Synthesis and crystal structures of substituted benzenes and benzoquinones as tissue factor VIIa inhibitors

Article abstract of DOI:10.1021/jm030233b

Several multistep syntheses of substituted benzenes are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S₁, S₂, and S₃ pockets of the tissue Factor VIIa enzyme. A variety of chemical transformations including nucleophilic additions, reductive aminations, Stille couplings, and polymer-assisted solution-phase (PASP) techniques were used to prepare key intermediates and final products. The initial analogues identified some weakly active compounds which ultimately led to a 340 nM (IC₅₀) tissue Factor VIIa inhibitor with selectivity over other related enzymes. The structure-activity relationship of these inhibitors and the synthetic progression from the discovery of the lead compound to the development of potent analogues will be discussed. The X-ray crystal structures of fluorobenzene 50c and benzoquinone 54 inhibitors complexed with the TF/VIIa enzyme will also be described.

Full text of DOI:10.1021/jm030233b

J . Med. Chem. 2003, 46, 4297-4312
4297
Syn th esis a n d Cr ysta l Str u ctu r es of Su bstitu ted Ben zen es a n d Ben zoqu in on es
a s Tissu e F a ctor VIIa In h ibitor s
J ohn J . Parlow,*,† Anna M. Stevens,§ Roderick A. Stegeman,§ William C. Stallings,§ Ravi G. Kurumbail,§ and
Michael S. South†
Department of Medicinal and Combinatorial Chemistry, Pharmacia Corporation, 800 North Lindbergh Boulevard,
St. Louis, Missouri 63167, and Structure and Computational Chemistry, Pharmacia Corporation,
700 Chesterfield Village Parkway, St. Louis, Missouri 63198
Received May 15, 2003
Several multistep syntheses of substituted benzenes are reported. The benzene analogues were
designed such that their substitution pattern would occupy and interact with the S1, S2, and
S3 pockets of the tissue Factor VIIa enzyme. A variety of chemical transformations including
nucleophilic additions, reductive aminations, Stille couplings, and polymer-assisted solution-
phase (PASP) techniques were used to prepare key intermediates and final products. The initial
analogues identified some weakly active compounds which ultimately led to a 340 nM (IC50)
tissue Factor VIIa inhibitor with selectivity over other related enzymes. The structure-activity
relationship of these inhibitors and the synthetic progression from the discovery of the lead
compound to the development of potent analogues will be discussed. The X-ray crystal structures
of fluorobenzene 50c and benzoquinone 54 inhibitors complexed with the TF/VIIa enzyme will
also be described.
In tr od u ction
Acute coronary syndromes (ACS) consisting of un-
stable angina, myocardial infarction, and sudden death
are the most frequent cause of mortality in the United
States and Western countries.1 ACS is associated with
acute thrombus formation, often as the result of plaque
rupture. Thrombosis occurs in transient ischemic attack,
stroke, peripheral occlusive arterial disease, deep vein
thrombosis, pulmonary embolism, abrupt closure fol-
F igu r e 1. Pyrazinone and benzene core structures.
lowing angioplasty, and the disseminated intravascular
coagulation associated with sepsis and certain cancers.
We previously reported the preparation of pyrazinone
Effective and safe antithrombotics are needed to combat
analogues as tissue Factor VIIa inhibitors.5 These
these diseases. Most research has focused on thrombin
pyrazinone compounds are active-site inhibitors for TF/
and Factor Xa inhibitors as potentially valuable thera-
VIIa exhibiting potency at the single-digit nanomolar
peutic agents for these diseases.2 More recently, small
level with excellent selectivity over thrombin (IIa) and
molecule inhibitors of tissue Factor (TF) VIIa have been
Factor Xa. In an effort to increase the potency and
the point of much research effort because of their
influence the pharmocokinetic properties, other core
potential to inhibit the coagulation cascade while less-
ring systems were evaluated. Depicted in Figure 1 is
ening the risk of bleeding side effects.3 The extrinsic
the lead pyrazinone structure I. Focusing on the central
coagulation cascade is triggered by the binding of Factor
ring, one of the exercises was to replace the nitrogens
VIIa to cell surface TF. This cascade is critical in normal
with carbons on the pyrazinone ring, resulting in
hemostasis, but is also involved in the pathogenesis of
benzene II and benzoquinone as central ring systems.6
various thrombotic diseases. Under normal conditions,
On the basis of the X-ray crystal structures of TF/VIIa,5,7
TF expressed in the subendothelium of healthy blood
several key interactions were crucial for both potency
vessels is not exposed to blood. However, in a disease
on TF/VIIa and selectivity over thrombin and Factor Xa.
state or during injury, TF comes in contact with Factor
It was found in the pyrazinone series that the benz-
VIIa and the ensuing TF/VIIa complex activates Factors
amidine binds, as expected, mediated by an ion-pair
X and IX to Xa and IXa, respectively. The complex of
between the basic amidine moiety and the carboxylate
Factor Xa and Factor Va on a membrane surface
of Asp 189. The substituted m-aminophenyl ring oc-
converts prothrombin to thrombin, leading to fibrin
cupies the S2 pocket, and the substituted amino group
formation, deposition, and subsequent thrombus forma-
at the 3-position of the pyrazinone occupies and inter-
tion.4
acts with the S3 pocket. In addition, the ketone at the
2-position on the pyrazinone ring forms a hydrogen bond
with the peptide backbone of Gly 216. The hypothesis
was that the benzene ring could orient the substituents
in the correct spatial arrangement to probe the S1, S2,
* To whom correspondence should be addressed. Phone: 314-274-
3494. Fax 314-274-1621. e-mail: john.j.parlow@pfizer.com.
† Department of Medicinal and Combinatorial Chemistry.
§ Department of Crystallography and Molecular Modeling.
