Design, synthesis, and biological activity of potent and selective inhibitors of blood coagulation factor Xa

Article abstract of DOI:10.1021/jm0497491

Factor Xa (FXa) has materialized as a key enzyme for the intervention of the blood coagulation cascade and for the development of new antithrombotic agents. FXa is the lone enzyme responsible for the production of thrombin and therefore is an attractive t

Full text of DOI:10.1021/jm0497491

J . Med. Chem. 2004, 47, 4089-4099
4089
Design , Syn th esis, a n d Biologica l Activity of P oten t a n d Selective In h ibitor s of
Blood Coa gu la tion F a ctor Xa
J . Adam Willardsen,†,‡ Danette A. Dudley,† Wayne L. Cody,† Liguo Chi,§ Thomas B. McClanahan,§
Thomas E. Mertz,§ Ronald E. Potoczak,§ Lakshmi S. Narasimhan,| Debra R. Holland,|
Stephen T. Rapundalo, and J eremy J . Edmunds*,†
Departments of Chemistry, Cardiovascular Therapeutics, Discovery Technologies, and Cardiovascular Molecular Sciences,
Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received March 30, 2004
Factor Xa (FXa) has materialized as a key enzyme for the intervention of the blood coagulation
cascade and for the development of new antithrombotic agents. FXa is the lone enzyme
responsible for the production of thrombin and therefore is an attractive target for the control
of thrombus formation. We have designed and synthesized a unique series of quinoxalinone
FXa inhibitors. This series resulted in 3-{4-[5-((2S,6R)-2,6-dimethylpiperidin-1-yl)pentyl]-3-
oxo-3,4-dihydroquinoxolin-2-yl}benzamidine (35) with 0.83 nM activity against FXa and
excellent selectivity over similar serine proteases. An X-ray crystal structure of compound 35
bound to trypsin along with molecular modeling has led to a predicted binding conformation
of compound 35 in FXa. Compound 35 has also been proven to be efficacious in vivo in both
the rabbit veno-venous shunt and dog electrolytic injury models. In addition, it was shown
that compound 35 did not significantly increase bleeding times in a rabbit model except at the
highest doses and plasma concentrations were elevated in a dose dependent manner following
a bolus dose and continuous intravenous infusion.
In tr od u ction
phospholipid membrane to create the prothrombinase
complex. This complex is then responsible for the
conversion of prothrombin (FII) into thrombin (FIIa).6
Thrombin proceeds to catalyze the formation of the
fibrin clot. The inhibition of FXa provides for a small,
but hemostatically important, amount of thrombin to
be produced.7 This provides evidence for favorable
benefit-to-risk properties for FXa inhibitors and makes
the development of a direct FXa inhibitor an appealing
method for the control of thrombotic diseases.
We have previously discussed a new class of potent
and selective inhibitors of FXa, which originated from
a high-throughput screen (HTS) of our chemical library.8
Herein, we will report on modifications to the benzoxa-
zinone skeleton, which was identified from our HTS
screen, along with the in vitro and in vivo data that led
to the clinical candidate compound 35, a quinoxalinone
based FXa inhibitor, with a p-hydroxybenzamidine
binding in the S1 pocket.9
Cardiovascular disease is the leading cause of death
in the United States, and within the cardiovascular
disease class, heart disease is the primary source of
mortality.1 In 1996, myocardial infarction, stroke, deep
vein thrombosis, and pulmonary embolism accounted
for approximately two million deaths in the United
States alone.2 Inappropriate thrombus formation is a
common cause of the above clinical conditions, and
therefore there has been a large effort devoted to the
discovery of new antithrombotic therapies.3 The coagu-
lation cascade is composed of a complex series of plasma
proteins, serine proteases, and cellular receptors. Each
of these helps play a delicate balancing act, through
positive and negative feedback controls, between throm-
bosis and hemostasis.
Recently, significant effort has been put forth to
develop inhibitors of the key serine proteases that are
part of the coagulation cascade. Thrombin was the
initial target, and many small molecule inhibitors have
been reported.4 Being that Factor Xa (FXa) is at the
convergence of the intrinsic and extrinsic pathways of
the coagulation cascade, it represents an attractive
target for the control of thrombus formation and inhibi-
tors for this enzyme have also been pursued.5 In the
route to the physiological formation of a clot, FXa
combines with Factor Va (FVa) and calcium ions on a
Ch em istr y
The molecules of interest replaced the oxygen atom
of the benzoxazinone ring of 18 and 210 with sulfur,
nitrogen, and carbon (Figure 1). The desired sulfur
analogues 22a and 22b of the previously reported
compounds 1 and 2 were synthesized as outlined in
Schemes 1, 2, and 6. 3-Cyanobenzaldehyde was treated
with aluminum foil and lead(II) bromide in carbon
tetrachloride to furnish the trichlorocarbinol 4 (Scheme
1).11 In the presence of base and 2-aminothiophenol, 4
was readily cyclized to the benzothiazinone 5a .11,12 The
desired target compound was then obtained via the
standard route outlined in Scheme 6. The amide nitro-
gen was first alkylated with 1,5-dibromopentane to yield
* To whom correspondence should be addressed. Phone: (734) 622-
4984, fax: (734) 622-3909, e-mail: jeremy.edmunds@pfizer.com.
† Department of Chemistry.
‡ Present address: Myriad Genetic Pharmaceuticals Inc., 320
Wakara Way, Salt Lake City, UT 84108.
§ Department of Cardiovascular Therapeutics.
| Department of Discovery Technologies.
Department of Cardiovascular Molecular Sciences.
10.1021/jm0497491 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/26/2004

4090 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Willardsen et al.
Sch em e 3a
F igu r e 1.
Sch em e 1a
a
Reagents and conditions: (a) 3-Hydroxyphenylacetic acid,
Ac2O, Et3N, 150 °C; (b) H2, Pd/C, MeOH; (c) N-phenyltrifluo-
romethanesulfonimide, NaH, THF; (d) Zn(CN)2, Pd(PPh3)4, DMF,
100 °C.
Sch em e 4a
a
Reagents and conditions: (a) Al foil, PbBr2, CCl4, DMF; (b)
2-(HS)C6H4NH2, NaH, DMF.
Sch em e 2a
a
Reagents and conditions: (a) 2-Hydroxyphenylacetic acid,
Ac2O, Et3N, 150 °C; (b) H2, Pd/C, MeOH; (c) Br2, CS2, CH2Cl2; (d)
BnBr, Cs2CO3, DMF; (e) CuCN, DMF, 150 °C.
Sch em e 5a
a
Reagents and conditions: (a) Al foil, PbBr2, CCl4, DMF; (b)
2-HSC6H4NH2, NaH, DMF; (c) CuCN, DMF, 160 °C.
20a . In the cases outlined in Scheme 6, only a small
amount of O-alkylated product was observed, which was
easily removed via column chromatography. Displace-
ment of the alkyl bromide of 20a with cis-2,6-dimeth-
ylpiperidine resulted in 21a in excellent yields.
Although there are many reported methods available
for the formation of amidines,13,14 it was found that
proceeding through the amidoxime was usually the most
advantageous. The amidoxime was formed by treating
the benzonitrile with hydroxylamine, followed by acy-
lation with trifluoroacetic anhydride. Acylation resulted
in cyclization to produce an oxadiazole, which when
subjected to hydrogenation over palladium on carbon
revealed the amidine 22a .15 The p-hydroxyamidine
derivative of 22a was obtained in a similar manner;
trichlorocarbinol 7 was converted to the benzothiazinone
8 as discussed, previously (Scheme 2). By heating the
arylbromide 8 in a mixture of cuprous cyanide and
DMF, the bromide was displaced to give the nitrile 5b.
The intermediate 5b was taken on using the same
procedure as described before, with one note: as seen
in Scheme 6, the benzyl ether was cleanly removed
during the hydrogenation of the oxadiazole to give the
second desired dihydrobenzothiazinone analogue 22b.For
the tetrahydroquinolinone series 22c and 22d, the
carbon skeleton was assembled in a slightly different
manner (Schemes 3 and 4). Treating 3-hydroxypheny-
lacetic acid with 2-nitrobenzaldehyde (9) and acetic
anhydride gave compound 10 (Scheme 3).16 Reduction
a
Reagents and conditions: (a) 1,2-Phenylenediamine, KOH,
H2O, 60 °C; (b) DDQ, toluene, 120 0C; (c) CuCN, DMF, 150 °C.
of the aryl nitro group resulted in spontaneous cycliza-
tion to yield 11 as a mixture of enantiomers. Phenol 11
was converted to the corresponding triflate and coupled
with zinc cyanide using Pd(PPh3)4 to give the core
structure of 5c.17 For the 4-hydroxyamidine analogue,
a mixture of 2-hydroxyphenylacetic acid and 2-nitroben-
zaldehyde gave the lactone 14 (Scheme 4).16 Again,
reduction of the nitro group of 14 yielded the cyclized
product 15. Electrophilic bromination resulted in the
bromide 16, which was converted to the nitrile 5d with
cuprous cyanide as described previously for 5b. Tet-
rahydroquinolinone analogues 5c,d were transformed
to the desired amidine analogues 22c,d as shown in
Scheme 6.
For the dihydroquinolinone series, 27a and 27b,
conversion of 3-bromo-1H-quinolin-2-one (23)18 to the
nitrile 25a via a palladium(0)-mediated coupling fol-
lowed by alkylation of the amide nitrogen, as previously
outlined for other analogues, led to complex reaction

Inhibitors of Blood Coagulation Factor Xa
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4091
Sch em e 6a
Sch em e 8a
a
Reagents and conditions: (a) DDQ, toluene 80 °C.
Sch em e 9a
a
Reagents and conditions: (a) Br(CH2)5Br, NaH, DMF, 70 °C;
(b) cis-2,6-dimethylpiperidine, DMF, 70 °C; (c) H2NOH‚HCl, DIEA,
MeOH; (d) TFAA; (e) H2, Pd/C, TFA.
Sch em e 7a
a
Reagents and conditions: (a) cis-2,6-Dimethylpiperidine, DMF,
75 °C; (b) H2NOH‚HCl, DIEA, MeOH; (c) TFAA; (d) H2, Pd/C, TFA.
plished with cuprous cyanide as shown previously
(Scheme 2).
