Molecular models of the isoxazolines and isoxazoles in
the active site of the enzyme were compared using
Insight II6 and a model of fXa based on the X-ray
crystal structure of fXa determined by Tulinsky.7 Both
molecules have bidentate hydrogen bonds and electro-
static binding to the Asp189 residue. Since these
molecules do not interact with Ser 195, this interaction
is crucial for activity. Compound 7, in magenta, and
SA862, in white, take di€erent pathways to the S4
region of the active site with SA862 more closely
approaching the solvent accessible surface near Tyr99
and Gly216. This closer approach to the enzyme may
explain the enhanced potency of SA862. Each molecule
is able to position the terminal phenyl ring for a strong
edge-to-face interaction with Trp215, a consistent feature
Table 1 shows a comparison of the fXa inhibition for
the 3,4,5-trisubstituted isoxazolines (2) relative to the
3,5,5-trisubstituted isoxazolines (1).4 For two pairs of
entries, the methyl (4) and methoxymethyl (6) derivatives
show improved fXa inhibition relative to their 3,5,5-tri-
substituted isomers. The tetrazoylmethyl derivatives (8
*Corresponding author. Tel.: +1-302-695-8341; fax: +1-302-695-
3404; e-mail: james.r.pruitt@dupontpharma.com
yPresented in part at the 216th ACS National Meeting, Boston, MA,
23±27 August, 1998, MEDI-077.
0960-894X/00/$ - see front matter # 2000 DuPont Pharmaceuticals Company.
Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00097-4

686
J. R. Pruitt et al. / Bioorg. Med. Chem. Lett. 10 (2000) 685±689
Table 1. Comparison of the fXa activity of isoxazoline 4- and 5-carboxamides
Compound
Structure
R
fXa Ki (nM)
Thrombin Ki (nM)
Trypsin Ki (nM)
4
A
B
A
B
A
B
Me
Me
1.8
11.0
0.55
3.4
2.3
1.6
4500
5800
4600
2400
5700
900
110
120
190
70
180
91
53
6
CH2OMe
CH2OMe
CH2-tetrazole
CH2-tetrazole
73
8
93
of all our inhibitors. The overall modeled orientation of
SA862 is maintained in the X-ray crystal structure
determined in bovine trypsin. However, the amide bond
is not in conjugation with the isoxazole ring and is
oriented towards His57 rather than Gly216 in the model
(Figs. 1 and 2).
were administered to dogs intravenously and their pro-
®le was observed (Table 4). The two isoxazoles tested
had higher clearance and volumes of distribution than
the representatives from the isoxazoline class. SA862
had a signi®cantly longer half-life than the other three
compounds tested.
The 3,5,5-trisubstituted isoxazolines showed enhanced
fXa potency when the internal phenyl ring was replaced
with a pyridyl ring.4 Table 3 describes the results of
these same changes with the isoxazole core. Unlike the
3,5,5-trisubstituted isoxazolines, the phenylpyridine (14)
does not improve fXa potency relative to the biphenyl
(12). However, as with the 3,5,5-trisubstituted iso-
xazolines,4 replacing the sulfonamide (SA862) with a
methyl sulfone (15) resulted in a compound with similar
potency.
In the rabbit A-V shunt model of thrombosis (Table 5),
the isoxazoles were compared to the 3,4,5- and 3,5,5-
trisubstituted isoxazolines. In this model,8 a plastic
tubular shunt, containing a thrombogenic silk thread, is
placed between the femoral artery and vein. After 40
min, the resulting clots are removed and their weights
determined. An ID50 can be derived by determining the
doses required to diminish the clot size by 50%. These
data were compared to the relative fXa Ki in rabbit. The
potencies of each of the inhibitors for fXa in the rabbit
blood were within a 2-fold window. This explains the
In a pharmacokinetic comparison of the 3,5,5-trisub-
stituted isoxazolines with the isoxazoles, compounds
Table 2. SAR of isoxazole 4-carboxamides
Compound
R
fXa
Ki (nM)
Thrombin
KI (nM)
Trypsin
Ki (nM)
10
11
12
13
NH2
CF3
CH2OMe
CH2OH
H
3.4
9.5
0.63
0.50
0.15
4000
11000
3800
1900
2000
117
400
76
50
21
Figure 1. Modeling of 7 vs SA862 in factor Xa.
SA862