10.1021/jm030233b CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/06/2003

4298 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Sch em e 1. Synthesis of Substituted Benzamidesa
Parlow et al.
a
Reagents: (a) TEA, THF, 65 °C; (b) (ClCO)2, DCM; (c) MeOH, pyridine; (d) Fe, MeCO2H, 80 °C; (e) 6, DIEA, DCM; (f) NaOH, MeOH,
65 °C; (g) 4 N HCl/dioxane.
and S3 pockets. In addition, the benzene ring could be
substituted at the 2-position with various hydrogen
bond accepting groups to engage Gly 216. Reported
herein is the discovery, synthesis, X-ray crystal struc-
tures, and biological activity of a series of novel substi-
tuted benzene analogues as tissue Factor VIIa inhibi-
tors.
relative to thrombin and Factor Xa, a phenyl ring was
selected as a potential group to probe the S2 pocket of
TF/VIIa with the hope of providing selectivity. To allow
for these types of substitutions, commercially available
2-fluoro-5-nitrobenzoic acid 1 was used as the starting
material. Nucleophilic displacement of 1 with thiophenol
2 afforded the triethylamine salt of 3. The salt 3 was
converted to the acid chloride using oxalyl chloride, and
quenching with methanol formed the methyl ester 4.
An iron reduction of the nitro compound 4 in acetic acid
afforded the aniline 5. The aniline 5 was reacted with
R-toluenesulfonyl chloride 6 to afford a mixture of the
mono-sulfonamide 7, bis-sulfonamide 8, and starting
material 5. Excess R-toluenesulfonyl chloride 6 was
added to consume the remaining starting material
affording both the mono- and bis-sulfonamide 7 and 8,
respectively. Hydrolysis of the methyl ester of the mono-
sulfonamide 7 with sodium hydroxide afforded the free
carboxylic acid 9. Hydrolysis of the bis-sulfonamide 8
resulted in both hydrolysis of the methyl ester and
hydrolysis of one of the sulfonamides, affording the same
Resu lts a n d Discu ssion
Early in the program, the first set of compounds was
designed in an attempt to occupy and interact with the
S1, S2, and S3 pockets of the enzyme. With little
knowledge of what was required for the key interactions,
the first set of analogues was prepared as shown in
Scheme 1. These compounds were designed with a
benzylsulfonamide at the 3-position of the benzene ring
to probe the S3 pocket. This moiety was chosen as a
starting point based on the structure-activity relation-
ship (SAR) from the thrombin literature and the simi-
larity of the S3 pocket of TF/VIIa with thrombin.
Considering the larger size of the S2 pocket of TF/VIIa

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4299
Sch em e 2. Thiophenol Displacementa
a
Reagents: (a) Fuming HNO3, -10 °C; (b) (ClCO)2, DCM; (c) MeOH, pyridine; (d) TEA, THF, reflux.
Ta ble 1. IC50 Values
desired acid 9. The intermediate compound 9 possessed
IC50 (µM)
the unsubstituted thiophenyl ring to occupy the S2
pocket and the benzylsulfonamide to occupy the S3
region. It was desirable at this stage of the synthesis to
rapidly prepare a variety of amides to occupy the S1
pocket. The carboxylic acid 9 was coupled with different
amines 10, each containing a basic moiety in an attempt
to engage the Asp 189. The amide coupling was ac-
complished in a parallel format using polymer-assisted
solution-phase (PASP) technology8 by treating the
acid with polymer-bound carbodiimide, hydroxybenzo-
triazole, and N-methylmorpholine as base followed by
addition of the amine 10 to afford the products 12. Upon
completion of the reaction, the polyamine and aldehyde
sequestering resins were added to each reaction vial
to remove any remaining benzoic acid/ester, hydroxy-
benzotriazole, and primary amine. Simple filtration and
rinsing with dichloromethane yielded a filtrate, where-
upon evaporation of the solvents afforded the desired
products 12a -d . The BOC protecting group of com-
pounds 12a and 12d was removed/deprotected using 4
N hydrochloric acid in dioxane followed by evaporation
to afford the desired products 13a and 13d , respectively.
The target compounds 13a -d were screened for potency
on tissue Factor VIIa, and for other enzymes affecting
coagulation to determine specificity including Factor Xa
and thrombin (Table 1). This set of compounds 13a -d
was inactive against the various enzymes with the
exception of 13a showing slight activity on thrombin.
The next set of analogues was designed with an
acetate linker between the benzene ring and the amide.
This would permit more flexibility and allow the basic
group on the amide to engage with the Asp 189, which
is thought to be the anchor point and essential for
biological activity. In addition to the acetate linker, a
fluorine in the 2-position of the benzene ring was
designed to hydrogen bond with Gly 216, allowing for
another point of interaction. Thus, a second synthesis
using 2,6-difluorophenylacetic acid 14 as the starting
material was considered as shown in Scheme 2. The
nitration of 2,6-difluorophenylacetic acid 14 using fum-
ing nitric acid afforded a single regioisomer 15 distin-
guished by proton and fluorine NMR. The phenylacetic
acid 15 was converted to the methyl ester 16 by forming
compd
VIIa
>30a
Xa
thrombin
13a
13b
13c
13d
29a
29b
29c
29d
29e
29f
29g
30b
50a
50b
50c
50d
50e
51
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
22% @30
>30
>30
>30
>30
26.9
>30
>30
>30
>30
>30
1.56
7.0
10
0.95
9.5
7.9
6.8
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
16.4
3.98
0.50
0.34
25
14.7
2.7
53
54
2.5
2.8
a
No inhibition was observed at a concentration of 30 µM.
the acid chloride with oxalyl chloride and reacting the
resulting acid chloride with methanol. Nucleophilic
displacement of the difluoro compound 16 with thio-
phenol 17 resulted in two isomers, compounds 18 and
19, with the major (undesired) product 18 resulting from
displacement of the fluorine at the 2-position.
Since nucleophlic displacement of the difluoro com-
pound 16 led to the undesired regioisomer 18, it was
decided to maintain the 6-fluoro and address the ability
to substitute at the 6-position at a later time. Continu-
ing with the synthesis in Scheme 3, reduction of the
nitro compound 16 using iron in acetic acid afforded the
aniline 21. Derivatization of the aniline nitrogen was
achieved using two different electrophiles. The first
involved reacting aniline 21 with an excess of R-tolu-
enesulfonyl chloride 6 to afford exclusively the bis-
sulfonamide 23. An excess of 6 was used to avoid
forming a mixture of both mono- and bis-sulfonamide.