The final quinoxalinone derivative 35 was synthesized
in a slightly different manner to the above examples as
shown in Scheme 10. The benzaldehyde analogue 28
was hydrocyanated with potassium cyanide to yield
compound 29.20 Under Pinner conditions (HCl, ethanol
followed by NH3), 29 was converted into the R-hydroxy
ester 30.14 Swern conditions were used to oxidize the
hydroxyl functionality to the R-ketoester 31. Condensa-
tion of the ester 31 and 1,2-phenylenediamine resulted
in the parent quinoxalinone 32. Due to the previously
mentioned cuprous cyanide methodology leading to
many byproducts, a palladium(0)-mediated coupling
between aryl bromide 32 and zinc cyanide was used to
install the aryl nitrile functionality of 33. Since the
previously mentioned methods for alkylation of the
quinoxalinone nucleus resulted in poor yields, it was
found that using cesium carbonate and DMF resulted
in a nearly quantitative yield of a 1:1 mixture on N-
and O-alkylated products. The N-alkylated component
was separated via column chromatography and carried
on to the piperidine derivative 34. Formation of the
amidine via the hydroxylamine method vida infra
resulted in a complex reaction mixture; therefore, Pin-
ner conditions were used to introduce the amidine
functionality. Finally, the phenolic benzyl ether was
removed by hydrogenation to give the desired quinox-
alinone derivative 35.
a
Reagents and conditions: (a) Br(CH2)5Br, NaH, DMF, 70 °C;
(b) (3-cyanophenyl)boronic acid, (Ph3P)4Pd, NaHCO3, toluene H2O,
EtOH.
mixtures. When the amide nitrogen of 23 was first
alkylated with dibromopentane to give 24, the subse-
quent palladium(0) coupling proceeded cleanly to give
25a (Scheme 7).19 As shown in Scheme 8, an intermedi-
ate from the tetrahydroquinolinone series 20d was
treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) to give 25b for the dihydroquinolinone series of
compounds. Again, 25a ,b were carried on to the desired
products by addition of cis-2,6-dimethylpiperidine and
formation of the amidine to give 27a ,b (Scheme 9).
The quinoxalinone, nitrogen analogue, 5e was ob-
tained via the addition of 1,2-phenylenediamine to a
solution of 17 (Scheme 5). Upon treatment of intermedi-
ate 18 with DDQ, the bromide 19 was obtained. Con-
version of the bromide 19 tothe nitrile 5e was accom-

4092 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Willardsen et al.
Sch em e 10a
Ta ble 1. Selectivity Data for Specified Compounds as
Evidenced by the Inhibition of Various Serine Proteases
FXa
thrombin
trypsin
compd
X
Y
Ki (nM)
IC50 (µM)
IC50 (µM)
1
2
22a
22b
22c
22d
O
O
S
S
CH2
CH2
H
OH
H
OH
H
OH
3.0 (IC50
0.16
67
2.3
60.5
12.9
)
2.0
1.0
1.3
3.6
0.9
4.8
0.5
0.4
1.0
0.7
0.3
0.7
Ta ble 2. Selectivity Data for Specified Compounds as
Evidenced by the Inhibition of Various Serine Proteases
FXa
thrombin
trypsin
compd
X
Y
Ki (nM)
IC50 (µM)
IC50 (µM)
22e
27a
27b
35
N
H
H
OH
OH
18.9
177.0 (IC50
23.5
2.9
10.7
4.8
2.0
0.3
0.7
2.5
CH
CH
N
)
0.8
3.5
carbon and nitrogen, this would allow for the design of
inhibitors lacking the troublesome asymmetric center.
For the benzothiazinone derivatives 22a and 22b, the
affinity for FXa was somewhat diminished (Table 1).
Interestingly, X-ray crystallography analysis in the
trypsin active site revealed that the R enantiomer was
the preferred configuration for this molecule. The
increased radius of the sulfur resulted in van der Waals
interactions with the Cys192-Cys220 disulfide bridge
and caused repulsion of 22a and 22b from the active
site. Inversion of the asymmetric center enables the
phenyl amidine to maintain the two-on-two hydrogen
bond with Asp189. In the case of the quinolinone
derivatives 22c and 22d , the binding to FXa was
reduced, as was the selectivity over other serine pro-
teases. Crystallization of 22d with trypsin reveled that
it was bound in the active site in a fashion very similar
to 2. To our surprise, this crystallization of 22d with
trypsin and modeling studies in FXa did not reveal
reasonable indications as to why there was a decrease
in affinity or selectivity. It is assumed that entropic
factors associated with these saturated species are
responsible for the loss of affinity for FXa. Although,
the SAR on the previous compounds was important,
they still contained the problematic asymmetric center,
and therefore this problem still needed to be addressed.
When the oxygen atom in the core structure of 1 and
2 is replaced by a methine, dihydroquinoxalinone 27a ,b,
it was shown that there is at least a 150-fold loss of
activity against FXa (Table 2). These compounds also
show a large loss in selectivity over similar serine
proteases. Exchanging the oxygen for nitrogen to give
the quinoxalinones 22e and 35, a loss in activity of 20-
and 5-fold respectively was seen. These results were
promising; quinoxalinone 35, although less potent than
2, still maintained sub-nanomolar activity. More im-
portantly, this molecule lacks the asymmetric center.
Another important consideration is that 35 maintains
a 390- and 275-fold selectivity over thrombin and
trypsin, respectively.
a
Reagents and conditions: (a) KCN, AcOH, MeOH; (b) HCl,
Dioxane, MeOH, Et2O, 0 °C; (c) H2O, dioxane; (d) DMSO, (COCl)2,
Et3N, CH2Cl2, -78 °C; (e) 1,2-phenylenediamine, MeOH, 65 °C;
(f) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C; (g) Br(CH2)5Br, Cs2CO3,
DMF, 0 °C-RT; (h) cis-2,6-dimethylpiperidine, DMF, 75 °C; (i)
HCl, EtOH; (j) NH3, MeOH; (k) H2, Pd/C, TFA.
Resu lts a n d Discu ssion
The aim of this program was to develop an anti-
coagulant therapy that utilized a selective inhibitor of
coagulation FXa. This research was initiated by con-
ducting an in vitro high throughput screen of our
chemical library. The results of this high throughput
screen and subsequent design of benzoxazinone 1 have
been previously reported.8 Although 1 was highly potent
(Ki ) 0.885 nM against FXa) and selective for FXa over
other similar serine proteases, development would be
hindered by the easily epimerized asymmetric center.
Subsequent studies on the benzoxazinone template
resulted in incorporation of the p-hydroxyphenylamidine
moiety9 to provide racemic 2, which further improved
potency (Ki ) 0.160 nM against FXa). While an asym-
metric synthesis of 2 resulted in the demonstration that
the S-isomer had the greatest affinity for FXa (Ki ) 0.08
nM); however, this enantiomer was easily racemized at
pH > 8.10
Due to the difficulty of obtaining crystal structures
of a small molecule bound to FXa, in these studies,
trypsin was used as a surrogate to predict the binding
conformations in FXa. Using this rationale, molecular
modeling suggested that the high potency of this series
of compounds could be retained if the oxygen atom of
the benzoxazinone nucleus was replaced with sulfur,
carbon, or nitrogen. Advantageously, in the cases of
Being that 35 still had some affinity for trypsin, X-ray
crystallographic experiments were conducted to deter-
mine its binding mode in these trypsin-like serine
proteases (Figure 2). As expected, the amidine was
positioned in the S1 pocket of the protein and forms a
strong two-on-two hydrogen bond with Asp189. The

Inhibitors of Blood Coagulation Factor Xa
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4093
Ta ble 4. Results of Compound 35 in the Rabbit Veno-venous
Shunt Model21
dose
(bolus µg/kg +
maintenance µg/kg/min)
incidence:
occluded/total
time to occlusion,
min
saline
7/7
5/6
3/6
1/6
10 ( 1
34 ( 18
71 ( 22
100 ( 19
30 + 1
60 + 2
480 + 16
A second in vivo test involved the electrolytic injury
of the femoral artery (or vein) of a dog in the presence
of restricted blood flow.22 The electrolytic injury induced
the formation of a thrombus and in control studies all
vessels occluded in approximately 100 min. Upon con-
stant infusion of a solution of 35, a dose dependent
response was seen in both the arterial and venous
systems (Table 5) with most vessels in the two highest
doses remaining patent for the duration of the study.
At the highest dose, it actually appears that the time
to occlusion decreased, but there is a great deal of
variability in the formation of an occlusive clot in this
model that could account for this. In addition to the
extension in the time for a vessel to occlude any thrombi
were also analyzed. In the arteries, the thrombus weight
was decreased from 46 ( 10 mg to 15 ( 5 mg and in
the vein from 136 ( 26 mg to 10 ( 2 mg at the highest
doses. In addition, it was shown that compound 35 did
not significantly increase bleeding times in a continuous
infusion rabbit model except at the highest dose (Table
6) and plasma drug concentrations were elevated in a
dose dependent manner after a bolus dose and followed
by continuous intravenous infusion (Table 7).
F igu r e 2. Interactions of compound 35 with trypsin.