J. R. Pruitt et al. / Bioorg. Med. Chem. Lett. 10 (2000) 685±689
687
tight range of activity seen in the A-V shunt model,
implying that each of these compounds is a potent
antithrombotic.
Scheme 1 shows the synthetic route used to prepare
the 3,4,5-trisubstituted isoxazolines. The oxime of
the commercially available 3-cyanobenzaldehyde was
chlorinated9 and the crude product reacted with a
3-substituted acrylate. This [3+2] cycloaddition deter-
mined the relative stereochemistry.3 Trimethylalumi-
num promoted amide bond formation10 was followed
by amidine formation via the methyl imidate.11
In Scheme 2, the general synthetic route to the iso-
xazoles is outlined. A b-ketoester was initially reacted
Figure 2. SA862 X-ray structure in bovine trypsin.
Table 3. Substituent e€ects on biphenyl system of isoxazole
Compound
R
X
Y
fXa Ki (nM)
Thrombin Ki (nM)
Trypsin Ki (nM)
12
14
SA862
15
CH2OMe
CH2OMe
H
N
CH
CH
SO2NH2
SO2NH2
SO2NH2
SO2Me
0.63
0.75
0.15
0.10
3800
12000
2000
540
76
200
21
H
H
25
Table 4. Isoxazole versus isoxazolinea dog pharmacokinetic data
Compoundb
Structure
R
fXa
Ki (nM)
CL
(L/h/kg)
t1/2
(h)
Vdss
(L/g)
12
SA862
6
8
A
A
B
B
CH2OMe
H
CH2OMe
CH2-tetrazole
0.63
0.15
0.55
2.3
1.7
1.5
0.99
0.49
1.2
4.3
1.2
1.0
0.85
1.7
0.38
0.24
aAll isoxazolines are racemic.
bCompounds infused at 1 mg/kg/h iv.
Table 5. Comparison of the rabbit A-V-shunt antithrombotic e€ect
a
Compound
Structure
Human fXa Ki (nM)
Rabbit fXa Ki (nM)
ID50 (mmol/kg/h)
SA862
6
A
B
C
0.15
0.55
3.4
0.40
0.64
0.80
0.31
0.21
0.26
73b
aID50s' were determined as described in refs 4a,b.

688
J. R. Pruitt et al. / Bioorg. Med. Chem. Lett. 10 (2000) 685±689
Scheme 1. General synthesis of isoxazolines.
Scheme 2. General synthesis of isoxazoles.
with diazomethane,12 followed by triethylamine and
the chlorooxime (formed from the NCS reaction with
the oxime in Scheme 1) to provide the appropriate
regioisomeric isoxazole in moderate yield.13 Amide
bond formation promoted by trimethylaluminum and
amidine formation proceeded as in Scheme 1.
References and Notes
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to a planar isoxazole core further enhances anity but
substitution on the heterocycle is not well tolerated. In
dogs, while the clearance was moderate, the half-life for
SA862 was attractive. All series were shown to have
comparable antithrombotic activity in the A-V-shunt
rabbit model. Further elaboration of these ®ndings is
ongoing at DuPont Pharmaceuticals.
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Acknowledgements
The authors would like to acknowledge Dr. Richard S.
Alexander for determining the X-ray crystallographic
structure. The authors would also like to thank Dale E.
McCall, Joseph M. Luettgen and Andrew W. Leamy for
obtaining compound binding data, and Carol Watson
and Earl J. Crain for performing in vivo studies.
7. Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.;
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