Rational design, synthesis, and biological activity of benzoxazinones as novel factor Xa inhibitors

Article abstract of DOI:10.1021/jm000074l

Inappropriate thrombus formation within blood vessels is the leading cause of mortality in the industrialized world. Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade and represents an attractive targ

Full text of DOI:10.1021/jm000074l

J . Med. Chem. 2000, 43, 4063-4070
4063
Ra tion a l Design , Syn th esis, a n d Biologica l Activity of Ben zoxa zin on es a s Novel
F a ctor Xa In h ibitor s
Danette A. Dudley,* Amy M. Bunker, Liguo Chi, Wayne L. Cody, Debra R. Holland, Diane P. Ignasiak,
Nancy J aniczek-Dolphin, Thomas B. McClanahan, Thomas E. Mertz, Lakshmi S. Narasimhan,
Stephen T. Rapundalo, J ulia A. Trautschold, Chad A. Van Huis, and J eremy J . Edmunds
Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received February 21, 2000
Inappropriate thrombus formation within blood vessels is the leading cause of mortality in
the industrialized world. Factor Xa (FXa) is a trypsin-like serine protease that plays a key
role in the blood coagulation cascade and represents an attractive target for anticoagulant
drug development. From a high-throughput in vitro mass screen of our chemical library, we
identified 4-[5-[(2R,6S)-2,6-dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-2-phenyl-2H-1,4-ben-
zoxazin-3(4H)-one (1a ) as an inhibitor of FXa with an IC50 of 27 µM. Through a combination
of SAR studies and molecular modeling, we synthesized 3-(4-[5-[(2R,6S)-2,6-dimethyltetrahydro-
1(2H)-pyridinyl]pentyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)-1-benzenecarboximida-
mide (1n ) which was a potent FXa inhibitor with an IC50 of 3 nM. This compound exhibited
high selectivity for FXa over other related serine proteases and was efficacious when dosed
intravenously in rabbit and dog antithrombotic models.
In tr od u ction
Ample evidence exists for the role of FXa inhibitors
as anticoagulants. Antistatin, a potent inhibitor of blood
coagulation FXa isolated from the Mexican leech (Hae-
menteria offcinalis), displayed antithrombotic activity
in various models of arterial and venous thrombosis.3
Other protein or polypeptide FXa inhibitors include
recombinant tick anticoagulant peptide (rTAP), which
exhibited potent antithrombotic efficacy in various
experimental models and was equal or superior to
herapin and hirudin in its action when administered
parenterally.4 In addition to these relatively high-
molecular-weight proteins which are not suitable as oral
antithrombotic agents, there also exist examples of low-
molecular-weight FXa inhibitors.5 In particular the bis-
amidine DX-9065a (Figure 1), a low-molecular-weight
synthetic FXa inhibitor, has been shown to possess
antithrombotic potential in various experimental throm-
bosis models.6 In both arteriovenous shunt and venous
stasis models, inhibition of thrombus formation was
achieved at doses that had little effect on activated
partial thromboplastin time (aPTT), indicating that DX-
9065a was effective in preventing thrombosis and hence
has therapeutic antithrombotic potential.7
Abnormal coagulation and inappropriate thrombus
formation within blood vessels precipitates many acute
cardiovascular disease states. The resulting clinical
ramifications of occlusive thrombus development, in-
cluding myocardial infarction, deep-vein thrombosis,
pulmonary embolism, and stroke, annually affect mil-
lions of people worldwide and are the leading causes of
mortality and morbidity in the industrialized world.1
While a variety of plasma proteins such as fibrinogen,
serine proteases, and cellular receptors are involved in
hemostasis, it is the abnormal regulation that disrupts
the fine balance between hemostasis and thrombosis
that leads to cardiovascular disease. Thrombin (factor
IIa) is the final serine protease in the pathway to
generate a fibrin clot and can be considered the principal
regulatory enzyme in the coagulation cascade. It serves
a pluralistic role as a positive and negative feedback
regulator in normal hemostasis. However, in some
pathologic conditions, the former is amplified through
catalytic activation of cofactors required for thrombin
generation such as factor Xa (FXa).
Factor Xa is a trypsin-like serine protease that plays
a key role in the blood coagulation cascade by ultimately
regulating the generation of thrombin by proteolysis of
prothrombin. The catalytic activity of FXa is present in
the prothrombinase complex, which is also composed of
nonenzymatic cofactor Va and calcium ions assembled
on the phospholipid membrane surface of activated
platelets or inflammatory cells adhering to the site of
vascular damage.2 This prothrombinase complex is
located at the convergence of both the intrinsic and
extrinsic coagulation pathways, and because of this key
position in the enzymatic cascade, FXa represents an
attractive target for anticoagulant drug development.
We have designed and systematically synthesized a
new class of low-molecular-weight benzoxazinone FXa
inhibitors of structural type 1, which originated from
high-volume screening of our chemical library. In this
report, we will describe the synthesis and in vitro and
8
in vivo activity of this novel series of FXa inhibitors.
Ch em istr y
The synthesis of the compounds in Table 1 proceeded
by linear stepwise processes as shown in Schemes 1-3.
Of the routes shown in Scheme 1 the alkylation of the
o-nitrophenol or o-aminophenol by the R-bromoacetic
acid derivatives 3 was the most favored approach.9,10
In general for step b, the yields were modest (∼55%)
when an excess of the sodium salt of the appropriate
* To whom correspondence should be addressed. Phone: (734) 622-
5416. Fax: (734) 622-7879. E-mail: Danette.Dudley@pfizer.com.
10.1021/jm000074l CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/27/2000

4064 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Dudley et al.
Sch em e 2. Synthesis of 3-(4-[5-[(2R,6S)-2,6-Dimethyl-
tetrahydro-1(2H)-pyridinyl]pentyl]-3-oxo-3,4-dihydro-2H-
1,4-benzoxazin-2-yl)-1-N-hydroxybenzenecarboximid-
amide (1n )a
F igu r e 1. DX-9065a and structural formula for a new class
of benzoxazinones.