Ta ble 3. Concentration of Compound 35 (micromolar) Needed
to Double the Desired Coagulation Parameter in the Specified
Species
species
PT
aPTT
rat
dog
human
1.3
0.9
0.3
5.2
1.6
0.4
Ser190 hydroxyl and the Gly219 carbonyl also form
hydrogen bonds with the amidine. The phenol, which
lies para to the amidine, makes use of a water molecule
to form hydrogen bonds with the Ser195 hydroxyl and
the His57 imidazole residues. This interaction alone was
responsible for the greater than 20-fold increase in
affinity over 22e. Another interaction, which plays a key
role in the high affinity of compound 35 for FXa, was
the hydrogen bond formed between the amide carbonyl
and the Gly216 amine. Also, the phenyl ring of the
quinoxalinone template makes significant nonbonding
interactions with Cys191, Cys220, and Gly219. The
X-ray structure revealed that the dimethylpiperidine
fragment does not have hydrogen bonding interactions
to trypsin, but modeling of this fragment into FXa shows
that it made nonbonded (van der Waals) interactions
with the side chains of Trp215, Gln175, and Leu99 (see
Figure 2).8
With these encouraging in vitro results, 35 was tested
ex vivo in rabbit, dog, and human plasma to determine
the effect it had on the standard coagulation parameters
(Table 3). As seen with compound 1, quinoxalinone 35
was most effective in human plasma and showed species
dependent inhibition of coagulation FXa. To determine
the effects that 35 had on thrombosis and hemostasis,
two in vivo studies were also conducted. First, anesthe-
tized rabbits were subjected to a veno-venous shunt
model, which has previously been described.21 A bolus
injection of 35 was followed by a constant infusion of
35 for 120 min as outlined in Table 4. In control animals,
all vessels occluded in approximately 10 min. With the
introduction of 35 at the highest doses (480 + 16), five
out of the six vessels remained patent for the duration
of the experiment (120 min). Table 4 also shows an
expected dose dependent increase in the average time
for the vessels to occlude in this experiment. Thus,
providing more evidence that 35 may be useful in
treating coagulation disorders.
Con clu sion s
In summary, we have designed and synthesized a
novel and selective quinoxalinone inhibitor of coagula-
tion FXa. As previously discussed, compound 35 origi-
nated from a high-throughput screen of our chemical
library. Through the use of molecular modeling and a
Topliss tree analysis approach,23 and structure-activity
relationships (SAR) from other FXa inhibitors,10 the 6
µM high throughput-screening lead ultimately led to the
design of the sub-nanomolar compound 35. Compound
35 has been shown to be efficacious in two antithrom-
botic animal models and shows a dose dependent
response in each. Quinoxalinone 35 shows promise for
the parental treatment of thrombotic diseases and is
being investigated for these uses.
Exp er im en ta l Section
Gen er a l Ch em istr y. All starting materials were obtained
from commercial sources and were 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 Robertson Microlit, Inc. (Madison, NJ ), and the results were
within 0.4% of the theoretical values for the elements indi-
cated. Mass spectral data were obtained on a VG Analytical
7070 E/HF mass spectrometer. Flash column chromatography
was performed on Merck silica gel 60, 230-400 mesh, pur-
chased from Mallinckrodt. Reactions were monitored by thin-
layer chromatography (TLC) on Merck glass plates precoated
with 0.25 mm of silica gel. Standard analytical high perfor-
mance liquid chromatography (HPLC) conditions unless stated
otherwise are Beckman 235328 C18 column, 5 µm, 4.6 mm ×
25 cm, eluted with a gradient of (80:20 to 10:90 H2O: CH3CN)

4094 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Willardsen et al.
Ta ble 5. Results of Compound 35 in the Canine Electrolytic Injury Model22
time to
time to
mg/kg/min
occlusion (vein, min)
occluded/total
occlusion (artery, min)
occluded/total
saline
1
3
5
86 ( 15
91 ( 21
175 ( 25
240 ( 0
205 ( 22
7/7
5/5
5/5
0/5
1/5
106 ( 21
101 ( 23
130 ( 18
240 ( 0
14/14
10/10
9/10
0/5
10
225 ( 15
3/10
Ta ble 6. Effects of Compound 35 on the Bleeding Time in
Cr ysta l Str u ctu r e Deter m in a tion a n d Refin em en t. The
Seconds in Rabbitsa
structure was solved by difference electron density methods
and the published coordinates for the bovine pancreatic trypsin
structure.24 Difference electron density maps using data to
1.7Å resolution revealed the positions and conformations of a
single bound 35 ligand located in the active site of the trypsin
molecule (Figure 1). Compound 35 was fit to the difference
electron density using QUANTA and the complex structure
including 80 water molecules was refined to 1.7 Å resolution
using XPLOR.25 The final crystallographic R-factor was 0.200
using diffraction data from 8.0 to 1.7 Å.
baseline
15 min
60 min
120 min
control
30 µg/kg + 1 µg/kg/min
60 µg/kg + 2 µg/kg/min
122 ( 14 117 ( 17 112 ( 15 120 ( 27
113 ( 19 169 ( 67
99 ( 6 84 ( 27
93 ( 12
96 ( 25
78 ( 3
68 ( 8
206 ( 42
480 µg/kg + 16 µg/kg/min 116 ( 14 170 ( 36 170 ( 7
a
Values expressed as mean ( SEM; n ) 6 all groups except
480+16 group, n ) 5. The ear bleeding time was determined at
baseline, 15, 60, and 120 min of compound infusion.
3-(2,2,2-Tr ich lor o-1-h yd r oxyeth yl)ben zon itr ile (4). To
a mixture of lead(II) bromide (1.69 g, 4.60 mmol) and finely
cut aluminum foil (1.25 g, 45.8 mmol) in DMF (226 mL) were
added 3-cyanobenzaldehyde (6.01 g, 45.8 mmol) and carbon
tetrachloride (8.84 mL, 91.7 mmol.). The mixture was stirred
at ambient temperature for 3 h before being quenched with
HCl (1 N, 100 mL). The aqueous mixture was extracted with
ethyl acetate (3×), and the combined organic extracts were
washed with brine, dried over magnesium sulfate, filtered, and
evaporated in vacuo to give the trichloromethyl carbinol (4)
as a brown oil in quantitative yield. 1H NMR (CDCl3, 300
MHz): δ 7.94 (m, 1H), 7.86 (m, 1H), 7.66 (m, 1H), 7.48 (m,
1H), 5.24 (s, H), 4.98 (bs, 1H).
2-(3-Cya n op h en yl)-3,4-d ih yd r o-2H-1,4-ben zoth ia zin -3-
on e (5a ). To 2-aminothiophenol (2.3 mL, 22.3 mmol) in DMF
(60 mL) was added sodium hydride (3.61 g, 90.3 mmol). After
bubbling and the evolution of heat ceased, a solution of 4 (5.67
g, 22.6 mmol) in DMSO (40 mL) was added dropwise over 60
s. After the evolution of heat ceased, the mixture was stirred
at 50 °C for 6 h. and at room temperature for 16 h. The reaction
mixture was cooled, diluted with water and extracted with
ethyl acetate (3×). The combined organic extracts were washed
with brine, dried over magnesium sulfate, filtered, and evapo-
rated in vacuo. The residue was absorbed onto silica gel and
purified by gradient chromatography (1% to 4% methanol in
dichloromethane). The residue was crystallized from ethyl
acetate and hexanes to provide 1.71 g (29%) of the title
compound 5a as a tan solid. 1H NMR (300 MHz, CDCl3): δ
10.97 (s, 1H), 7.75 (m, 1H), 7.57 (m, 2H), 7.32 (m, 1H), 7.20
(m, 1H), 7.00 (m, 3H), 5.10 (s, 1H).
1-(2-Ben zyloxy-5-b r om op h en yl)-2,2,2-t r ich lor oet h a -
n ol (7). Prepared using the same procedure as to synthesize
4. 1H NMR (300 MHz, CDCl3) δ 7.80 (d, 1H), 7.30-7.40 (m,
6H), 6.85 (d, 1H), 5.71 (d, 1H), 5.15 (d, 2H).
2-(2-Ben zyloxy-5-br om op h en yl)-4H-ben zo[1,4]th ia zin -
3-on e (8). Prepared via the same route to Compound 4. 1H
NMR (300 MHz, CDCl3) δ 7.18-7.43 (m, 9H), 7.02 (t, 1H), 6.98
(d, 1H), 6.80 (d, 1H), 5.16 (s, 1H), 5.11 (s, 2H). MS (CI) m/z
426/428 [M + H]. Anal. (C21H16BrNO2S) C, H, N.
4-Ben zyloxy-3-(3-oxo-3,4-dih ydr o-2H-ben zo[1,4]th iazin -
2-yl}ben zon itr ile (5b). To a solution of 8 (1.11 g, 2.6 mmol)
in DMF (10 mL) was added CuCN (460 mg, 5.2 mmol), and
the mixture was warmed to 160 °C for 12 h or until HPLC
showed disappearance of starting 8. The mixture was diluted
with water and extracted with ethyl acetate (3×). The com-
bined organics were washed with brine and dried over MgSO4.
After concentration, the title compound was obtained in
quantitative yield and used as is without further purification.
1H NMR (300 MHz, CDCl3) δ 7.55 (dd, 1H), 7.20-7.75 (m, 9H),
6.96-7.40 (m, 1H), 6.89 (d,1H), 5.19 (s, 1H), 5.16 (s, 1H). MS
(CI) m/z 373 [M + H].
+ 0.1% TFA over 23 min with a flow rate of 1.5 mL/min and
detected at 214 and 280 nm.
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 1.0 nM for FXa and
plasmin) was combined with 2 µL of inhibitor dilutions
(creating a final concentration range of 1.0 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 (Pharmacia Hepar) for FXa, CHROMOZYM TH (Boe-
hringer Mannheim) 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 an n ) 2 was performed
on each compound such that data reported is an average of
the two measurements.
Cr ysta lliza tion . Bovine pancreatic trypsin and benzami-
dine were obtained from Sigma Chemical Company. Crystals
of the trypsin-benzamidine complex were obtained by the
hanging drop vapor diffusion method. 8 µL drops of protein
solution containing trypsin (20 mg/mL), benzamidine (0.5 M),
calcium chloride (0.01 M), Tris-HCl (0.05 M), and ammonium
sulfate (1.25 M) at pH 7.0 were allowed to equilibrate against
a reservoir containing ammonium sulfate (600 µL, 2.5 M) and
Tris-HCl (0.1 M) at pH 6.5. Rectangular rod shaped crystals
of the trypsin-benzamidine complex appeared after 48 h and
continued to grow for several weeks. Crystals of the trypsin-
benzamidine complex are orthorhombic, space group P212121
with unit cell dimensions of a ) 63.70, b ) 63.40, and c )
69.10. Crystals of the complex of trypsin with 35 were obtained
by soaking pregrown trypsin-benzamidine crystals in solution
containing: 35 (10 mM), calcium chloride (0.01 M), Tris-HCl
(0.10 M), and ammonium sulfate (3.0 M) at pH 7.0 and 24 °C
for 72 h.