Hydrolysis of the bis-sulfonamide 23 resulted in both
hydrolysis of the methyl ester and hydrolysis of one of
the sulfonamides, affording the desired acid 25. The
carboxylic acid 25 was coupled with a variety of amines,

4300 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Sch em e 3. Synthesis of 2,6-Difluorobenenzene Analoguesa
Parlow et al.
a
Reagents: (a) Fe, MeCO2H, 80 °C; (b) 6, DIEA, DCM; (c) NaHB(OAc)3, DCM, THF; (d) NaOH, MeOH, 65 °C.
Sch em e 4. Cbz Deprotection Protocola
each containing a basic moiety to engage Asp 189. The
amide coupling was run in a parallel format using PASP
technology as previously mentioned to afford the desired
amide products 27/29. Another compound was designed
with phenethylamine as the substituent at the 3-posi-
tion of the benzene ring. Reductive amination of aniline
21 with phenylacetaldehyde 22 using sodium triacetoxy-
borohydride afforded the desired phenethylamine prod-
uct 24. Reaction of 24 with sodium hydroxide at 65 °C
in methanol resulted in the desired carboxylic acid 26.
On the basis of the biological data generated with the
products 29, the carboxylic acid 26 was coupled with
only one of the amines, benzyl amino[4-(aminomethyl)-
phenyl]methylcarbamate, to afford the Cbz protected
amide 28b.
Several of the amine inputs for the amide coupling
were protected with either a BOC or Cbz protecting
group. The BOC protecting group was easily removed
by incubating the compound with 4 N hydrochloric acid
in dioxane followed by evaporation to afford the desired
products 29e and 29f (Scheme 5). A deprotection
protocol for the Cbz protecting group was designed
allowing for the reaction to be run in a parallel format
using PASP technology. The Cbz protecting group was
removed using an in-situ trimethylsilyl idodide (TMSI)
deprotection protocol.9 The procedure (Scheme 4) gener-
ates TMSI by stirring the compound in acetonitrile with
an excess of sodium iodide and adding trimethylsilyl
a
Reagents: (a) TMSCl, NaI, MeCN, 55 °C; (b) MeOH.
chloride followed by heating. Upon completion of the
reaction, methanol is added to quench the reaction and
react with the remaining TMSI, affording volatile
byproducts. Polyvinyl pyridine or a resin containing a
tertiary amine group is then added to sequester the
benzyl iodide byproduct 31 and remove the salts. It was
found that if a polymer-bound primary or secondary
amine is used as a sequestering resin with products that
contain an amidine group, the resin reacts with the
amidine group resulting in sequestration of the product.

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4301
Sch em e 5. 2,6-Difluorobenzene Products
synthesis of such analogues is shown in Scheme 7. The
hydroxy compounds 32 and 36 were reacted with triflic
anhydride to afford the triflates 40 and 41, respectively.
The triflates 40 and 41 underwent Stille couplings
with tri-n-butylphenylstannanes 39a and 39b using
tetrakis(triphenylphosphine)palladium(0) and lithium
chloride in dioxane at 65 °C to afford the 6-phenyl
derivatives 42a -c.11 Iron reduction of the nitro group
afforded the desired amines 43a -c. The Stille reaction
involving the 2-fluoro derivative 40 and stannane 39b
afforded the 3-aminobenzene 43b as the major product,
as reduction of the nitro group occurred during the Stille
coupling. Reductive amination of the amino compounds
43a -c with benzaldehyde 44, phenylacetaldehyde 45,
or acetone 46 using sodium triacetoxyborohydride af-
forded the substituted amines 47a -e. Hydrolysis of the
methyl ester using sodium hydroxide afforded the
carboxylic acids 48a -e. The acids 48a -e were coupled
with benzyl amino[4-(aminomethyl)phenyl]methylcar-
bamate to afford the Cbz protected products 49a -e.
Hydrogenation deprotection of the Cbz group was
used to afford the desired products 50a ,c-e. However,
hydrogenation of compound 49b resulted in debenzyl-
ation of the amine. As a result, hydrogen bromide in
acetic acid was used to afford the desired product 50b.
The in-situ trimethylsilyl iodide deprotection protocol
afforded the desired products 29a ,b and 30b as shown
in Scheme 5.
With the exception of 29b, which exhibited potency
on thrombin, the sulfonamide compounds 29a -g were
inactive. Of all of the amides tested thus far, only the
analogues with the p-benzamidine moiety have exhib-
ited low levels of activity. As a result, that amine input
was used for the amide coupling with the 3-phenethyl-
amine acid 26, followed by deprotection to afford the
desired product 30b (Scheme 5). Compound 30b exhib-
ited activity against TF/VIIa and thrombin (Table 1).
This was the first benzene analogue of the series that
demonstrated activity (IC50 ) 16.4 µM) against TF/VIIa.
With the TF/VIIa activity exhibited from the 6-fluoro
analogue 30b, a synthesis was required that would
allow for a phenyl ring substitution at the 6-position of
the benzene ring in an attempt to improve the binding
affinity by interacting with the S2 pocket. The reaction
of 2,6-difluorobenzeneacetate 16 with thiophenol 17
afforded the wrong regioisomer 18 as the major product
(Scheme 2). However, when 2,6-difluoroacetate 16 was
reacted with pivalic acid 31 using potassium carbonate
as the base in dimethyl sulfoxide at 80 °C (Scheme 6),10
the product mixture contained a 1:2 mixture of the
6-hydroxy 32 to 2-hydroxy 33 compounds as determined
by NMR. It is believed that the steric crowding of the
pivalic acid 31 influenced the regioselectivity. Both
regioisomers 32 and 33 can be utilized as starting
materials. The 6-hydroxybenzene 32 allows for substitu-
tion at the 6-position while maintaining the fluorine at
the 2-position. The 2-hydroxybenzene 33 permits sub-
stitution at the 6-position and derivatization at the
2-position.
In an effort to obtain an analogue with a hydroxy in
the 2-position for hydrogen bonding with the Gly 216,
the methyl ether 49d was reacted using the in-situ
trimethylsilyl idodide protocol (Scheme 8). Upon puri-
fication, the desired hydroxy product 51 was isolated
as the minor component with the lactone 52 as the
major product. Under the reaction conditions, intra-
molecular cyclization occurs with the hydroxy attacking
the amide carbonyl, followed by loss of the benzamidine
to afford the lactone product 52.