Sch em e 1. Synthesis of Benzoxazinonesa
a
(a) Pd(PPh3)4, Zn(CN)2, DMF, 100 °C; (b) 1,5-dibromopentane,
NaH, DMF; (c) cis-2,6-dimethylpiperidine, DMF, 70 °C; (d)
H2NOH‚HCl, N,N-diisopropylethylamine, MeOH; (e) trifluoroacetic
anhydride; (f) H2, trifluoroacetic acid, 20% Pd/C.
Scheme 2 depicts the subsequent steps that were
employed to elaborate the intermediate benzoxazinone
9 to the final compounds 1m -1n . The bromide 9 was
converted to the nitrile 10 by treatment with a transi-
tion metal, such tetrakis(triphenylphosphine)palladium-
(0) and zinc cyanide, in DMF, typically to a temperature
of 100 °C for 24 h to afford the required product in 75%
yield.14 Alkylation of the lactam at N-1 proceeded in
reasonably good yield by generating the sodium salt
with sodium hydride in DMF and then treating with
an excess of 1,5-dibromopentane to give 11. Addition of
cis-2,6-dimethylpiperidine and warming of the reaction
mixture to 70 °C afforded the piperidine derivative after
a period of 16 h. Conversion of the nitrile 12 to the
amidine in 1n was achieved by first generating the
amidoxime 1m with an excess of hydroxylamine at room
temperature.15 In situ activation of the amidoxime by
formation of the trifluoroacetate ester 13 and then
hydrogenation readily afforded the required compound
1n . This route also demonstrates the alkylation of
benzoxazinones and the further derivatization with
amines which is applicable to the preparations of 1a -
1h and 1r -1y.
a
(a) NBS, VAZO52, CCl4; (b) o-nitrophenol sodium salt, DMSO;
(c) H2, RaNi, MeOH; (d) PBr3, Br2, MeOH; (e) o-aminophenol
hydrochloride, NaH, DMF; (f) CCl4, PbBr2, Al foil, DMF, rt; (g)
o-aminophenol hydrochloride, NaH, DMF or DMSO.
o-nitrophenol was employed in DMSO, at a slightly
elevated temperature (60-70 °C). These ethers were
then reduced with hydrogen and Raney nickel in
methanol which provided the corresponding aniline that
underwent intramolecular cyclization to directly afford
the benzoxazinone 8. In some instances, as a slightly
modified procedure, the o-aminophenol was treated
directly with the R-bromoacetic acid derivative 3 such
that the benzoxazinone 8 was generated directly, as
shown by step e.11 This sequence was particularly useful
to circumvent undesirable overreduction, which resulted
in debenzylation or in some situations dehalogenation,
for some of the derivatives of Table 1. While an alterna-
tive approach of treating the trichloromethyl carbinol
derivative 7 with o-aminophenol benefited from a large
variety of aldehydes that could easily be converted to
the carbinol, the route suffered from modest yields that
were obtained upon formation of the benzoxazinone 8
and hence was not generally employed.12 Interestingly,
while a number of reaction conditions were attempted,
the yields of the benzoxazinones never met those
described for this transformation in the literature.13
While it is apparent that nitriles may be converted
to a variety of functional groups, as depicted in Scheme
3, the generation of the imino ether with dry hydrogen
chloride in methanol was the preferred procedure for
preparing the substituted amidines 1p -1q. The well-
known methodology of proceeding to the amidine via the
thioamide followed by alkylation and treatment with
ammonia was not efficient in this series.16

Benzoxazinones as Novel FXa Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4065
Sch em e 3a
comparison of the screening lead with a known FXa
inhibitor, DX-9065a.17
Each of the analogues required multiple synthetic
steps, which did not proceed through any common
intermediates, severely restricting the number of ana-
logues that could be prepared. Therefore, the selection
of substituents was initially guided by the application
of the Topliss tree approach with the goal of gaining as
much information from the least numbers of analogues.
The Topliss approach takes into account the electronic,
lipophilic, and steric factors for substitution on a phenyl
ring using basic Hansch principles in a noncomputerized
manner.18 Fortunately, when 1f was synthesized among
others (1b-1h ), it displayed a modest improvement in
inhibitory activity suggesting that substituents on this
ring significantly affected in vitro FXa activity (IC50’s
4.9-26 µM for 1a -1h ). Further analysis of the GASP
generated overlays of 1f and DX-9065a suggested that
the 4-methoxyphenyl moiety may occupy the S1 pocket
of FXa. In fact, the first disclosure of a lipophilic moiety
which occupied the S1 pocket was described in the X-ray
of rTAP (tyrosine).19 More recently, a 4-methoxyphenyl
moiety was described as an S1 element.20 Therefore,
given the fact that an amidine substituent was common
to nearly all FXa inhibitors, we replaced the methoxy
group to afford the amidine analogue 1k . Surprisingly,
this analogue was even less active than the parent
compound 1a . However, preparation of the m-amidine
derivative 1n resulted in an almost 200-fold increase
in in vitro activity with good selectivity toward a variety
of other serine proteases (Table 2).
We then modified the chemically most accessible 2,6-
dimethyl-1-pentylpiperidine substituent of the benzox-
azinone. With the goal of retaining the basic nature of
the region, we introduced amines, alkylamines, and
various heterocycles (1t-1y) but did not elicit any
improvement in activity. We also varied the length of
the alkyl chain (1r and 1s) that attached this basic
substituent to the benzoxazinone and observed a loss
in activity, suggesting that the five-carbon chain was
optimal and implying that the cis-2,6-dimethyl-1-pen-
tylpiperidine afforded a rather unique pharmacophore.
a
(a) (i) HCl, EtOH, (ii) NH3/EtOH; (b) (i) HCl, EtOH, (ii)
morpholine; (c) (i) HCl, EtOH, (ii) 2 M methylamine in toluene;
(d) H2S, pyridine; (e) H2, RaNi, Et3N, MeOH.