Cr ysta l Da ta Collection . For X-ray data collection, crys-
tals of the trypsin-35 complex were sealed in thin walled glass
capillaries in equilibrium with soaking solution. X-ray diffrac-
tion data to 1.7 Å resolution were collected at 24 °C using a
MarResearch Image Plate X-ray detector and a Rigaku Ru-
200B rotating anode X-ray generator operating at 50 kV and
120 mA. The crystals of the complex are of the orthorhombic,
space group P212121, with unit cell dimensions of a ) 63.70,
b ) 63.40, c ) 69.40Å. A total of 28352 reflections (out of a
possible 28523) with I/(I) g 2.0 were measured to 1.7 Å
resolution.
(E)-2-(3-Hyd r oxyp h en yl)-3-(2-n itr op h en yl)-2-p r op en o-
ic Acid (10). To a mixture of 2-nitrobenzaldehyde (19.22 g,
127 mmol) and 3-hydroxyphenylacetic acid (19.30 g, 127 mmol)

Inhibitors of Blood Coagulation Factor Xa
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4095
Ta ble 7. Plasma Concentrations of Compound 35 (ng/mL) Following Intravenous Administration to Rabbits
time postdose (min)
dose
0
5
15
60
120
140
30 µg/kg + 1 µg/kg/min.
60 µg/kg + 2 µg/kg/min.
480 µg/kg + 16 µg/kg/min.
BLQa
BLQ
BLQ
80.0 ( 17.8
135 ( 14.4
830 ( 116.5
80. 3( 15.5
119 ( 16.7
784 ( 60.7
70.8 ( 12.1
188 ( 43.7
907 ( 75.9
79.7 ( 10.5
187 ( 37.1
1009 ( 55.8
102 ( 17.1
176 ( 18.4
1012 ( 90.6
a
BLQ ) Below the limit of quantitation.
in acetic anhydride (87 mL) was added triethylamine (17.5 mL,
126 mmol), and the reaction mixture was stirred and heated
at reflux (150 °C) for 45 min. The mixture was diluted with
water (200 mL), cooled, diluted with NaOH (2 N, 200 mL),
and washed with ether. The aqueous solution was then
acidified with HCl (6 N) to pH 3 and stirred for 3 h. The solid
was collected and dried under high vacuum at 45 °C to give
23.1 g (64%) of the desired compound 10. H NMR (300 MHz,
DMSO): δ 8.09 (m, 1H), 7.93 (s, 1H), 7.48 (m, 2H), 7.00 (m,
2H), 6.59 (m, 1H), 6.48 (m, 2H). MS (CI) m/z 285 [M + H].
(d, 1H), 8.15 (1H, s), 8.00-7.80 (m, 3H), 7.42 (t, 1H), 7.31 (d,
1H), 7.04 (m, 2H). MS (CI) m/z 267 [M + H].
3-(2-H yd r oxyp h en yl)-3,4-d ih yd r o-2(1H )-q u in olin on e
(15). To 3-[(E)-(2-nitrophenyl)methylidene]-1-benzofuran-2-one
(14) (2.18 g, 7.65 mmol) in methanol (70 mL) and THF (30
mL) was added Pd/C 20% (50 mg), and the mixture was
hydrogenated for 5 h. Filtration and flash chromatography
(30-50% ethyl acetate in hexanes) afforded the desired product
15 (0.913 g, 49%). 1H NMR (300 MHz, DMSO): δ 9.52 (s, 1H),
7.15 (m, 2H), 7.05 (m, 1H), 6.97 (m, 1H), 6.89 (d, 2H), 6.83 (d,
1H), 6.67 (t, 1H), 3.96 (dd, 1H), 3.19 (dd, 1H), 2.99 (dd, 1H).
MS (CI) m/z 240 [M + H]. Anal. (C15H13N1O2) C, H, N.
3-(5-Br om o-2-h yd r oxyp h en yl)-3,4-d ih yd r o-2(1H)-qu in -
olin on e (16). To 3-(2-hydroxyphenyl)-3,4-dihydro-2(1H)-
quinolinone (15) (0.650 g, 2.72 mmol) in carbon disulfide (10
mL) and dichloromethane (10 mL) was slowly added (10 min)
a solution of bromine (0.17 mL, 3.30 mmol) in carbon disulfide
(5 mL). After 2 h, a precipitate had formed which was collected
and washed with ethyl ether. This afforded the title compound
16 (0.738 g, 85%). 1H NMR (400 MHz, DMSO): δ 10.29 (s,
1H), 7.21 (m, 2H), 7.16 (m, 2H), 6.91 (m, 2H), 6.78 (d, 1H),
3.93 (dd, 1H), 3.19 (dd, 1H), 2.97 (dd, 1H). MS (CI) m/z 318
[M - H], 320 [M + H].
1
3-(3-H yd r oxyp h en yl)-3,4-d ih yd r o-2(1H )-q u in olin on e
(11). To (E)-2-(3-hydroxyphenyl)-3-(2-nitrophenyl)-2-propenoic
acid (10) (26.29 g, 92.0 mmol) in methanol (600 mL) was added
20% Pd/C (1.5 g), and the mixture was hydrogenated at 45
psi of hydrogen and 30 °C for 3.5 h. The mixture was filtered
through Celite and the filter pad washed with methanol. The
combined filtrate and washings were concentrated in vacuo,
and the product was crystallized from methanol in water to
give 17.9 g (82%) as a colorless solid. 1H NMR (400 MHz,
DMSO): δ 10.30 (s, 1H), 9.31 (s, 1H), 7.14 (m, 2H), 7.05 (m,
1H), 6.90 (m, 2H), 6.61 (m, 3H), 3.70 (m, 1H), 3.20-3.06 (m,
2H).
3-(2-Oxo-1, 2,3,4-tetr a h yd r o-3-qu in olin yl) p h en yl tr i-
flu or om eth a n esu lfon a te (12). To 3-(3-hydroxyphenyl)-3,4-
dihydro-2(1H)-quinolinone (11) (3.01 g, 12.6 mmol) in THF (40
mL) was added sodium hydride (0.55 g, 13.8 mmol), and the
mixture was stirred at room temperature for 5 min. To this
mixture was added N-phenyltrifluoromethanesulfonimide (4.92
g, 13.8 mmol), and the reaction mixture was stirred at room
temperature for 1 h. The mixture was cooled, diluted with H2O,
and extracted with EtOAc (3×). The combined organic extracts
were washed with brine (2×), dried with MgSO4, filtered, and
evaporated in vacuo. The residue was purified via silica gel
chromatography (20% to 40% ethyl acetate in hexanes). The
product 12 (4.29 g, 92%) was isolated as a yellow solid. 1H
NMR (400 MHz, CDCl3): δ 8.06 (s, 1H), 7.40-7.15 (m, 6H),
7.00 (m, 1H), 6.76 (m, 1H), 3.87 (m, 1H), 3.22 (m, 2H). MS
(CI) m/z 372 [M + H].
3-(2-Oxo-1,2,3,4-t et r a h yd r o-3-qu in olin yl)b en zen eca r -
bon itr ile (5c). To 3-(2-oxo-1,2,3,4-tetrahydro-3-quinolinyl)-
phenyl trifluoromethanesulfonate (12) (0.410 g, 1.11 mmol) in
DMF (5 mL) were added zinc cyanide (0.078 g, 0.690 mmol)
and tetrakis(triphenylphosphine)palladium(0) (Pd(Ph3P)4 (0.128
g, 0.11 mmol). The reaction mixture was stirred and heated
at 100 °C for 1 h, followed by cooling, dilution with water (200
mL) and 2 M sulfuric acid (20 mL), and extraction with EtOAc
(3×). The combined organic extracts were washed with brine
(2×), dried over magnesium sulfate, filtered, and evaporated
in vacuo. The product 5c (0.222 g, 81%) was crystallized from
ethyl acetate and hexanes to provide a colorless solid. 1H NMR
(300 MHz, CDCl3): δ 8.23 (s, 1H), 7.70-7.18 (m, 6H), 7.04 (m,
1H), 6.81 (m, 1H), 3.89 (m, 1H), 3.24 (m, 2H). MS (CI) m/z
249 [M + H].
3-[(E)-(2-Nit r op h en yl)m et h ylid en e]-1-b en zofu r a n -2-
on e (14). 2-Hydroxyphenylacetic acid (1.83 g, 10.0 mmol) was
added to a mixture of 2-nitrobenzaldehyde (1.83 g, 10.0 mmol),
acetic anhydride (10 mL), and triethylamine (1.7 mL, 12.0
mmol). The reaction mixture was heated at 140 °C for 15 min.
The mixture was allowed to cool to 80 °C at which point water
(10 mL) was carefully added. The precipitated material was
filtered, washed with methanol, and dried in vacuo to afford
the required product 14 (0.905 g, 28%). A further amount of
product (1.523 g) was recovered from the filtrate and recrystal-
lized from methanol/water. 1H NMR (400 MHz, DMSO): δ 8.36
3-[2-(Ben zyloxy)-5-b r om op h en yl]-3,4-d ih yd r o-2(1H )-
qu in olin on e. To 3-(5-bromo-2-hydroxyphenyl)-3,4-dihydro-2-
(1H)-quinolinone (16) (3.16 g, 9.94 mmol) in DMF (15 mL) was
added cesium carbonate (5.2 g, 31.0 mmol) followed by benzyl
bromide (1.18 mL, 9.92 mmol). The reaction mixture was
stirred at room temperature for 16 h, diluted with ethyl acetate
(200 mL), washed with brine, dried over magnesium sulfate,
combined, and concentrated under reduced pressure. Flash
chromatography (20% ethyl acetate in hexanes) afforded the
1
title compound (2.77 g, 68%). H NMR (300 MHz, CDCl3): δ
8.40 (brs, 1H), 7.30 (m, 7H), 7.17 (d1, H), 7.11 (d, 1H), 6.98 (t,
1H), 6.84 (d, 1H), 6.78 (d, 1H), 5.07 (s, 2H), 4.09 (dd, 1H), 3.38
(dd, 1H), 2.99 (dd, 1H). MS (CI) m/z 408 [M - H], 410 [M +
H].