A benzoquinone ring system was prepared as shown
in Scheme 9. The benzoquinone maintains the ketone
in the same position as the pyrazinone I for hydrogen
bonding with Gly 216. The precursor 49e was prepared
as shown in Scheme 7. Compound 49e was designed
with the isopropylamine at the 3-position and the
unsubstituted phenyl ring at the 6-position. The un-
substituted phenyl ring was chosen over the amino-
phenyl ring at the 6-position to avoid quinone formation
of that phenyl ring. The synthesis was carried out as
shown in Scheme 9. The methoxybenzene 49e was
treated with boron tribromide to afford the phenol 53.
Under these conditions no sign of lactone formation was
observed. Reacting the phenol 53 with Fremy’s salt12
afforded the benzoquinone 54. The benzoquinone ana-
logues proved to be unstable and, as a result, further
synthetic efforts with benzoquinone as the core ring
were not pursued.
Both the 2-hydroxybenzene 33 and its 2-methoxy
derivative have the potential to hydrogen bond to Gly
216. The 2-methoxy starting material was prepared as
shown in Scheme 6. The 2-hydroxybenzene 33 was
reacted with methyl iodide to afford the methyl ether
34. Nucleophilic displacement of the 6-fluoro with
sodium hydoxide and concomitant hydrolysis of the ester
afforded the acetic acid 35. The acid 35 was converted
back to the methyl ester 36 by forming the acid chloride
and reacting with methanol. The methyl ester 36 was
desired due to the ease of purification over the acid in
the subsequent reactions.
All of the compounds were screened against several
serine protease enzymes involved in the coagulation
cascade, including TF/VIIa, thrombin (IIa), and Factor
Xa. Each enzyme assay consisted of the specific enzyme
and the chromogenic substrate for that enzyme. Re-
ported are the inhibition data as IC50 values for TF/VIIa
and thrombin (Table 1). The structure-activity rela-
tionships of the benzene series can be divided into four
parts: The amide which occupies the S1 pocket and
interacts with Asp 189, the 3-amino substituent that
occupies the S3 pocket, the substituent at the 6-position
The intermediates 32 and 36 allow for substitution
at the 6-position with (substituted) phenyl rings via
Stille couplings. They also allow for varying the
amines at the 3-position via reductive amination. The

4302 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Sch em e 6. Displacement Using Pivalic Acida
Parlow et al.
a
Reagents: (a) K2CO3, DMSO, 80 °C; (b) MeI, K2CO3, THF, 65 °C; (c) NaOH, THF, 90 °C; (d) (ClCO)2, DCM; (e) MeOH, pyridine.
that occupies the S2 pocket, and the group at the
2-position that hydrogen bonds with the peptide back-
bone. In general, these compounds were selective against
Factor Xa with no activity observed at concentrations
of 30 µM. Factor Xa has a tyrosine at position 60 and
the S2 pocket of Factor Xa is occluded by the side chain
of Tyr 99. Thus, selectivity against Factor Xa is probably
due to a collision of the P2 phenyl ring with the Tyr 99
of Factor Xa.
The first set of compounds 29 and 30 identified that
p-benzamidine as the amide was required for any type
of biological activity and that the p-benzamidine needed
to be connected by the acetate linker (Table 1).
Having identified compound 30b as showing promis-
ing activity, further enhancements were made to probe
the S2 pocket, leading to compound 50a . Compound 50a
maintained the substituents of 30b (2-fluoro-3-phen-
ethylamine), but added an unsubstituted phenyl ring
at the 6-position. This resulted in an increase in potency
on TF/VIIa (IC50 ) 3.98 µM) with selectivity against
Factor Xa and 2-fold selectivity against thrombin. The
S2 pocket of TF/VIIa is larger than that of thrombin and
Factor Xa and contains a key residue, Asp 60, which is
not present in thrombin and Factor Xa. In an attempt
to engage the Asp 60, an amine was placed on the
F igu r e 2. Crystal structure of compound 50c (fluorobenzene)
bound in the active site of TF/VIIa complex. Crystals of TF/
VIIa complex were obtained by slight modification of the
6-phenyl ring with compounds 50b and 50c. An increase
in potency was observed with 50c, which is the most
potent compound of the series (IC50 ) 340 nM) on TF/
VIIa. In addition to the potency, the amine substituent
enhanced the selectivity profile of both 50b and 50c.
There was no activity on Factor Xa at 30 µM and a 20-
fold selectivity factor over thrombin with 50b.
procedure described by Banner et al. (ref 7a). The structure
has been refined to an Rfree of 29.1% at 2.4 Å resolution (Rcrystal
23.7%). Some of the key side chains of Factor VIIa are
displayed (C: green, N: dark blue, O: red, S: yellow, and H:
orange). The inhibitor is represented with carbon, nitrogen,
oxygen, and fluorine atoms displayed in gold, blue, red, and
purple, respectively. The hydrogen bonds formed by the
inhibitor are shown in dotted white lines.
:
An X-ray crystal structure of compound 50c bound
to TF/VIIa is shown in Figure 2. The bound conforma-
tion of fluorobenzene inhibitor 50c in the active site of
VIIa resembles that of pyrazinone inhibitors that have
been reported previously.5 The benzamidine moiety
engages the carboxylate of Asp 189 in the S1 site, similar
to the interactions observed in the crystal structures of
other serine proteases. The peptide nitrogen of the
acetate linker forms a hydrogen bond (3.2 Å) with the
carbonyl oxygen of Ser 214. The anilino nitrogen at-
tached to the central fluorobenzene core donates a
hydrogen bond (3.4 Å) to the main chain oxygen of Gly
216. As anticipated, the fluorine atom in the central ring
accepts a hydrogen bond (3.4 Å) from the amide nitrogen
of Gly 216. Some of these hydrogen bonds are longer
and perhaps indicate nonoptimized interactions of the
inhibitor in the active site. This might explain the
relatively weaker binding affinity of this class of inhibi-
tors compared to that of pyrazinone inhibitors. The
amino group attached to the P2 phenyl ring forms tight
interactions with Asp 60, Tyr 94, and the carbonyl
oxygen of Thr 98. Potential collision of the phenyl group
of the inhibitor at P2 with the side chain of Tyr 99 in
Factor Xa is probably why it does not inhibit Factor Xa.