Fortunately, 1n displayed marginal affinity for trypsin,
which allowed for the X-ray crystallographic determi-
nation of the binding mode of this compound. Trypsin
was used as a surrogate protein for FXa due to its
structural similarity and because crystals of FXa had
previously been shown to be very difficult to obtain.21
From the trypsin crystal structure it was observed that
the amidino moiety binds in the S1 pocket making the
classical twin-twin hydrogen bonds to Asp189 as shown
schematically in Figure 2 and stereographically in
Figure 3.22 The carbonyl group of the benzoxazinone
accepts a hydrogen bond from Gly216NH, and the aryl
ring of the benzoxazinone stacks against Gly219 and
makes van der Waals contact with the Cys191-Cys220
disulfide bridge. While the dimethylpiperidine substitu-
ent lacked any definitive interactions with trypsin, a
minimal conformational change of the flexible pentyl
chain and modeling with FXa orientated the dimeth-
ylpiperidine in the “aryl-binding site” of FXa.23 It is
presumed therefore that the piperidine ring makes a
cationic-π interaction with the protein affording en-
hanced binding. It was apparent that the trypsin/
Resu lts a n d Discu ssion
The discovery program oriented toward the identifica-
tion of antithrombotic FXa inhibitors was initiated by
conducting a high-throughput in vitro screen of our
chemical library against FXa at fixed inhibitor concen-
trations of 10 and 100 µM. Although several active
series of compounds emerged from this analysis, the
benzoxazinone 1a with an IC50 of 27 µM was selected
for further chemical exploration. The compound had a
relatively unique chemical structure that was readily
amenable to SAR studies. It was also apparent that the
compound did not contain a moiety capable of interact-
ing with the FXa Asp189 (chymotrypsinogen number-
ing) in the S1 pocket. This is an interaction typical of
known FXa inhibitors and suggested to us that the
introduction of such a pharmacophore could be expected
to improve activity. Thus, a systematic investigation of
the lead (1a ) was undertaken to determine substituents
that would improve FXa inhibitory activity. These
investigations were guided, in part, by the application
of a molecular modeling program (GASP) which allowed

4066 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Ta ble 1. Benzoxazinone Analoguesa
Dudley et al.
IC50 (µM) or % inhibition at 100 µM
compd
X
Y
Z
FXa
27
26
23
16
28
4.9
31%
30%
34
66
7.9
1.7
5.6
0.0030
0.84
2.8
thrombin
trypsin
plasmin
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
H
3,4-Cl
4-Cl
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)6
(CH2)4
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
(CH2)5
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,6-diMe-piperidinyl
cis-2,5-diMe-pyrrolidinyl
piperidinyl
19%
47%
38%
65
33%
29%
0%
30%
32%
18%
1.7
20%
19%
0.89
64%
68%
15%
5.6
2.8%
14%
46%
39
18%
2.3%
0%
12%
7.8%
8%
26%
25%
30%
23%
13%
19%
0%
20%
44%
10%
80%
29%
11%
8.4
25
32%
0%
12
38
85%
21
70%
64%
72%
2.2
2-Cl
4-CH3
4-OCH3
3,4-OCH3
3,4,5-OCH3
4-C(dS)NH2
4-C(dNH)NHOH
4-C(dNH)NH2
3-C(dS)NH2
3-C(dNH)NHOH
3-C(dNH)NH2
3-CH2NH2
3-C(dNH)NHCH3
3-C(dNH)NHmorpholinyl
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
3-C(dNH)NH2
13
5.2%
21%
0.33
7.8
32%
58%
2.1
0.30
0.76
0.99
1.1
1.5
0.068
0.012
0.048
0.053
0.24
6.3
1s
1t
1.1
0.91
3.4
2.2
60%
1.2
1u
1v
1w
1x
1y
morpholinyl
NH2
4.3
1.2
2.5
diisopropylamino
dihexylamino
0.12
1.6
1.9
a
The in vitro FXa, thrombin, trypsin, and plasmin assays were performed typically in duplicate using 14 concentrations of test substance
that bracketed the IC50
.
Ta ble 2. Selectivity of Compound 1n Against Various Serine
Proteasesa
serine protease
FXa
prothrombinase
thrombin
trypsin
plasmin
IC50 (µM) or % inhibition at 100 µM
0.0030
0.0040
0.89
0.33
8.4
activated protein c
chymotrypsin
factor VIIa
23
24%
32%
a
The in vitro FXa, thrombin, trypsin, and plasmin assays were
performed typically in duplicate using 14 concentrations of test
substance that bracketed the IC50
.
inhibitor crystal structure provided a conformation of
the inhibitor that would be expected to make similar
interaction with FXa, and thus the previously described
interactions account for the observed in vitro activity.
F igu r e 2. Schematic representation of the interaction of
compound 1n with the trypsin active site.
The in vivo studies were conducted in anesthetized
rabbits using a veno-venous shunt model which has
previously been described.26 In this thrombosis model
the test compounds were administered intravenously as
a bolus followed by a continuous infusion in the presence
of a cotton thread thrombotic stimulus. The main
endpoint of this study was the time to occlusion of the
vessel containing the cotton threads, which is typically
about 20 min in control animals. Clot weight was
significantly reduced when the test compound 1n was
administered at the highest dose associated with plasma
concentration of approximately 300 ng/mL, and only one
out of five animals occluded within a 2-h time frame
(Table 4). Thus, 1n was effective at relatively low doses
To evaluate the potential efficacy of 1n , in vitro tests
were conducted using human, dog, and rabbit plasma
to determine the effect of the compounds in the standard
coagulation tests.24 The anticoagulant status of patients
on heparin is frequently monitored ex vivo using the
aPTT assay, while the prothrombin time (PT) assay is
used for patients on oral coumarins. Interestingly, 1n
is a more potent inhibitor of FXa in human rather than
rabbit or dog plasma (Table 3). This species-dependent
inhibition of FXa is a common observation that has been
seen previously with the prototypical FXa inhibitors,
such as DX9065a.25

Benzoxazinones as Novel FXa Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4067
F igu r e 3. Stereographical representation of the interaction of compound 1n with the trypsin active site.
Ta ble 3. In Vitro aPTT and PT Assaya
Ta ble 5. Effect of Compound 1n on the Time to Occlusion in
the Dog Electrolytic Injury Modela
concn (µM) of 1n for 2-fold
prolongation of PT and aPTT
time to occlusion (min)
dose of 1n
incidence
(µg/kg/min)
(occluded/total)
artery
vein
species
PT
aPTT
ratio of PT/aPTT
0 (control)
24/24
7/8
9/10
1/10
0/10
87 ( 12
148 ( 33
167 ( 27
240 ( 0
240 ( 0
120 ( 15
125 ( 12
144 ( 22
220 ( 18
240 ( 0
rabbit
dog
human
0.33
0.57
0.17
0.15
0.19
0.34
2.2
3.0
0.5
1.25
2.5
5
a
The in vitro PT and aPTT assays were done by using pooled
plasma, and values were duplicated.