4-(Ben zyloxy)-3-(2-oxo-1,2,3,4-tetr ah ydr o-3-qu in olin yl)-
ben zen eca r bon itr ile (5d ). The title compound was synthe-
sized as per compound 5b and isolated as a colorless oil (1.077
g, 45%). 1H NMR (300 MHz, DMSO): δ 10.40 (s, 1H), 7.77
(dd, 1H), 7.69 (d, 1H), 7.40-7.10 (m, 8H), 6.89 (m, 2H), 5.22
(m, 2H), 4.04 (dd, 1H), 3.31(dd, 1H), 2.93 (dd, 1H). MS (CI)
m/z 355 [M + H].
3-(3-Br om op h en yl)-3,4-d ih yd r o-1H -q u in oxa lin -2-on e
(18). To 2-bromo-(3-bromophenyl)acetic acid (4.95 g, 16.9
mmol) as a suspension in water (40 mL) was added powdered
NaOH (0.65 g, 16.7 mmol) to yield a red solution. 2-Phenylene-
diamine (1.83 g, 16.9 mmol) was added, and the mixture was
stirred at 60 °C for 30 min. The formed solid was filtered,
washed with water, and dried in vacuo. The remaining residue
was recrystallized from ethanol and water to afford 1.88 g
(37%) of the title compound. 1H NMR (400 MHz, DMSO) δ
7.40-7.50 (m, 2H), 7.20-7.40 (m, 2H), 6.65-6.80 (m, 2H),
6.58-6.63 (m, 2H). MS (CI) m/z 303/305 [M + H] and 300/303
[M - H].
3-(3-Br om op h en yl)-1H-qu in oxa lin -2-on e (19). To a solu-
tion of 3-(3-bromophenyl)-3,4-dihydro-1H-quinoxalin-2-one (1.73
g, 5.71 mmol) in toluene (50 mL) was added 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone (1.30 g, 5.73 mmol) and the mixture
was warmed to reflux for 3 h. The formed solid was filtered to
afford the title compound (2.49 g, 66%), which was used
without further purification. 1H NMR (400 MHz, DMSO) d 8.45
(s, 1H), 8.25 (d, 1H), 7.80 (d, 1H), 7.66 (d, 1H), 7.41 (t, 1H),

4096 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Willardsen et al.
7.30 (t, 1H), 7.28 (m, 2H). MS CI m/z 300/302 [M + H] and
298/300 [M - H]. Anal.(C14H9N2OBr) C, H, N.
2H), 2.35-2.72 (m, 4H), 0.75-1.65 (m, 20H). MS (CI) m/z 554
[M + H]. HPLC 17.4 min.
3-(3-Oxo-3,4-d ih yd r oqu in oxa lin -2-yl)ben zon itr ile (5e).
Following the preparation for 5b gave the title compound in
92% yield. 1H NMR (400 MHz, DMSO) δ 8.76 (t, 1H), 8.62 (dt,
1H), 7.91 (m, 1H), 7.80 (d, 1H), 7.71 (t, 1H), 7.55 (t, 1H), 7.20-
7.35 (m, 2H). MS CI m/z 248 [M + H] and 247 [M-]. Anal.
(C15H9N3O) C, H, N.
3-{1-[5-[(2R,6S)-2,6-Dim et h ylp ip er id in -1-yl]p en t yl-2-
oxo-1,2,3,4-tetr a h yd r o-3-qu in olin -2-yl)ben zon itr ile (21c).
Prepared via the same route as compound 21a , and the title
compound was isolated in quantitative yield as a yellow oil.
1H NMR (300 MHz, CDCl3): δ 7.50-6.96 (m, 8H), 3.91 (m,
2H), 3.80 (m, 1H), 3.13 (m, 2H), 2.67 (s, 2H), 2.36 (s, 2H), 1.66-
1.15 (m, 12H), 1.01 (m, 6H). HPLC 14.2 min.
4-Ben zyloxy-3-{1-[5-[(2R,6S)-2,6-d im eth ylp ip er id in -1-
yl]p en t yl-2-oxo-1,2,3,4-t et r a h yd r o-3-q u in olin yl}b en zo-
n itr ile (21d ). Prepared via the same route as compound 21a ,
but the title compound was not isolated, but rather taken
directly on to 22d .
3-{4-[5-((2S,6R)-2,6-Dim et h ylp ip er id in -1-yl)p en t yl]-3-
oxo-3,4-d ih yd r oqu in oxa lin -2-yl}ben zon itr ile (21e). Pre-
pared via the same route as compound 21a and wa s used
without further purification. MS CI m/z 429 [M + H] and 428
[M-]. HPLC 14.6 min.
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 zoth ia zin -3-on e (20a ). To the benzothiazinone 5a
(0.75 g, 2.82 mmol) in DMF (5 mL) was added sodium hydride
(0.124 g, 3.09 mmol), and the solution was stirred at 70 °C for
15 min, at which point, the bubbling had stopped. To this
solution was added 1,5-dibromopentane (1.54 mL, 11.2 mmol),
and the reaction mixture was stirred at 70 °C for an additional
16 h. The reaction mixture was cooled, diluted with water, and
extracted with ethyl acetate (3×). The combined organic
extracts were washed with brine (2×), dried over magnesium
sulfate, filtered, and evaporated in vacuo. The residue was
purified via gradient chromatography (20% to 30% ethyl
acetate in hexanes). The title compound (20a ) was isolated
(0.66 g, 52%) as a yellow oil. 1H NMR (300 MHz, CDCl3): δ
7.57 (m, 3H), 7.38 (m, 2H), 7.25 (m, 1H), 7.11-7.01 (m, 2H),
4.64 (s, 1H), 4.09 (m, 2H), 3.40 (m, 2H), 1.91 (m, 2H), 1.70 (m,
2H), 1.53 (m, 2H).
4-Ben zyloxy-3-[4-(5-b r om op en t yl)-3-oxo-3,4-d ih yd r o-
2H-ben zo[1,4]th ia zin -2-yl}ben zon itr ile (20b). Prepared via
the same route as compound 20a . 1H NMR (300 MHz, CDCl3)
δ 7.60 (dd, 1H), 7.20-7.74 (m, 9H), 6.95-7.05 (m, 2H), 5.18
(s, 2H), 5.04 (s, 1H), 4.03-4.19 (m, 2H), 3.40 (t, 2H), 1.83-
1.98 (m, 2H), 1.66-1.80 (m, 2H), 1.47-1.58 (m, 4H). MS (CI)
m/z 521/523 [M + H].
3-(4-[5-[(2R,6S)-2,6-Dim et h ylp ip er id in -1-yl]p en t yl]-3-
oxo-3,4-d ih yd r o-2H-1,4-ben zoth ia zin -2-yl)-1-ben zen eca r -
boxim id a m id e (22a ). To 21a (0.30 g, 0.67 mmol) in methanol
(10 mL) were added N-hydroxylamine hydrochloride (0.116 g,
1.67 mmol) and diisopropylethylamine (DIEA, 0.12 mL, 0.67
mmol). The reaction mixture was stirred at room temperature
for 24 h. The solvent was evaporated in vacuo, and the
remaining residue was dried under high vacuum. To the
residue was added trifluoroacetic anhydride (2 mL), and the
resulting solution was stirred at room temperature for 2 h.
The reaction mixture was diluted with acetic acid, and the
mixture was concentrated in vacuo to give a yellow oil. To the
yellow oil in acetic acid (2 mL) was added 20% Pd/C (45 mg),
and the mixture was hydrogenated at 23 °C for 2 h. The
mixture was filtered through Celite and the filter pad washed
with acetic acid. The combined filtrate and washings were
evaporated in vacuo and purified by preparative HPLC (Vydac
218TP1022 C-18, 95:5 (H2O:CH3CN + 0.1% TFA) to 60:40
(H2O:CH3CN + 0.1% TFA) over 90 min), and appropriate
fractions were combined and lyophilized to provide 50 mg
3-[1-(5-Br om op en tyl)-2-oxo-1,2,3,4-tetr a h yd r o-3-qu in o-
lin yl]ben zen eca r bon itr ile (20c). Prepared via the same
route as 20a and the product 20c was isolated (1.59 g, 36%)
1
as a yellow oil. H NMR (300 MHz, CDCl3): δ 7.58-7.28 (m,
5H), 7.20 (m, 1H), 7.05 (m, 2H), 3.99 (m, 2H), 3.85 (m, 1H),
3.41 (m, 2H), 3.19 (m, 2H), 1.91 (m, 2H), 1.70 (m, 2H), 1.54
(m, 2H). MS (CI) m/z 397/399 [M + H], 395/396 [M - H].
4-(Ben zyloxy)-3-[1-(5-br om op en tyl)-2-oxo-1,2,3,4-tetr a -
h yd r o-3-qu in olin yl]ben zen eca r bon itr ile (20d ). Prepared
via the same route as compound 20a and isolated as an oil
1
(16%) of the title compound 22a as a fluffy off-white solid. H
NMR (400 MHz, DMSO): δ 9.33 (s, 1H), 9.18 (s, 1H), 7.69 (m,
3H), 7.55 (m, 1H), 7.41 (m, 2H), 7.33 (m, 1H), 7.07 (m, 1H),
5.11 (s, 1H), 4.09 (m, 2H), 3.38 (m, 2H), 3.24 (m, 2H), 3.10 (m,
2H), 2.91 (m, 2H), 1.84 (m, 2H), 1.66-1.33 (m, 6H), 1.24 (m,
6H). MS (CI) m/z 465 [M + H] 464 [M - H]. HPLC 8.9 min.
3-{4-[5-((2S,6R)-2,6-Dim eth ylp ip er id in -1-yl)-p en tyl]-3-
oxo-3,4-d ih yd r o-2H -b en zo[1,4]t h ia zin -2-yl}-4-h yd r oxy-
ben za m id in e (22b). Prepared via the same synthetic route
as compound 22a . 1H NMR (300 MHz, CDCl3) δ 7.61 (dd, 1H),
7.40 (M, 4h), 7.05 (m, 2H), 4.88 (s, 1H), 4.00-4.22 (m, 4H),
2.80-3.20 (m, 4H), 1.20-1.80 (m, 16H). MS (CI) m/z 481 [M
+ H]. HPLC 8.77 min.