The S3 pocket was accommodating to all 3-amino
substituents (phenethylamine, benzylamine, and iso-
propylamine). In the 2-fluoro series, the 3-isopropyl-

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4303
Sch em e 7. Synthesis of Substituted Benzene TF/VIIa Inhibitorsa
a
Reagents: (a) (CF3SO2)2O, TEA, DCM, -10 °C; (b) (Ph3P)4Pd, LiCl, dioxane, 65 °C; (c) Fe, MeCO2H, 80 °C; (d) NaHB(OAc)3, DCM,
THF; (e) NaOH, MeOH, 65 °C; (f) H2, Pd/C, MeOH; (g) HBr, MeCO2H.
amine (50c) was ∼2-fold more potent than the 3-benzyl-
amine (50b). In the 2-methoxy series, the 3-isopropyl-
amine (50e) was ∼2-fold more potent than the 3-phen-
ethylamine (50d ). Thus, the small aliphatic isopropyl
group was the most potent 3-amino substituent of the
series.
compound exhibited modest potency on TF/VIIa with
selectivity over both thrombin and Factor Xa.
The crystal structure of the benzoquinone compound
54 bound to TF/VIIa is shown in Figure 3. The binding
orientation of the benzoquinone inhibitor 54 is similar
to that of the fluorobenzene 50c. The ion-pair that is
formed by the amidine moiety of the inhibitor with the
carboxylate of Asp 189 functions as the main anchor for
the inhibitor in the enzyme active site. In addition to
this, the amidine group also forms hydrogen bonds to
the main chain carbonyl of Gly 219 and the hydroxyl
group of the side chain of Ser 190. Three other hydrogen
bonds are formed by the inhibitor with the peptide
backbone of residues Ser 214 -Gly 216 of VIIa. The
amide nitrogen of the acetate linker interacts with the
main chain oxygen of Ser 214 (3.2 Å) while the second-
The ketone at the 4-position on the pyrazinone ring
hydrogen bonds with Gly 216 of the peptide backbone.
In an attempt to maintain this key interaction with Gly
216, the benzene ring was substituted with various
hydrogen bond accepting groups at the 4-position in-
cluding fluorine, hydroxy, and methoxy. The fluoro and
hydroxy analogues had roughly the same potency on TF/
VIIa, whereas the methoxy was ∼10-fold less active.
Included in this set was a benzoquinone 54 which
maintained the ketone from the pyrazinone core. This

4304 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Sch em e 8. Synthesis of 2-Hydroxy Analoguea
Parlow et al.
a
Reagents: (a) TMSCl, NaI, MeCN, 55 °C.
Sch em e 9. Benzoquinone Synthesisa
F igu r e 3. Crystal structure of compound 54 (benzoquinone)
bound in the active site of TF/VIIa. The structure was refined
to an Rfree of 28.0% at 2.2 Å resolution (Rcrystal: 22.5%). The
atoms are colored as in Figure 2. Some of the key side chains
of Factor VIIa are displayed. The hydrogen bonds are shown
as dotted lines (magenta). One of the quinone oxygen atoms
accepts a hydrogen bond from the amide nitrogen of Gly 216
as observed in the structures of pyrazinone inhibitors.
zene 50c and benzoquinone 54 inhibitors bound to TF/
VIIa were obtained. The crystal structure of the fluo-
robenzene 50c analogue clearly shows that the fluorine
acts as the hydrogen bond acceptor and engages with
Gly 216. Similarly, one of the ketones of the benzo-
quinone 54 registers with the peptide backbone of Gly
216 via a hydrogen bond. These compounds exhibit
modest potency for TF/VIIa and some of them display
selectivity over Factor Xa and thrombin.
a
Reagents: (a) 1 M BBr3, DCM, -10 °C; (b) ON(SO3K)2, THF,
Exp er im en ta l Section
H2O.
Gen er a l. Solvents and chemicals were reagent grade or
better and were obtained from commercial sources. All poly-
meric reagents and sequestering resins were obtained from
ary nitrogen attached to the quinone scaffold donates a
hydrogen bond (3.0 Å) to the carbonyl oxygen of Gly 216.
One of the quinone oxygen atoms accepts a hydrogen
bond (3.3 Å) from the peptide nitrogen of Gly 216 as
anticipated in our design. The other quinone oxygen
forms van der Waals interactions (3.5 Å) with the
carbonyl oxygen of Gly 97.
In summary, we have prepared a novel series of tissue
Factor VIIa inhibitors. The initial set of analogues
identified some weakly active compounds. Optimization
of the initial lead was accomplished by novel synthesis
of benzene analogues to occupy and interact with the
S1, S2, and S3 pockets of the TF/VIIa enzyme. The
substituted benzene analogues were successfully pre-
pared via multistep synthesis using various chemical
transformations and regioselective additions with a
benzene nucleus to afford novel benzene derivatives. In
addition, a novel substituted benzoquinone analogue
was prepared. The X-ray crystal structures of fluoroben-
1
commercial sources. H and 13C NMR spectra were recorded
using a 300 or 400 MHz NMR spectrometer. Sample purities
were determined by HPLC analysis equipped with a mass spec
detector using a C18 3.5 um 30 × 2.1 mm column, eluting with
a gradient system of 5/95 to 95/5 acetonitrile/H2O with a buffer
consisting of 0.1% TFA over 4.5 min at 1 mL/min and detected
by UV at 254 and 210 nm using a diode array detector. Column
chromatography was performed on a preparative liquid chro-
matography instrument using silica gel columns and on a
HPLC system using a 15 µm 100A, C18 column (25 mm I.D.
× 100 mm length). Reported yields are not optimized with
emphasis on purity of products rather than quantity.
5-Nitr o-2-(p h en ylth io)ben zoic Acid (3). Triethylamine
(8.3 mL, 0.060 mol) was added to a solution of 2-fluoro-5-
nitrobenzoic acid 1 (5.0 g, 0.027 mol) and thiophenol 2 (2.8
mL, 0.027 mol) in tetrahydrofuran. After stirring at reflux for
20 h, a saturated solution of ammonium chloride was added
until solution became neutral. The solution was extracted with
dichloromethane, and the organic layer was washed with

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4305
water, brine, dried over magnesium sulfate, and filtered. The
solvent was removed by evaporation to afford 8.85 g (87%) of
a yellow solid of product 3; H NMR ppm: 1.40 (t, 9H), 3.21
solution of the methyl ester (1.0 mmol) in methanol and the
resulting solution stirred at 65 °C for one to several hours.