10
a
All numbers are mean ( SEM where n ) 12 in the control
group, n ) 4 in the 1.25 µg/kg/min group, and n ) 5 in the 2.5, 5,
and 10 µg/kg/min groups for both artery and vein.
Ta ble 4. Effect of Compound 1n on the Time to Occlusion in
the Rabbit Veno-Venous Shunt Model
dose of 1n
(iv bolus µg/kg +
infusion µg/kg/min)
1.25 and 2.5 µg/kg/min groups. In the group treated with
5 µg/kg/min compound 1n , 1 out of 10 vessels occluded,
while all vessels remained patent in the 10 µg/kg/min
group for the full duration of the study. Loss of blood
from the surgical incisions was significantly elevated
only at the highest dose of 10 µg/kg/min.
incidence
(occluded/total)
time to occlusiona
(min)
30 + 1
60 + 2
90 + 3
5/5
4/5
1/5
27 ( 7
37 ( 21
98 ( 22
a
All numbers are mean ( SE where n ) 5 for each of three
dose groups.
Pharmacokinetic data for an oral/intravenous cross-
over study with 1n in rats or dogs indicated oral
bioavailabilities were quite poor (e6-13%, respectively)
with terminal half-lives less than 0.5 h. While these
characteristics are unfavorable for oral administration
of an antithrombotic for chronic use, they are however
suitable for development as a parenteral for the treat-
ment of acute coronary syndromes. However, there is
also interest in developing FXa inhibitors for the treat-
ment or prevention of chronic maladies, such as deep-
vein thrombosis. While there are a limited number of
orally active FXa inhibitors, or even antithrombins, it
is readily apparent from the published SAR that oral
activity is limited in part by highly basic P1 substitu-
ents.28 In an attempt to address this issue, we prepared
a number of benzoxazinones (1m and 1o-1q) containing
P1 groups with modest pKa (10 ( 2) as calculated from
commercial software.29 Unfortunately, we observed a
substantial loss of in vitro activity with each replace-
ment of the amidine moiety, and thus further work in
this area represents a current area of focus.
supporting the potential utility of this agent as a
parenteral antithrombotic.
Compound 1n was also investigated in anesthetized
dogs using an electrolytic injury to the endothelium as
a thrombotic stimulus. In this model the efficacy of the
test compound was assessed in both arteries and veins
and was measured, in part, by recording the time to
occlusion of the vessels under study (Table 5).27 For
example, when the compound was infused continuously
at 1.25, 2.5, 5, and 10 µg/kg/min, the time to occlusion
of the femoral artery varied in a dose-dependent manner
from 150 min at the lowest dose to the maximal
duration of 240 min at the higher dose. In the vein a
similar dose response was observed. Administration of
compound 1n significantly extended the time to occlu-
sion with the two highest doses in the vein (mean
plasma concentration g 400 ng/mL) and the three
highest doses in the artery (mean plasma concentration
g 250 ng/mL). Most vessels occluded in the control and

4068 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Dudley et al.
Meth yl 2-Br om o-2-(3-br om op h en yl)a ceta te. To 3-bro-
mophenylacetic acid (10 g, 47 mmol) under argon was added
PBr3 (11.2 mL, 118 mmol) and the suspension stirred at room
temperature for 45 min. Bromine (11.1 mL, 216 mmol) was
added dropwise over 5 min. The mixture was stirred at 100
°C for 3 h and then cooled. Anhydrous MeOH (35 mL) was
added dropwise over 30 min and then the reaction mixture
was diluted with Et2O (400 mL), washed with 5% NaHCO3
(800 mL), brine (200 mL), and then dried over MgSO4. The
mixture was filtered and concentrated in vacuo to afford 13.9
It should also be noted that in addition to the low
bioavailability, this class of agents also presents a chiral
center. Throughout this work these compounds were
prepared and analyzed as racemic mixtures, and it can
be assumed that the potency of the pure enantiomer
could potentially be twice that of the mixture. In fact,
the trypsin crystal structure of 1n revealed that the S
isomer bound preferentially in the active site. Currently,
several approaches are being assessed to prepare chiral
benzoxazinones and will be described in detail in future
publications.
1
g (96%) of pure material. H NMR (CDCl3, 400 MHz): δ 7.71-
(1H, m), 7.48(2H, m), 7.25(1H, m), 5.29(1H, s), 3.80(3H, s). CI
MS M - 1 ) 304/306/308. Anal. (C9H8Br2O2) C, H, N, Br.
In summary a high-throughput screening lead was
optimized from approximately 27 µM to a low-nanomo-
lar lead. Critical to the success of this process was the
application of molecular modeling and the Topliss tree
approach, which enabled us to rationally design a series
of novel benzoxazinone inhibitors of FXa. While the
molecular modeling tools were unable to uniquely place
the amidine moiety, sufficient guidance was provided
to ultimately synthesize the lead 1n . This compound
exhibits high selectivity for the target protein FXa over
other related serine proteases such as trypsin. Finally
this compound is efficacious in dog and rabbit anti-
thrombotic models. Discovery of this novel, selective,
potent, efficacious inhibitor of FXa incorporating the
amidine moiety at S1 was a key step in our search for
a clinical candidate. Subsequent publications will de-
scribe further results in this area.
2-(3-Br om op h en yl)-3,4-d ih yd r o-2H -1,4-b en zoxa zin -3-
on e (9). To o-aminophenol hydrochloride (4.25 g, 38.9 mmol)
in DMF (30 mL) was added NaH (1.55 g, 38.9 mmol). After
bubbling and evolution of heat ceased, a solution of methyl
2-bromo-2-(3-bromophenyl)acetate (3.00 g, 9.74 mmol) in DMF
(10 mL) was added dropwise over 15 min. After evolution of
heat ceased, the mixture was stirred at room temperature for
24 h. The reaction mixture was diluted with water, and
extracted with EtOAc (3 × 200 mL). The combined organic
extracts were washed with brine (200 mL), dried with MgSO4,
filtered, and evaporated in vacuo. The residue was purified
on a silica gel column eluted with 20% EtOAc in hexane. The
product 9 was isolated 1.63 g (55%) as a solid. 1H NMR (CDCl3,
300 MHz): δ 8.76(1H, s), 7.63(1H, m), 7.43(2H, m), 7.23(1H,
m), 7.01(3H, m), 6.81(1H, m), 5.66(1H, s). CI MS M + 1 ) 306,
M - 1 ) 304. Anal. (C14H10Br1N1O2) C, H, N.