3-(1-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id i-
n yl]p en t yl]-2-oxo-1,2,3,4-t et r a h yd r o-3-q u in olin yl)b en -
zen eca r boxim id a m id e (22c). Prepared via the same syn-
thetic route as compound 22a and converted to an HCl salt
by filtration through Amberlite 400(Cl) resin to give 619 mg
(52%) of 22c as an off-white solid. 1H NMR (400 MHz,
DMSO): δ 9.35 (s, 1H), 9.07 (s, 2H), 7.66 (m, 2H), 7.49 (m,
2H), 7.21 (m, 3H), 6.97 (m, 1H), 3.95 (m, 3H), 3.42 (s, 4H),
3.35-2.86 (m, 6H), 1.75-1.26 (m, 8H), 1.20 (m, 6H). MS (CI)
m/z 447 [M + H]. HPLC 9.6 min.
1
(0.230 g, 60%). H NMR (300 MHz, CDCl3): δ 7.57 (dd, 1H),
7.42 (d, 1H), 7.40-7.20 (m, 6H), 7.13 (d, 1H), 7.02 (m, 3H),
5.15 (m, 2H), 4.04 (dd, 1H), 3.98 (m, 2H), 3.39 (t, 2H), 3.30
(dd, 1H), 2.96 (dd, 1H), 1.88 (m, 2H), 1.75-1.45 (m, 4H). MS
(CI) m/z 505 [M + H], 503 [M - H].
3-[4-(5-Br om op en t yl)-3-oxo-3,4-d ih yd r oq u in oxa lin -2-
yl]ben zon it r ile (20e). Prepared via the same route as
compound 20a to yield 46% along with 29% of the O-alkylated
1
analogue. H NMR (400 MHz, DMSO) δ 8.65 (t, 1H), 8,42 (dt,
1H), 8.18 (d, 1H), 7.85 (d, 1H), 7.60-7.80 (m, 4H), 4.61 (t, 2H),
3.46 (t, 2H), 1.88-2.05 (m, 4H), 2.65-2.78 (m, 2H). MS (CI)
m/z 396/398 [M + H] and 395/397 [M - H].
5-[(2R,6S)-2,6-Dim et h ylp ip er id in -1-yl]p en t yl-2-(3-cy-
a n op h en yl)-3,4-d ih yd r o-2H-1,4-ben zoth ia zin -3-on e (21a ).
To 20a (0.66 g, 1.59 mmol) was added cis-2,6-dimethylpiperi-
dine (6.0 mL, 44.0 mmol), and the reaction mixture was stirred
at 70 °C for 48 h. A small amount of DMF was added to obtain
a homogeneous mixture before heating. The solution was
cooled, diluted with water, and extracted with ethyl acetate
(3×). The combined organic extracts were washed with satu-
rated sodium bicarbonate (2×), brine (2×), dried with mag-
nesium sulfate, filtered, and evaporated in vacuo. The residue
was coevaporated with toluene and dried under high vacuum
to give 0.69 g (97%) of 21a as a yellow oil. 1H NMR (300 MHz,
CDCl3): δ 7.55 (m, 3H), 7.37 (m, 2H), 7.24 (m, 1H), 7.10-7.00
(m, 2H), 4.63 (s, 1H), 4.07 (m, 2H), 2.74 (m, 2H), 2.47 (m, 2H),
1.76-1.22 (m, 12H), 1.08 (m, 6H). HPLC 14.2 min.
3-(1-[5-[(2R,6S)-2,6-Dim eth yltetr a h yd r o-1(2H)-p yr id i-
n yl]p en t yl]-2-oxo-1,2,3,4-t et r a h yd r o-3-q u in olin yl)-4-h y-
d r oxyb en zen eca r boxim id a m id e (22d ). Prepared via the
same synthetic route as compound 22a and isolated as a
1
colorless powder (0.123 g, 50%). H NMR (400 MHz, DMSO):
δ 10.89 (s, 1H), 10.20 (brs, 1H), 9.09 (s, 2H), 8.80 (s, 1H), 7.62
(m, 2H), 7.20 (m, 3H), 7.00 (m, 2H), 3.90 (m, 3H), 3.40 (m,
1H), 3.20 (m, 2H), 3.00 (m, 1H), 2.85 (m, 2H), 1.80-1.20 (m,
18H). MS (CI) m/z 463 [M + H]. TOF HRMS calcd 463.3073,
found 463.3069. HPLC 10.1 min.
4-Ben zyloxy-3-{1-[5-((2S,6R)-2,6-d im eth ylp ip er id in -1-
yl)p en tyl]-3-oxo-3,4-d ih yd r o-2H-ben zo[1,4]th ia zin -2-yl}-
ben zon itr ile (21b). Prepared via the same route as compound
21a . 1H NMR (300 MHz, CDCl3) δ 7.55 (dd, 1H), 7.05-7.40
(m, 9H), 6.90-7.00 (m, 2H), 5.12 (s, 2H), 4.97 (s, 1H), 4.04 (m,
3-{4-[5-((2S,6R)-2,6-Dim et h ylp ip er id in -1-yl)p en t yl]-3-
oxo-3,4-d ih yd r oqu in oxa lin -2-yl}ben za m id in e (22e). Pre-

Inhibitors of Blood Coagulation Factor Xa
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4097
pared via the same synthetic route as compound 22a . 1H NMR
(400 MHz, DMSO) δ 8.70 (m, 1H), 8.68(d, 1H), 7.95 (t, 2H),
7.78 (m, 3H), 7.48 (m, 1H), 4.44 (m, 2H), 3.00-3.40 (m, 2H),
1.40-1.80 (m, 12H), 1.28 (d, 6H). MS CI m/z 446 [M + H].
TOF HRMS calcd 466.2920, found 466.2911. HPLC 8.5 min.
3-Br om o-1-(5-br om op en tyl)qu in olin -2-on e (24). To a
solution of 23 (2.0 g, 8.92 mmol) in DMF (6 mL) were added
NaH (390 mg of a 60% oil dispersion, 9.75 mmol) and
1,5-dibromopentane (2.6 mL, 18 mmol). The reaction mixture
was stirred for 3 h at ambient temperature before being diluted
with water and extracted with ethyl acetate (3×). The com-
bined organics were washed with brine and dried over mag-
nesium sulfate. After evaporation, the residue was purified
by column chromatography (20% ethyl acetate in hexanes) to
1H), 4.25-4.41 (m, 2H), 3.00-3.25 (m, 2H), 1.22-1.82 (m,
14H), 0.85 (m, 6H). MS (CI) m/z 461 [M + H]. HPLC 9.6 min.
5-Ben zyloxy-2-br om oben za ld eh yd e (28). 5-Bromosali-
cylaldehyde (100 g, 496 mmol) was dissolved in ethanol (300
mL), and a solution of potassium hydroxide (27.8 g, 496 mmol)
in water (80 mL) was added. After the mixture was stirred at
ambient temperature for 30 min, benzyl bromide (71.2 mL,
596 mmol), which was previously passed through a plug of
neutral alumina, was added. The resulting mixture was
warmed to reflux for 18 h, at which time a colorless precipitate
formed. After the mixture was cooled to room temperature,
water (50 mL) was added and the title compound was filtered.
After the mixture was washed with water, 28 was recrystal-
lized from ethanol to provide 122 g (85%) of a colorless solid.
1H NMR (400 MHz, CDCl3) δ 10.46 (d, 1H); 7.90 (d, 1H); 7.56
(dd, 1H); 7.30 (m, 5H); 6.91 (d, 1H); 5.14 (s, 2H). MS (CI) m/z
292 [M+], 261 [M - CHO]. HPLC 20.4 min.
1
yield 1.76 g (33%) of the desired product. H NMR (300 MHz,
DMSO) δ 8.13 (s, 1H), 7.48-7.63 (m, 2H), 7.36 (d, 1H), 7.25
(t, 1H), 4.35 (t, 2H), 3.43 (t, 2H), 1.92 (m, 2H), 1.80 (m 2H),
1.63 (m, 2H). MS (CI) m/z 371/373/375 [M + H].
(2-Ben zyloxy-5-b r om op h en yl)-2-h yd r oxya cet on it r ile
(29). Aldehyde 28 (20 g, 69 mmol) was dissolved in methanol
(150 mL) followed by the addition of potassium cyanide (20.1
g, 308 mmol). Acetic acid (6.3 mL, 109 mmol) was added
dropwise over 20 min at room temperature. The resulting
mixture was stirred at ambient temperature for 3 h. Acetic
acid (2 mL) was added, and the mixture was stirred for an
additional 60 min. Additional acetic acid (30 mL) was added,
and the mixture was diluted with water (60 mL). After
concentration at reduced pressure, the aqueous residue was
extracted with ethyl acetate (3×). The combined organics were
washed with brine and dried over magnesium sulfate. Filtra-
tion and concentration left a pale yellow oil, which was used
without further purification. The title compound could be
isolated in pure form by gradient column chromatography (10
3-[1-(5-Br om op en tyl)-2-oxo-1,2-d ih yd r oqu in olin -3-yl]-
ben zon itr ile (25a ). To a mixture of 24 (720 mg, 1.93 mmol)
in toluene (6 mL) and ethanol (6 mL) was added saturated
aqueous sodium bicarbonate (6 mL). Pd(PPh3)4 was added
followed by 3-cyanophenylboronic acid, and the mixture was
stirred at 100 °C for 4 h. The mixture was diluted with and
washed with water and brine. After the organics were dried
over magnesium sulfate, the residue was purified by column
chromatography (20% ethyl acetate in hexanes) to provide 560
1
mg of 25a (74%). H NMR (400 MHz, DMSO) δ 8.08 (s, 1H),
8.00 (d, 1H), 7.85 (s, 1H), 7.62 (m, 3H), 6.48 (t, 1H), 6.40 (d,
1H), 7.25 (d, 1H), 4.39 (t, 2H), 3.42 (t, 2H), 1.98 (m, 2H), 1.82
(m, 2H), 1.64 (m, 2H). MS (CI) m/z 397/399 [M + H].
4-Ben zyloxy-3-[1-(5-b r om op en t yl)-2-oxo-1,2-d ih yd r o-
qu in olin -3-yl}ben zon itr ile (25b). To a solution of 20d (600
mg, 1.19 mmol) in toluene (5 mL) was added DDQ (540 mg,
2.38 mmol). The mixture was warmed to 80 °C in a sealed tube.