The solution was acidified with 2 N hydrochloric acid and
extracted with diethyl ether. The solution was washed with
brine, dried over magnesium sulfate, and filtered. The solvent
was removed by evaporation to afford the product. When
necessary, the product was purified by column chromatogra-
phy.
1
(q, 6H), 6.75 (d, 1H, J ) 8.7 Hz), 7.47 (m, 3H), 7.57 (m, 2H),
7.85 (d, 1H, J ) 8.7 Hz), 8.85 (d, 1H, J ) 2.4 Hz); HRMS calcd
for C13H9O4N1S1 (M+ + NH4) 293.0596, found 293.0602.
Gen er a l P r oced u r e A. P r ep a r a t ion of Ca r b oxyla t e
Ester s. Oxalyl chloride (5.0 mmol) was added to a solution of
the carboxylic acid (1.0 mmol) in dichloromethane followed by
a drop of dimethylformamide. After being stirred at room
temperature for one to several hours, the solvent was removed
by evaporation to afford the acid chloride. The acid chloride
was redissolved into dichloromethane and an excess of metha-
nol (50 mL) was added followed by addition of pyridine (1.4
mmol). The solution stirred at room temperature for one to
several hours. The solution was washed with water, brine,
dried over magnesium sulfate, and filtered. The solvent was
removed by evaporation to afford the crude product. When
necessary, the product was purified by column chromatogra-
phy.
5-[(Ben zylsu lfon yl)a m in o]-2-(p h en ylth io)ben zoic Acid
(9). General procedure C. Using sodium hydroxide (3.0 mL,
7.5 mmol) and methyl ester 7 (0.70 g, 1.7 mmol) to afford 0.66
g (98%) of a white solid of product 9;
1H NMR ppm: 3.86 (s,
2H), 6.31 (dd, 1H), 6.83 (m, 6H), 6.97 (m, 3H), 7.06 (m, 2H),
7.52 (d, 1H), 9.35 (s, 1H); HPLC purity (retention time): 93.9%
(3.69 min); HRMS calcd for C20H17O4N1S2 (M+ + NH4) 417.0943,
found 417.0933.
Gen er a l P r oced u r e D. Am id e Cou p lin g. Under condi-
tions of parallel reaction synthesis, 1-hydroxybenzotriazole
(0.10 mmol) was added to a solution or slurry of the acid (0.10
mmol) and PS-carbodiimide (1.00 mmol/g) (0.20 mmol) in a
mixture of 3 mL of dichloromethane and 0.5 mL of dimethyl-
formamide. The suspension was agitated for 15 min. The
amine (0.10 mmol) was added, along with N-methylmorpholine
(0.12 mmol) when the amine is a hydrochloride salt. The
suspension was agitated for one to several hours. Upon
completion of the reaction, the polyamine resin (2.69 mmol/g)
(1.0 mmol) and polymer-bound aldehyde (2.3 mmol/g) (1.0
mmol) was added, and the suspension was agitated for one to
several hours. The solution was filtered and the polymer was
rinsed with dimethylformamide and dichloromethane until no
more UV activity was seen in the dichloromethane washing.
The combined filtrate and washings were evaporated to afford
the product. When necessary, the product was purified by
column chromatography.
Meth yl 5-Nitr o-2-(p h en ylth io)ben zoa te (4). General pro-
cedure A. Using oxalyl chloride (7.7 mL, 0.088 mol), acid 3
(6.68 g, 0.017 mol), and pyridine (2.0 mL, 0.024 mol) to afford
the crude product. The product was purified by column
chromatography (30% ethyl acetate-hexane) to afford 4.21 g
(82%) of a yellow solid of product 4; 1H NMR ppm: 4.05 (s,
3H), 6.89 (d, 1H, J ) 9.0 Hz), 7.61 (m, 5H), 8.05 (dd, 1H, J )
8.7, 2.4 Hz), 8.88 (d, 1H, J ) 2.4 Hz); HRMS calcd for
C
14H11O4N1S1 (M+ + H) 290.0487, found 290.0491.
Gen er a l P r oced u r e B. Ir on R ed u ct ion of t h e Nit r o
Gr ou p . The substituted nitrobenzene (1.0 mmol) was stirred
in glacial acetic acid (8 mL). Powdered iron (5.0 mmol) was
added, and the solution was heated to 80 °C with vigorous
stirring. The solution was stirred at 80 °C for 15-60 min at
which point the iron had turned gray. The reaction mixture
was filtered through Celite, and the solid was washed with
diethyl ether. The resultant organic layer was washed with
water, stirred with saturated sodium bicarbonate until basic,
and washed with water again. The solution was dried over
magnesium sulfate and filtered, and the solvent was removed
by evaporation to afford the product. When necessary, the
product was purified by column chromatography.
ter t-Bu tyl [4-({[5-[(Ben zylsu lfon yl)a m in o]-2-(p h en yl-
t h io )b e n zo y l]a m in o }m e t h y l)p h e n y l](im in o )m e t h y l-
ca r ba m a te (12a ). General procedure D afforded 89.9 mg
(71%) of a clear oil of product 12a ; 1H NMR ppm: 1.56 (s, 9H),
4.31 (s, 2H), 4.52 (d, 2H), 7.12-7.68 (m, 19H); HPLC purity
(retention time): 66.4% (3.41 min).
5-[(Ben zylsu lfon yl)a m in o]-2-(p h en ylth io)-N-(p yr id in -
4-ylm eth yl)ben za m id e (12b). General procedure D afforded
1
30.5 mg (62%) of a white solid of product 12b; H NMR ppm:
Meth yl 5-Am in o-2-(p h en ylth io)ben zoa te (5). General
procedure B. Using the nitro compound 4 (3.71 g, 0.012 mmol)
and powdered iron (3.58 g, 0.064 mmol) to afford 2.48 g (75%)
4.35 (s, 2H), 4.55 (d, 2H), 7.13 (d, 2H), 7.26 (m, 13H), 7.66 (s,
2H), 8.39 (d, 2H); HPLC purity (retention time): >99% (3.00
min); HRMS calcd for C26H23O3N3S2 (M+ + H) 490.1259, found
490.1222.