2-(3-Cya n op h en yl)-3,4-d ih yd r o-2H -1,4-b en zoxa zin -3-
on e (10). To a solution of 2-(3-bromophenyl)-3,4-dihydro-2H-
1,4-benzoxazin-3-one (9) (3.00 g, 9.87 mmol) in DMF (20 mL)
were added zinc cyanide (0.68 g, 5.79 mmol) and then tetrakis-
(triphenylphosphine)palladium(0) (0.96 g, 8 mol %). The
nitrogen-degassed solution was heated at 100 °C or 5 h, cooled
to room temperature and treated with water (50 mL). The
product was extracted into EtOAc (2 × 100 mL), washed with
brine (50 mL), dried over MgSO4, and then purified by silica
gel chromatography, eluent 50% EtOAc in hexane. This
process afforded the title compound (1.86 g, 76%) that was
Exp er im en ta l Section
Bioch em istr y. Compounds were evaluated for their ability
to inhibit the serine proteases, FXa, thrombin, trypsin, and
plasmin. In vitro kinetic assays were conducted at 37 °C using
a kinetic spectrophotometric plate reader. An optimized con-
centration of human enzyme in buffer (0.5 nM final concentra-
tion for thrombin and trypsin, and 1 nM for FXa and plasmin)
was combined with 2 µL of inhibitor dilutions (creating a final
concentration range of 1 nM to 100 µM) and preincubated for
1 h at room temperature. The assay was initiated by the
addition of an appropriate synthetic substrate (S-2765 (Phar-
macia Hepar) for FXa, CHROMOZYM TH (Boehringer Man-
nheim) for thrombin, S-2222 (Pharmacia Hepar) for trypsin,
and S-2403 (Pharmacia Hepar) for plasmin), at predetermined
2 × Km. Absorbance at 405 nm was determined over 10 min,
and percent inhibition was calculated from the slope of the
progress curves during the linear part of the time course at
each concentration. The IC50 was defined as that concentration
of test substance that inhibited 50% of the respective protease
activity. Typically a n ) 2 was performed on each compound
such that data reported is an average of the two measure-
ments.
Ch em istr y. All starting materials were obtained from
commercial sources and used without further purification
unless otherwise specified in the experimental. Proton NMR
spectra were obtained on a Varian Unity 400- or 300-MHz
spectrometer. Elemental analyses were determined by Rob-
ertson Microlit, Inc. (Madison, NJ ), and the results were within
0.4% of the theoretical values for the elements indicated. Mass
spectral data were obtained on a VG Analytical 7070 E/HF
mass spectrometer. Flash column chromatography was per-
formed on Merck silica gel 60, 230-400 mesh, purchased from
Mallinckrodt. Reactions were monitored by thin-layer chro-
matography (TLC) on Merck glass plates precoated with 0.25
mm of silica gel.
1
recrystallized from EtOH/water. H NMR (CDCl3, 300 MHz):
δ 8.50 (1H, s), 7.80-7.72(2H, m), 7.64(1H, m), 7.49(1H, m),
7.11-6.97(3H, m), 6.82(1H, m), 5.70(1H, s). CI MS M + 1 )
251, M - 1 ) 250. Anal. (C15H10N2O2) C, H, N.
4-(5-Br om op en tyl)-2-(3-cya n op h en yl)-3,4-d ih yd r o-2H-
1,4-ben zoxa zin -3-on e (11). To 2-(3-cyanophenyl)-3,4-dihydro-
2H-1,4-benzoxazin-3-one (10) (0.72 g, 2.87 mmol) in DMF (5
mL) was added NaH (0.126 g, 3.15 mmol) and the solution
was stirred at 70 °C for 15 min until bubbling stopped. To this
solution was added 1,5-dibromopentane (1.57 mL, 11.5 mmol)
and the solution was stirred at 70 °C for additional 3 h. The
solution was cooled, diluted with water, and extracted with
EtOAc (5 × 200 mL). The combined organic extracts were
washed with brine (2 × 100 mL), dried with MgSO4, filtered,
and evaporated in vacuo. The residue was purified on a silica
gel column eluted with 20% to 40% EtOAc in hexane. The
1
product 11 was isolated 0.64 g (56%) as a yellow oil. H NMR
(CDCl3, 300 MHz): δ 7.71(2H, m), 7.61(1H, m), 7.46(1H, m),
7.12-7.03(3H, m), 6.97(1H, m), 5.69(1H, s), 3.98(2H, m), 3.41-
(2H, m), 1.91(2H, m), 1.71(2H, m), 1.55(2H, m). CI MS M + 1
) 401/402. Anal. (C20H19N2O2Br1) C, H, N.
4-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id in yl]-
pen tyl]-2-(3-cyan oph en yl)-3,4-dih ydr o-2H-1,4-ben zoxazin -
3-on e (12). To 4-(5-bromopentyl)-2-(3-cyanophenyl)-3,4-dihydro-
2H-1,4-benzoxazin-3-one (11) (1.08 g, 2.70 mmol) was added
cis-2,6-dimethylpiperidine (8 mL, 60 mmol). The solution was
stirred at 70 °C for 16 h. The solution was cooled, diluted with
water, and extracted with EtOAc (3 × 200 mL). The combined
organic extracts were washed with saturated NaHCO3 (2 ×
100 mL), washed with brine (2 × 100 mL), dried with MgSO4,
filtered, evaporated in vacuo, coevaporated with toluene, and
dried under high vacuum to give 1.09 g (94%) of 12 as a yellow
oil. An analytical sample was prepared by purification by
preparative reverse phase HPLC (Vydac 218TP1022 C18,
Abbr evia tion s: DIEA, N,N-diisopropylethylamine; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOAc,
ethyl acetate; EtOH, ethanol; MeOH, methanol; NBS, N-
bromosuccinimide; RaNi, Raney nickel; TFA, trifluoroacetic
acid.