After 18 h, compound 20d still remained. Additional DDQ (540
mg, 2.38 mmol) was added, and the mixture was stirred at 80
°C for 72 h. The reaction mixture was diluted with water and
extracted with ethyl acetate (3×). The combined organics were
washed with brine and dried over magnesium sulfate. The
mixture was filtered, evaporated, and purified by flash chro-
matography (20% ethyl acetate in hexanes) to give 68% of the
1
to 20% ethyl acetate in hexanes). H NMR (400 MHz, CDCl3)
δ 7.53 (d, 1H); 7.24 (m, 6H); 6.86 (d, 1H); 5.52 (d, 1H); 5.13 (s,
2H); 3.38 (d, 1H). MS (CI) m/z 199, 201 [M - Bn - CN]. HPLC
20.4 min.
(2-Ben zyloxy-5-b r om op h en yl)-2-h yd r oxya cet ic Acid
Meth yl Ester (30). Crude 29, from above, was dissolved in
anhydrous p-dioxane (25 mL), ethyl ether (25 mL), and
methanol (6 mL). After the mixture was cooled to 0 °C, it was
saturated with HCl gas and warmed to room temperature. A
precipitate formed, the mixture was stirred for 90 min, and
anhydrous ethyl ether was added as necessary to maintain
viscousity. The reaction mixture was diluted with ethyl ether
(200 mL) and filtered. The colorless solid was washed with
ethyl ether and was used without further purification. The
solid was suspended in water (60 mL) and p-dioxane (60 mL)
and stirred vigorously for approximately 4 h. The reaction was
complete when all the solids had disappeared and an oily
residue remained. The mixture was poured into water (150
mL) and extracted with ethyl acetate (3×). The combined
organics were washed with brine and dried over magnesium
sulfate. Filtration and concentration yielded the crude title
compound 30, which was used without purification. The title
compound could be isolated in pure form by column chroma-
1
slightly impure desired product. H NMR (300 MHz, CDCl3)
δ 8.05 (s, 1H), 7.00-7.80 (m, 12H), 5.16 (s, 2H), 4.45 (t, 2H),
4.34 (t, 2H), 1.50-2.00 (m, 6H). MS (CI) m/z 501/503 [M + H].
3-{1-[5-((2S,6R)-2,6-Dim et h ylp ip er id in -1-yl)p en t yl]-2-
oxo-1,2-d ih yd r oqu in olin -3-yl}ben zon itr ile (26a ). Prepared
1
via the same synthetic route as compound 21a . H NMR (300
MHz, CDCl3) δ 7.99 (s, 1H), 7.92 (d, 1H), 7.78 (s, 1H), 7.40-
7.62 (m, 3H), 7.33 (d, 1H), 7.05-7.25 (m, 2H), 4.29 (t, 2H),
2.60-2.80 (m, 2H), 2.30-2.50 (m, 2H). 1.00-1.80 (m, 18H).
MS (CI) m/z 428 [M + H]. HPLC 14.2 min.
4-Ben zyloxy-3-{1-[5-((2S,6R)-2,6-d im eth ylp ip er id in -1-
yl)-p en t yl]-2-oxo-1,2-d ih yd r oqu in olin -3-yl}b en zon it r ile
(26b). Prepared via the same synthetic route as compound
1
1
21a . H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 7.00-7.80 (m,
tography (15% ethyl acetate in hexanes). H NMR (400 MHz,
12H), 5.09 (s, 2H), 4.41 (t, 2H), 4.26 (t, 2H), 2.70 (m, 2H), 2.40
(m, 2H), 1.00-1.90 (m, 16H). MS (CI) m/z 534 [M + H]. HPLC
16.7 min.
CDCl3) δ 7.4 (m, 7H), 6.82 (m, 1H), 5.32 (d, 1H), 5.90 (ABq,
2H), 3.70 (s, 3H), 3.63 (d, 2H). MS (CI) m/z 352 [M+]. HPLC
17.5 min.
3-{1-[5-((2S,6R)-2,6-Dim et h ylp ip er id in -1-yl)p en t yl]-2-
oxo-1,2-d ih yd r oq u in olin -3-yl}b en za m id in e (27a ). Pre-
pared via the same synthetic route as compound 21a The HCl
salt was formed by treating an aqueous solution of the TFA
salt with Amberlite IRA 400(Cl) resin followed by filtration
and lyopholization. 1H NMR (400 MHz, DMSO) δ 8.38 (s, 1H),
8.18 (s, 1H), 8.12 (d, 1H), 7.81 (t, 2H), 7.64 (m,3H), 7.32 (t,
1H), 4.23 (bs, 2H), 2.80-3.40 (m, 4H), 1.40-1.80 (m, 12H), 1.26
(d, 6H). MS (CI) m/z 445 (M + H). HPLC 9.2 min.
3-{1-[5-((2S,6R)-2,6-Dim eth yl-p ip er id in -1-yl)-p en tyl]-2-
oxo-1,2-d ih yd r oq u in olin -3-yl}-4-h yd r oxyb en za m id in e
(27b). Amidine was formed as previously mentioned for 22a ,
and the benzyl group was removed via hydrogenation. The HCl
salt was prepared as described for 27a 1H NMR (300 MHz,
DMSO) δ 8.35 (s,1H), 7.61-7.83 m, 5H), 7.36 (t, 1H), 7.16 (d,
(2-Ben zyloxy-5-br om op h en yl)-2-oxoa cetic Acid Meth yl
Ester (31). Dimethyl sulfoxide (DMSO, 12.4 mL, 164 mmol)
was added slowly to a solution of oxalyl chloride (7.2 mL, 82.4
mmol) in dichloromethane (100 mL) at -78 °C. After addition,
the mixture was stirred at -78 °C for 15 min. A solution of
the alcohol 30 (68.6 mmol) in dichloromethane (60 mL) was
added via a cannula. After the mixture was stirred for 30 min
at -78 °C, triethylamine (47.8 mL, 343 mmol) was added, and
the mixture was allowed to warm to room temperature. After
the mixture was stirred for 1 h at room temperature, it was
poured into water (200 mL). The layers were separated, and
the aqueous layer was washed with ethyl acetate (3×). The
combined organics were washed with brine and dried over
magnesium sulfate and filtered, and after concentration, the
title compound 31 was crystallized from ethyl acetate/hexanes

4098 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Willardsen et al.
1
to yield 13.5 g (60% from aldehyde 28) as a colorless solid. H
(100% water to 20% water 80% acetonitrile with 0.1%TFA over
120 min) to yield 35. The lyophilized solid from above was
dissolved in water (100 mL), and Amberlite 400(Cl) resin was
added. After being stirred for 3 h, the resin was filtered and
the aqueous solution was lyophilized. The lyophilized solid was
dissolved in methanol (5 mL), and the product was crystallized
by the slow addition of a vast excess of ethyl acetate. Filtration
yielded compound 35 as a yellow solid (630 mg, 30%). 1H NMR
(400 MHz, DMSO) δ 9.14 (s, 1H); 9.00 (s, 1H); 8.34 (d, 1H);
7.91 (d, 1H); 7.81 (dd, 1H); 7.71 (m, 2H); 7.47 (m, 1H); 7.11 (d,
1H); 4.31 (m, 2H); 3.0-3.8 (m, 2H); 1.2-1.8 (m, 20H). MS (CI)
m/z 462 [M + H]. HPLC 14.3 min. HPLC method: Vydac
218TP54 C18 column, 5 µm, 4.6 mm × 25 cm, eluted with a
gradient of (95:5 to 10:90 water:acetonitrile with 0.1% TFA).
NMR (400 MHz, CDCl3) δ 7.99 (d, 1H); 7.65 (dd, 1H); 7.40 (m,
5H); 6.95 (d, 1H); 5.06 (s, 2H); 3.34 (s, 3H). MS (CI) m/z 256,
258[M+]. HPLC 22.6 min.
3-(2-Ben zyloxy-5-br om oph en yl)-1-qu in oxalin -2-on e (32).
Ester 31 (7.0 g, 20.0 mmol) was dissolved in methanol (60 mL),
and 1,2-phenylenediamine was added (2.2 g, 20.0 mmol). The
mixture was warmed to reflux overnight, at which time a
precipitate was formed. The mixture was cooled to room
temperature, and water (10.mL) was added. The product was
filtered and washed with a small amount of cold methanol to
1
yield 7.7 g (94%) of the title compound as a colorless solid. H
NMR (400 MHz, DMSO) δ 12.54 (s, 1H); 7.73 (d, 1H); 7.65 (m,
2H); 7.23 (m, 8H); 7.10 (d, 1H); 5.08 (s, 2H). MS (CI) m/z 407,
409[M+]. HPLC 24.1 min.
4-Ben zyloxy-3-(3-oxo-3,4-d ih yd r oqu in oxa lin -2-yl)-ben -
zon itr ile (33). Bromide 32 (20.4 g, 50.0 mmol) was dissolved
in anhydrous DMF (200 mL) followed by the addition of zinc
cyanide (3.52 g, 30.0 mmol) and Pd(Ph3P)4 (5.78 g, 5.0 mmol).
The resulting mixture was warmed to 100 °C for 5 h. The dark
green solution was cooled to room temperature and stirred
overnight. The title compound formed as a precipitate, which
was separated by filtration and washed with ethyl ether to
yield 12.3 g (69%). 1H NMR (400 MHz, DMSO) δ 12.63 (s, 1H);
7.92 (dd, 1H); 7.87 (d, 1H); 7.79 (d, 1H); 7.58 (m, 1H); 7.2-7.4
(m, 8H); 5.24 (s, 2H). MS (CI) m/z 354 [M+]. HPLC 16.4 min.
Su p p or tin g In for m a tion Ava ila ble: Combustion analy-
sis of the final compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
Note Ad d ed a fter ASAP P ostin g. In the version of
the paper posted J une 26, 2004, “acetic anhydride” in
the synthetic procedure for 22a has been changed to
“trifluoroacetic anhydride”. The revised version of the
paper was posted J uly 7, 2004.