5-[(Ben zylsu lfon yl)a m in o]-N-[4-(d im eth yla m in o)ben -
zyl]-2-(p h en ylth io)ben za m id e (12c). General procedure D
afforded 30.0 mg (70%) of a white solid of product 12c; 1H NMR
ppm: 2.95 (s, 6H), 4.35 (s, 2H), 4.44 (d, 2H), 6.70 (d, 2H), 7.23
(m, 16H), 7.62 (s, 1H); HPLC purity (retention time): >99%
(3.15 min); HRMS calcd for C29H29O3N3S2 (M+ + H) 532.1729,
found 532.1681.
1
of a yellow oil of product 5; H NMR ppm: 3.90 (s, 3H), 6.89
(dd, 1H, J ) 8.4, 2.7 Hz), 6.96 (d, 1H, J ) 8.4 Hz), 7.22 (d, 1H,
J ) 2.7 Hz), 7.32 (m, 5H); HPLC purity (retention time): >99%
(2.81 min); HRMS calcd for C14H13O2N1S1 (M+ + H) 260.0745,
found 260.0718.
Meth yl 5-[(Ben zylsu lfon yl)a m in o]-2-(p h en ylth io)ben -
zoa te (7). Diisopropylethylamine (2.5 mL, 1.42 mmol) was
added to a solution of the aniline 5 (2.46 g, 9.48 mmol) and
R-toluenesulfonyl chloride 6 (3.17 g, 16.6 mmol) in dichlo-
romethane, and the resulting solution stirred at room tem-
perature for 3 h. The solution was washed with 2 N hydro-
chloric acid and brine, dried over magnesium sulfate, and
filtered. The solvent was removed by evaporation to afford a
mixture of two products. Fraction two of column chromatog-
raphy (30% ethyl acetate-hexane) afforded 1.16 g (30%) of a
ter t-Bu tyl 4-({[5-[(Ben zylsu lfon yl)am in o]-2-(ph en ylth io)-
ben zoyl]a m in o}m eth yl)p ip er id in e-1-ca r boxyla te (12d ).
General procedure D afforded 58.4 mg (98%) of a clear oil of
1
product 12d ; H NMR ppm: 1.21 (m, 2H), 1.45 (s, 9H), 1.62
(m, 3H), 2.63 (m, 2H), 3.25 (m, 2H), 4.03 (m, 2H), 4.35 (s, 2H),
7.27 (m, 13H), 7.66 (s, 1H); HPLC purity (retention time):
>99% (4.41 min).
1
white solid of product 7; H NMR ppm: 3.98 (s, 3H), 4.32 (s,
2H), 6.66 (s, 1H), 6.80 (d, 1H), 7.28 (dd, 1H), 7.29-7.73 (m,
11H); HPLC purity (retention time): 97.2% (4.20 min); HRMS
calcd for C21H19O4N1S2 (M+ + NH4) 431.1099, found 431.1069.
Met h yl 5-[Bis(b en zylsu lfon yl)a m in o]-2-(p h en ylt h io)-
ben zoa te (8). Following the same procedure described for 7,
fraction one of column chromatography (30% ethyl acetate-
hexane) afforded 2.64 g (49%) of a white solid of product 8; 1H
NMR ppm: 3.94 (s, 3H), 4.85 (s, 4H), 6.22 (dd, 1H, J ) 9.0,
2.4 Hz), 6.44 (d, 1H, J ) 9.0 Hz), 7.07 (d, 1H, J ) 2.4 Hz),
7.40 (m, 7H), 7.50 (m, 9H); HRMS calcd for C28H25O6N1S3 (M+
+ NH4) 585.1188, found 585.1141.
Gen er a l P r oced u r e E. BOC Dep r otection . Under condi-
tions of parallel reaction synthesis, hydrochloric acid in
dioxane (4 N) (3 mL) was added to the BOC protected
compound, and the solution was agitated at room temperature
for one to several hours. Evaporation of the solvents afforded
the product.
N-{4-[Am in o(im in o)m eth yl]ben zyl}-5-[(ben zylsu lfon yl)-
a m in o]-2-(p h en ylth io)ben za m id e (13a ). General procedure
E afforded 89.9 mg (71%) of a clear oil of product 13a ; 1H NMR
ppm: 4.85 (s, 2H), 4.90 (d, 2H), 7.52-8.22 (m, 18H), 9.31 (m,
1H), 9.82 (bs, 1H), 10.22 (s, 1H), 10.49 (s, 1H); HPLC purity
(retention time): >83.7%(3.15min);HRMS calcd for C28H26O3N4S2
(M+ + H) 531.1525, found 531.1583.
Gen er a l P r oced u r e C. Hyd r olysis of th e Meth yl Ester .
Aqueous sodium hydroxide (10%) (4.5 mmol) was added to a

4306 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Parlow et al.
5-[(Ben zylsu lfon yl)am in o]-2-(ph en ylth io)-N-(piper idin -
4-ylm eth yl)ben za m id e (13d ). General procedure E afforded
35.3 mg (71%) of a clear oil of product 13d ; 1H NMR ppm: 1.31
(m, 2H), 2.17 (m, 2H), 3.14 (m, 3H), 3.59 (m, 2H), 3.79 (m,
2H), 4.73 (s, 2H), 7.52 (m, 12H), 7.71 (s, 1H), 8.62 (bs, 1H),
9.60 (m, 1H), 10.34 (s, 1H); HPLC purity (retention time):
>99% (2.97 min); HRMS calcd for C26H29O3N3S2 (M+ + H)
496.1729, found 496.1710.