Benzoxazinones as Novel FXa Inhibitors
eluted with mixture of solvents consisting of (i) 0.1%
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4069
a
Su p p or tin g In for m a tion Ava ila ble: Characterization
data for compounds 1a -1y, average IC50, N size, standard
deviation, and crystallography data. This material is available
free of charge via the Internet at http://pubs.acs.org.
trifluoroacetic acid (TFA) in water and (ii) 0.1% TFA in CH3-
CN, gradient profile 95:5 i:ii to 60:40 i:ii over 90 min, flow rate
20 mL/min, λ ) 214 nm) and appropriate fractions were
combined and lyophilized. The powder was then dissolved in
CH3CN (2 mL) and water (1 mL) was added Amberlite IRA-
400(Cl) ion-exchange resin (5 g) and the mixture swirled for
30 min. The mixture was filtered through additional resin, and
the filtrate was lyophilized to give 31 mg of the HCl salt 12.
1H NMR (DMSO, 300 MHz): δ 7.83(2H, m), 7.70(1H, m), 7.60-
(1H, m), 7.26(1H, m), 7.08(3H, m), 5.93(1H, s), 3.97(2H, bs),
Refer en ces
(1) Vlasuk, G. P. Direct Factor Xa Inhibitors. In New Therapeutic
Agents in Thrombosis and Thrombolysis, 1st ed.; Sasahara, A.
A., Loscalzo, J ., Eds.; Marcel Dekker: New York, 1997; pp 261-
283.
(2) Rapaport, S. I.; Rao, L. V. Initiation and regulation of tissue
factor-dependent blood coagulation. Arterioscler. Thromb. 1992,
12, 1111-1121.
(3) Lapatto, R.; Krengel, U.; Schreuder, H. A.; Arkema, A.; De Boer,
B.; Kalk, K. H.; Hol, W. G. J .; Grootenhuis, P. D. J .; Mulders, J .
W. M.; Dijkema, R.; Theunissen, H. J . M.; Dijkstra, B. W. X-ray
structure of antistasin at 1.9.ANG. resolution and its modeled
complex with blood coagulation factor Xa. EMBO J . 1997, 16,
5151-5161.
2.94(4H, bm), 1.74-1.32(12H, bm), 1.23(6H, m). HPLC: tR
)
13.8 min (Beckman 235328 C18 5 µm 4.6 mm × 25 cm, eluted
with a mixture of solvents consisting of (i) 0.1% TFA in water
and (ii) 0.1% TFA in CH3CN, gradient profile 80:20 i:ii to 10:
90 i:ii over 23 min, flow rate 1.5 mL/min, λ ) 214 nm). CI MS
M + 1 ) 432. Anal. (C27H33N3O2‚HCl‚1.5H2O) C, H, N.
3-(4-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id i-
n yl]p en tyl]-3-oxo-3,4-d ih yd r o-2H-1,4-ben zoxa zin -2-yl)-1-
N-h ydr oxyben zen ecar boxim idam ide (1m ). To 4-[5-[(2R,6S)-
2,6-dim et h yt et r a h ydr o-1(2H )-pyr idin yl]pen t yl]-2-(3-cy-
anophenyl)-3,4-dihydro-2H-1,4-benzoxazin-3-one (12) (1.09 g,
2.53 mmol) in MeOH (30 mL) were added hydroxylamine
hydrochloride (0.438 g, 6.30 mmol) and DIEA (0.44 mL, 2.53
mmol). The solution was stirred at room temperature for 16
h. The solvent was evaporated in vacuo and the oil was dried
under high vacuum to give 1m in quantitative yield, which
was used without purification. HPLC: tR ) 8.4 min (Beckman
235328 C18 5 µm 4.6 mm × 25 cm, eluted with a mixture of
solvents consisting of (i) 0.1% TFA in water and (ii) 0.1% TFA
in CH3CN, gradient profile 80:20 i:ii to 10:90 i:ii over 23 min,
flow rate 1.5 mL/min, λ ) 214 nm).
3-(4-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id i-
n yl]p en tyl]-3-oxo-3,4-d ih yd r o-2H-1,4-ben zoxa zin -2-yl)-1-
N-h yd r oxytr iflu or oa cetoben zen eca r boxim id a m id e (13).
To 3-(4-[5-[(2R,6S)-2,6-dimethyltetrahydro-1(2H)-pyridinyl]-
pentyl]-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl)-1-N-hydroxy-
benzenecarboximidamide (1m ) (0.53 g, 1.14 mmol) was added
trifluoroacetic anhydride (7 mL) and the solution was stirred
at room temperature for 2 h. The solvent was removed in vacuo
(4) (a) Schaffer, L. W.; Davidson, J . T.; Vlasuk, G. P.; Siegl, P. K.
Antithrombotic efficacy of recombinant tick anticoagulant pep-
tide. A potent inhibitor of coagulation of factor Xa in a primate
model of arterial thrombosis. Circulation 1991, 84, 1741-1748.
(b) Vlasuk, G. P.; Ramjit, D.; Fujita, T.; Dunwiddie, C. T.; Nutt,
E. M.; Smith, D. E.; Shebuski, R. J . Comparison of the in vivo
anticoagulant properties of standard heparin and the highly
selective factor Xa inhibitors antistatin and tick anticoagulant
peptide (TAP) in a rabbit model of venous thrombosis. Thromb.
Haemost. 1991, 65, 257-262. (c) Sitko, G. R.; Ramjit, D. R.;
Stabilito, I. I.; Lehman, D.; Lynch, J . J .; Vlasuk, G. P. Conjunc-
tive enhancement of enzymatic thrombolysis and prevention of
thrombotic reocclusion with the selective factor Xa inhibitor, tick
anticoagulant peptide. Comparison to hirudin and heparin in a
canine model of acute coronary artery thrombosis. Circulation
1992, 85, 805-815.
(5) Al-Obeidi, F.; Ostrem, J . A. Factor Xa inhibitors. Exp. Opin.
Ther. Pat. 1999, 9, 931-953.