4-Ben zyloxy-3-[4[(5-b r om op en t yl)-3-oxo-3,4-d ih yd r o-
qu in oxa lin -2-yl]ben zon itr ile To a solution of 33 (2.0 g, 5.7
mmol) in DMF (50 mL) was added cesium carbonate (2.21 g,
6.8 mmol) at 0 °C. The mixture was stirred for 30 min before
the addition of 1,5-dibromopentane (4.62 mL, 40.0 mmol). The
reaction mixture was stirred overnight, while slowly warming
to room temperature, before being poured into water (100 mL).
The aqueous solution was extracted into ethyl acetate (3×).
The combined organics were washed with brine, dried over
magnesium sulfate, filtered, and concentrated under pressure.
The title compound (1.59 g, 53%, (Rf ) 0.15; 15% ethyl acetate
in hexanes)) was isolated by gradient column chromatography
(10 to 25% ethyl acetate in hexanes) along with the O-alkylated
analogue (1.17 g, 41%, (Rf ) 0.31; 15% ethyl acetate in
hexanes)). N-Alkylated: 1H NMR (400 MHz, CDCl3) δ 7.90
(d, 2H); 7.88 (d, 1H); 7.70 (dd, 1H); 7.57 (t, 1H); 7.2-7.4 (m,
6H); 7.03 (d, 1H); 5.14 (s, 2H); 4.24 (t, 2H); 3.36 (t, 2H); 1.8-
1.9 (m, 2H); 1.7-1.8 (m, 2H); 1.5-1.6 (m, 2H). MS (CI) m/z
502, 504 [M+]. HPLC 23.5 min.
4-Ben zyloxy-3-{4-[5-((2S,6R)-2,6-d im eth ylp ip er id in -1-
y l)p e n t y l]-3-o x o -3,4-d ih y d r o q u in o x o lin -2-y l}b e n zo -
n itr ile (34). 4-Benzyloxy-3-[4[(5-bromopentyl)-3-oxo-3,4-dihy-
droquinoxalin-2-yl]benzonitrile (1.59 g, 3.17 mmol) was dissolved
in anhydrous DMF (5 mL), and cis-2,6-dimethylpiperidine (15
mL) was added. The reaction mixture was warmed to 75 °C
overnight. The reaction mixture was diluted with water (50
mL) and extracted with ethyl acetate (2×). The combined
organics were washed with brine, dried over magnesium
sulfate, dried, and concentrated to yield a light yellow oil,
which was used without further purification. 1H NMR (400
MHz, DMSO) δ 7.95 (dd, 1H); 7.93 (d, 1H); 7.86 (d, 1H) 7.6-
7.7 (m, 2H); 7.2-7.4 (m, 7H); 5.22 (s, 2H); 4.23 (t, 2H), 0.9-
2.5 (m 22H). MS (CI) 535 [M + H]. HPLC 18.8 min.
4-Ben zyloxy-3-{4-[5-((2S,6R)-2,6-d im eth ylp ip er id in -1-
y l)p e n t y l]-3-o x o -3,4-d i h y d r o q u i n o x o li n -2-y l}b e n z -
a m id in e (35). A solution of crude 34 (assume 3.17 mmol), in
anhydrous ethanol (20 mL), at 0 °C was saturated with HCl
(gas). The gaseous HCl was bubbled through concentrated
sulfuric acid prior to the introduction to the reaction mixture.
The reaction mixture was sealed and warmed to room-
temperature overnight. The mixture was concentrated in vacuo
and used without further purification. The crude residue from
above was dissolved in ammonia (2 M in ethanol, 20 mL), and
the flask was sealed. After stirring for 18 h at room temper-
ature, HPLC showed complete consumption of starting materi-
als. The mixture was concentrated and the residue was
subjected to hydrogenation (Pd/C, hydrogen, TFA, 24 h). The
mixture was concentrated and purified via preparative HPLC
Refer en ces
(1) Anonymous. From the Centers for Disease Control and Preven-
tion. Decline in deaths from heart disease and stroke United
States, 1900-1999. J AMA, J . Am. Med. Assoc. 1999, 282, 724-
726.
(2) Bick, R. L.; Kaplan, H. Syndromes of thrombosis and hyperco-
agulability. Congenital and acquired causes of thrombosis. Med.
Clin. North Am. 1998, 82, 409-458.
(3) Mousa, S. A. Highlights of latest advances in antithrombotics.
Methods Mol. Med. 2004, 93, 1-7. Turpie, A. G. G. Antithrom-
botics and anticoagulants in coronary syndromes and stroke.
Semin. Thrombosis Hemostasis 2000, 26 (Suppl. 1), 79-83.
Agnelli, G.; Sonaglia, F. Perspectives on antithrombotic agents:
from unfractionated heparin to new antithrombotics. Haemato-
logica 2002, 87, 757-770.
(4) Kimball, D. Thrombin active site inhibitors. Curr. Pharm. Des.
1995, 1, 441-468. Steinmetzer, T., Hauptmann, J .; Sturze-
becher, J . Expert Opin. Invest. Drugs 2001, 10, 845-864.
(5) Ewing, W. R.; Pauls, H. W.; Spada, A. P. Progress in the design
of inhibitors of coagulation factor Xa. Drugs Future 1999, 24,
771-787. Sinha, J . Synthetic inhibitors of coagulation factor Xa.
Expert Opin. Invest. Drugs 1999, 8, 567-573.
(6) Rapaport, S. I.; Rao, L. V. M. Initiation and regulation of tissue
factor-dependent blood coagulation. Arterioscler. Thromb. 1992,
12, 1111-1121.
(7) Harker, L. A.; Maraganore, J . M.; Hirsh, J . Novel antithrombotic
agents. In Hemostasis and Thrombosis: Basic Priniciples and
Clinical Practice, 3rd ed.; Colman, R. W.; Hirsh, J .; Marder, V.
J .; Eds.; Lippincott Company: Philadelphia, PA, 1994; pp 1638-
1660.
(8) Dudley, D. A.; Bunker, A. M.; Chi, L.; Cody, W. L.; Holland, D.
R. et al. Rational Design, synthesis, and biological activity of
benzoxazinones as novel Factor Xa inhibitors. J . Med. Chem.
2000, 43, 4063-4070.
(9) Walsmann, P. Inhibition of serine proteinases by benzamidine
derivatives. Acta Biol. Med. Ger. 1977, 36, 1931-1937. Phillips,
G. B., Buckman, B. O., Davey, D. D. Eagen, K. A., Guilford, W.
J . et al. Discovery of N-[2-[5-[amino(imino)methyl]-2-hydro-1-
methyl-(1H-imidazol-2-yl)phenoxy]pyridin-4-yl]-N-methylgly-
cine (ZK-807834): A potent and orally active inhibitor of the
blood coagulation enzyme factor Xa. J . Med. Chem. 1998, 41,
3557-3562.
(10) Berryman, K. A.; Downing, D. M.; Dudley, D. A.; Edmunds, J .
J .; Narasimhan, L. S. et al. Preparation of benzoxazinones and
-thiazinones as serine protease inhibitors. PCT Int. Appl.;
(Warner-Lambert Company, USA).: WO 9250257, 1999; Down-
ing, D. M., Berryman, K. A., Dudley, D. A., Zhu, Z., Pamment,
M. et al. Chiral benzoxazinones as potent and selective FXa
inhibitors. J . Org. Chem. 2004, submitted.
(11) 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.

Inhibitors of Blood Coagulation Factor Xa
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4099
(12) Gukasyan, A. K.; Galstyan, L. K.; Avetisyan, A. A. Investigations
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.
(21) Chi, L.; Saganek, L.; Rogers, K.; Mertz, T.; Metz, A.; et al. A
novel model of venous thrombosis in the vena cava of rabbits.
J . Pharmacol. Toxicol. Methods 1988, 39, 193-202.
(22) McClanahan, T. B.; Ignasiak, D. P.; J uneau, P.; Finkle, C.;
Winocour, P. D. et al. Antithrombotic effects of BCH 2763, a new
direct thrombin inhibitor, in a canine model of venous throm-
bosis. J . Thromb. Thrombolysis 1999, 7, 301-306; Ignasiak, D.
P.; McClanahan, T. B.; Bousley, R. E.; J uneau, P. L.; Gallagher,
K. P. Effects of intravenous enoxaparin and intravenous inogat-
ran in an electrolytic injury model of venous thrombosis in the
dog. J . Thromb. Thrombolysis 1998, 6, 199-206.
(23) Topliss, J . G. A manual method for applying the hansch approach
to drug design. Med. Chem., Proc. Int. Symp., 5th 1977, 421-
426. Topliss, J . G. Utilization of operational schemes for
analogue synthesis in drug design. J . Med. Chem. 1972, 15,
1006-1011.
(24) 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.
(25) Brunger, A. T. Xplor A system for X-ray crystallography and
NMR; Yale University Press: New Haven, CT, 1992.
(13) Granik, V. G. Advances in chemistry of amidines. Usp. Khim.
1983 52, 669-703.
(14) Dunn, P. J . Amidines and N-substituted amidines. Compr. Org.
Funct. Group Transform. 1995, 5, 741-782, 1161-1308.
(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) Duclos, R. I., J r.; Tung, J . S.; Rapoport, H. A high-yield
modification of the Pschorr phenanthrene synthesis. J . Org.
Chem. 1984, 49, 5243-5246.
(17) Selnick, H. G.; Smith, G. R.; Tebben, A. J . An improved
procedure for the cyanation of aryl triflates:
a convenient
synthesis of 6-cyano-1,2,3,4-tetrahydroisoquinoline. Synth. Com-
mun. 1995, 25, 3255-3261.
(18) Timari, G.; Soos, T.; Hajos, G. A convenient synthesis of two
new indoloquinoline alkaloids. Synlett 1997, 1067-1068.
(19) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling
reactions of organoboron compounds. Chem. Rev. (Washington,
D. C.) 1995, 95, 2457-2483.
(20) Nitta, I.; Fujimori, S.; Ueno, H. The syntheses of the corticoid
side chain. I. An improved method for the preparation of
17.alpha.-hydroxyprogesterone from androst-4-ene-3, 17-dione.
Bull. Chem. Soc. J pn. 1985, 58, 978-980.
J M0497491