(6) Walenga, J . M.; J eske, W. P.; Hoppensteadt, D.; Kaiser, B. Factor
Xa inhibitors: Today and Beyond. Curr. Opin. Cardiovasc.,
Pulm. Renal Invest. Drugs 1999, 1, 13-27.
(7) Wong, P. C.; Crain, E. J .; Nguan, O.; Watson, C. A.; Racanelli,
A. Antithrombotic actions of selective inhibitors of blood coagula-
tion factor Xa in rat models of thrombosis. Thromb. Res. 1996,
83, 117-126.
(8) Berryman, K. A.; Downing, D. M.; Dudley, D. A.; Edmunds, J .
J .; Narasimhan, L. S.; Rapundalo, S. T. Preparation of benzox-
azinones and -thiazinones as serine protease inhibitors. Inter-
national Patent Application WO 99/50257, 1999.
(9) Othman, M.; Decroix, B. Synthesis of phthalimidine-3-carboxyl-
ate and benzopyrroloindolizine from N-(pyrrol-2-yl)phthalimi-
dine-3-carboxylate. Synth. Commun. 1996, 26, 2803-2809.
(10) Harwood, H. J . Reactions of the Hydrocarbon Chain of Fatty
Acids. Chem. Rev. 1962, 62, 99-154.
to give 13 as a yellow oil in quantitative yield. HPLC: tR
)
17.4 min (Beckman 235328 C18 5 µm 4.6 mm × 25 cm, eluted
with a mixture of solvents consisting of (i) 0.1% TFA in water
and (ii) 0.1% TFA in CH3CN, gradient profile 80:20 i:ii to 10:
90 i:ii over 23 min, flow rate 1.5 mL/min, λ ) 214 nm).
3-(4-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id i-
n yl]p en tyl]-3-oxo-3,4-d ih yd r o-2H-1,4-ben zoxa zin -2-yl)-1-
b en zen eca r b oxim id a m id e (1n ). To 3-(4-[5-[(2R,6S)-2,6-
dimethyltetrahydro-1(2H)-pyridinyl]pentyl]-3-oxo-3,4-dihydro-
2H -1,4-ben zoxa zin -2-yl)-1-N-h ydr oxyt r iflu or oa cet oben -
zenecarboximidamide (13) (0.57 g, 1.13 mmol) in TFA (16 mL)
was added 20% palladium on carbon (0.1 g) and the mixture
was hydrogenated at 23 °C for 48 h under 50 psi of hydrogen.
The mixture was filtered and the filter pad washed with TFA.
The combined filtrate and washings were evaporated in vacuo,
and the residue was purified by preparative reverse phase
HPLC (Vydac 218TP1022 C18, eluted with a mixture of
solvents consisting of (i) 0.1% TFA in water and (ii) 0.1% TFA
in CH3CN, gradient profile 95:5 i:ii to 60:40 i:ii over 90 min,
flow rate 20 mL/min, λ ) 214 nm) and lyophilized to give 251
mg (50%) of the bis-TFA salt of 1n . To 1n in CH3CN (2 mL)
and water (2 mL) was added Amberlite IRA-400(Cl) ion-
exchange resin (3.36 g) and the mixture swirled for 30 min.
The mixture was filtered, and the filtrate was lyophilized to
give 155 mg (26%) of the bis-HCl salt 1n . 1H NMR (DMSO,
300 MHz): δ 9.44(1H, s), 9.19(2H, s), 7.80(2H, m), 7.64(2H,
m), 7.28(1H, m), 7.11-7.01(3H, m), 5.93(1H, s), 3.97(2H, m),
3.18(2H, bs), 3.03(2H, bs), 1.75-1.36(12H, bm), 1.23(6H, m).
CI MS M + 1 ) 449. HPLC: tR ) 8.3 min (Beckman 235328
C18 5 µm 4.6 mm × 25 cm, eluted with a mixture of solvents
consisting of (i) 0.1% TFA in water and (ii) 0.1% TFA in CH3-
CN, gradient profile 80:20 i:ii to 10:90 i:ii over 23 min, flow
rate 1.5 mL/min, λ ) 214 nm). Anal. (C27H38N4O2‚2HCl‚H2O)
C, H, N, Cl.
(11) Olagbemiro, T. O.; Nyakutse, C. A.; Lajide, L.; Agho, M. O.;
Chukwu, C. E. Synthesis and reactions of 3-phenyl-3,4-dihydro-
1,4-quinoxalin-2(1H)-one and its heterocyclic analogues. Bull.
Soc. Chim. Belg. 1987, 96, 473-480.
(12) Tanaka, H.; Yamashita, S.; Yamanoue, M.; Torii, S. Reductive
addition of polyhalomethanes and their related compounds to
aldehydes and 1,2-elimination of the coupling products in a
palladium/aluminum bimetal redox system. J . Org. Chem. 1989,
54, 444-450.
(13) Gukasyan, A. Kh.; Galastyan, L. Kh.; Avetisyan, A. A. Investiga-
tions in the field of (trihalomethyl) carbinols. IV. Block-addition
reaction of aromatic 1,4-dinucleophiles to gem-dichloro epoxides
generated from aryl(trichloromethyl)carbinols. Arm. Khim. Zh.
1988, 41, 572-575.
(14) Tschaen, D. M.; Desmond, R.; King, A. O.; Fortin, M. C.; Pipik,
B.; King, S.; Verhoeven, T. R. An improved procedure for
aromatic cyanation. Synth. Commun. 1994, 24, 887-890.
(15) J udkins, B. D.; Allen, D. G.; Cook, T. A.; Evans, B.; Sardharwala,
T. E. A versatile synthesis of amidines from nitriles via ami-
doximes. Synth. Commun. 1996, 26, 4351-4367.
(16) Sall, D. J .; Afrsten, A. E.; Bastian, J . A.; Denney, M. L.; Harms,
C. S.; McCowan, J . R.; Morin, J . M., J r.; Rose, J . W.; Scarbor-
ough, R. M.; Smyth, M. S.; Um, S. L.; Utterback, B. G.; Vasileff,
R. T.; Wikel, J . H.; Wyss, V. L.; J akubowski, J . A. Use of
Conformationally Restricted Benzamidines as Arginine Sur-
rogates in the Design of Platelet GPIIb-IIIa Receptor Antago-
nists. J . Med. Chem. 1997, 40, 2843-2857.
(17) J ones, G.; Willett, P.; Glen, R. C. A genetic algorithm for flexible
molecular overlay and pharmacophore elucidation. J . Comput.-
Aided Mol. Des. 1995, 9, 532-549.
(18) (a) Topliss, J . G. Utilization of Operational Schemes for analogue
synthesis in drug design. J . Med. Chem. 1972, 15, 1006-1011.
(b) Topliss, J . G. A manual method for applying the Hansch
approach to drug design. J . Med. Chem. 1977, 20, 463-469.

4070 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Dudley et al.
(19) Wei, A.; Alexander, R. S.; Duke, J .; Ross, H.; Rosenfeld, S. A.;
Chang, C. H. Unexpected binding mode of tick anticoagulant
peptide complexed to bovine factor Xa. J . Mol. Biol. 1998, 283,
147-154.
(20) Herron, D. K.; Goodson, T., J r.; Wiley: M. R.; Weir, L. C.; Kyle,
J . A.; Ying, K. Y.; Tebbe, A. L.; Tinsley, J . M.; Mendel, D.;
Masters, J . J .; Franciskovich, J . B.; Sawyer, J . S.; Beight, D.
W.; Ratz, A. M.; Milot, G.; Hall, S. E.; Klimkowski, V. J .; Wikel,
J . H.; Eastwood, B. J .; Towner, R. D.; Gifford-Moore, D. S.; Craft,
T. J .; Smith, G. F. 1,2-Dibenzamidobenzene Inhibitors of Human
Factor Xa. J . Med. Chem. 2000, 43, 859-872.
(21) (a) Maignan, S.; Guilloteau, J .; Mikol, V.; Choi-Sledeski, Y. M.;
Becker, M. R.; Klein, S. I.; Ewing, W. R.; Pauls, H. W.; Spada,
A. P. Crystal structures of human factor Xa complexed with
potent inhibitors. Presented at the 218th American Chemical
Society National Meeting, New Orleans, LA, Aug 22-26, 1999;
Abstr. #20. (b) Renatus, M.; Bode, W.; Huber, R.; Sturzebecher,
J .; Stubbs, M. T. Structural and functional analyses of benza-
midine-based inhibitors in complex with trypsin: implications
for the inhibition of factor Xa, tPA, and urokinase. J . Med. Chem.
1998, 41, 5445-5456.
(22) (a) Bode, W.; Schwager, P. The refined crystal structure of bovine
beta-trypsin at 1.8 A resolution. II. Crystallographic refinement,
calcium binding site, benzamidine binding site and active site
at pH 7.0. J . Mol. Biol. 1975, 98, 693-717. (b) Singh, J .;
Thornton, J . M.; Snarey, M.; Campbell, S. F. The geometries of
interacting arginine-carboxyls in proteins. FEBS Lett. 1987, 224,
161-171.
19, 339-349. (b) Hara, T.; Yokoyama, A.; Morishima, Y.;
Kunitada, S. Species differences in anticoagulant and anti-Xa
activity of DX-9065a, a highly selective factor Xa inhibitor.
Thromb. Res. 1995, 80, 99-104.
(26) Rogers, K. L.; Chi, L.; Rapundalo, S. T.; Kramer, J . B.; Gallagher,
K. P. Effects of a factor Xa inhibitor, DX-9065a, in a novel rabbit
model of venous thrombosis. Basic Res. Cardiol. 1999, 94, 15-
22.
(27) (a) McClanahan, T. B.; Ignasiak, D. P.; J uneau, P.; Finkle, C.;
Winocour, P. D.; Gallagher, K. P. Antithrombotic effects of BCH
2763, a new direct thrombin inhibitor, in a canine model of
venous thrombosis. J . Thromb. Thrombolysis 1999, 7, 301-306.
(b) Ignasiak, D. P.; McClanahan, T. B.; Bousley, R. E.; J uneau,
P. L.; Gallagher, K. P. Effects of intravenous enoxaparin and
intravenous inogatran in an electrolytic injury model of venous
thrombosis in the dog. J . Thromb. Thrombolysis 1998, 6, 199-
206.
(28) (a) Al-Obeidi, F.; Ostrem, J . A. Factor Xa inhibitors. Exp. Opin.
Ther. Pat. 1999, 9, 931-953. (b) Kimball, S. D. Oral thrombin
inhibitors: Challenges and progress. Handbk. Exp. Pharmacol.
1999, 132, 367-396. (c) Rewinkel, J . B. M.; Lucas, H.; Van
Galen, P. J . M.; Noach, A. B. J .; Van Dinther, T. G.; Rood, A. M.
M.; J enneboer, A. J . S. M.; Van Boeckel, C. A. A. 1-aminoiso-
quinoline as benzamidine isostere in the design and synthesis
of orally active thrombin inhibitors. Bioorg. Med. Chem. Lett.
1999, 9, 685-690. (d) 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.
Aminoisoquinolines: design and synthesis of an orally active
benzamidine isostere for the inhibition of factor Xa. Bioorg. Med.
Chem. Lett. 1999, 9, 2539-2544.
(23) Brandstetter, H.; Kuhne, A.; Bode, W.; Huber, R.; von der Saal,
W.; Wirthensohn, K.; Engh, R. A.; X-ray structure of Active site-
inhibited clotting Factor Xa. Implications for drug design and
substrate recognition. J . Biol. Chem. 1996, 271, 29988-29992.
(24) Berryman, K. A.; Downing, D. M.; Dudley, D. A.; Edmunds, J .
J .; Narasimhan, L. S.; Rapundalo, S. T. Preparation of benzox-
azinones and -thiazinones as serine protease inhibitors. Inter-
national Patent Application WO 99/50257, 1999.
(29) Williams, A.; Kulkov, V. Web-based access to structure-based
prediction and databases for spectroscopy and physical proper-
ties. Presented at the 218th American Chemical Society National
Meeting, New Orleans, LA, Aug 22-26, 1999; Abstr. #64.
(25) (a) Tidwell, R. R.; Webster, W. P.; Shaver, S. R.; Geratz, J . D.
Strategies for anticoagulation with synthetic protease inhibitors.
Xa inhibitors versus thrombin inhibitors. Thromb. Res. 1980,
J M000074L