(2,6-Diflu or o-3-n itr op h en yl)a cetic Acid (15). 2,6-Dif-
luoropheylacetic acid 14 (5.0 g, 0.029 mol) was added in small
portions over a period of 20 min to fuming nitric acid chilled
to -10 °C (methanol/ice). The addition was closely monitored
to keep the temperature below 5 °C. Upon complete addition,
the solution stirred at -10 °C for 15 min. The solution was
poured over ice/water and extracted with diethyl ether. The
organic layer was washed with brine, dried over magnesium
sulfate, and filtered. The solvent was removed by evaporation
to afford 5.75 g (91%) of a white solid of product 15; 1H NMR
ppm: 3.89 (s, 2H), 7.11 (m, 1H), 8.16 (m, 1H); 19F NMR ppm:
-101.66 (m, 1F), -115.43 (m, 1F); HRMS calcd for C8H5O4N1F2
(M+) 217.0186, found 217.0166.
hydrochloric acid and brine, dried over magnesium sulfate,
and filtered. The solvent was removed by evaporation to afford
the crude product. The product was purified by column
chromatography (30% ethyl acetate-hexane) to afford 6.2 g
(61%) of a tan solid of product 23; 1H NMR ppm: 3.73 (s, 2H),
3.75 (s, 3H), 4.63 (d, 2H), 5.10 (d, 2H), 5.91 (m, 1H), 6.47 (m,
1H), 7.45 (m, 10H); 19F NMR ppm: -109.09 (m, 1F), -115.57
(m, 1F); HPLC purity (retention time): 85.3% (4.45 min);
HRMS calcd for C23H21O6N1S2F2 (M+ + NH4) 527.1122, found
527.1122.
Gen er a l P r oced u r e F . Red u ctive Am in a tion . Sodium
triacetoxyborohydride (4.0 mmol) was added to a solution of
the aniline (1.0 mmol), aldehyde or ketone (1.0 mmol), and a
drop of acetic acid in a tetrahydrofuran-dichloromethane
(1:1) solution. After being stirred at room temperature for one
to several hours, the solution was diluted with diethyl ether
and water. The organic layer was washed with brine, dried
over magnesium sulfate, and filtered. The solvent was removed
by evaporation to afford the product. When necessary, the
product was purified by column chromatography.
Meth yl {2,6-Diflu or o-3-[(2-ph en yleth yl)am in o]ph en yl}-
a ceta te (24). General procedure F. Using sodium triacetoxy-
borohydride (7.2 g, 0.033 mol), aniline 21 (1.72 g, 0.0085 mol),
and phenylacetaldehyde 22 (2.0 mL, 0.015 mol) to afford a
mixture of two products. Fraction two of column chromatog-
raphy (10% ethyl acetate-hexane) afforded 1.06 g (41%) of a
clear oil of product 24; 1H NMR ppm: 2.97 (t, 2H), 3.42 (t,
2H), 3.73 (s, 2H), 3.75 (s, 3H), 6.62 (m, 1H), 6.82 (m, 1H), 7.29
(m, 5H); 19F NMR ppm: -131.28 (m, 1F), -137.00 (m, 1F);
HPLC purity (retention time): 96.1% (3.93 min); HRMS calcd
for C17H17O2N1F2 (M+ + H) 306.1306, found 306.1309.
{3-[(B e n zy ls u lfo n y l)a m in o ]-2,6-d iflu o r o p h e n y l}-
a cetic Acid (25). General procedure C. Using sodium hydrox-
ide (10%) (10.3 mL, 0.025 mol) and methyl ester 23 (3.29 g,
0.0064 mol) to afford 2.1 g (95%) of a white solid of product
25; 1H NMR ppm: 3.87 (s, 2H), 4.60 (s, 2H), 7.11 (m, 1H), 7.51
(m, 6H), 9.77 (s, 1H); 19F NMR ppm: -119.10 (m, 1F), -123.54
(m, 1F); HPLC purity (retention time): >99% (2.86 min);
HRMS calcd for C15H13O4N1S1F2 (M+ + NH4) 359.0877, found
359.0867.
{2,6-Diflu or o-3-[(2-p h en ylet h yl)a m in o]p h en yl}a cet ic
Acid (26). General procedure C. Using sodium hydroxide
(10%) (5.5 mL, 13.7 mmol) and methyl ester 24 (1.06 g, 3.47
mmol) to afford 0.93 g (92%) of a white solid of product 26; 1H
NMR ppm: 2.64 (t, 2H), 3.02 (t, 2H), 3.19 (s, 2H), 6.46 (m,
1H), 6.75 (m, 6H), 7.10 (bd, 1H), 9.30 (bs, 1H); 19F NMR ppm:
-119.37 (m, 1F), -128.26 (m, 1F); HPLC purity (retention
time): >99% (3.26 min); HRMS calcd for C16H15O2N1F2 (M+
+ H) 292.1149, found 292.1150.
Meth yl (2,6-Diflu or o-3-n itr op h en yl)a ceta te (16). Gen-
eral procedure A. Using oxalyl chloride (12.6 mL, 0.144 mol),
acid 15 (5.75 g, 0.026 mol), and pyridine (3.5 mL, 0.043 mol)
to afford the crude product. The product was purified by
column chromatography (30% ethyl acetate-hexane) to afford
5.47 g (91%) of an orange oil of product 16; 1H NMR ppm: 3.78
(s, 3H), 3.84 (s, 2H), 7.09 (m, 1H), 8.12 (m, 1H); 19F NMR ppm:
-101.76 (m, 1F), -115.58 (m, 1F); HPLC purity (retention
time): >99% (2.88 min); HRMS calcd for C9H7O4N1F2 (M+
+
NH4) 249.0660, found 249.0687.
Meth yl [6-Flu or o-3-n itr o-2-(p h en ylth io)p h en yl]a ceta te
(18). Triethylamine (3.8 mL, 0.0270 mol) was added to a
solution of methyl 2,6-difluoro-3-nitrophenyl acetate 16 (5.4
g, 0.023 mol) and thiophenol 17 (2.4 mL, 0.023 mol) in
tetrahydrofuran. After stirring at reflux for 20 h, a 2 N
hydrochloric acid solution was added until solution became
acidic. The solution was extracted with diethyl ether, and the
organic layer was washed with water and brine, dried over
magnesium sulfate, and filtered. The solvent was removed by
evaporation to afford a mixture of two products. Fraction one
of column chromatography (10% ethyl acetate-hexane) af-
forded 4.41 g (60%) of a yellow oil of product 18; 1H NMR
ppm: 3.57 (s, 3H), 3.98 (d, 2H), 7.29 (m, 6H), 7.76 (m, 1H);
19F NMR ppm: -104.28 (m, 1F); 13C NMR ppm (deuteriochlo-
roform): 33.3, 52.5, 117.1, 124.9, 127.5, 129.1, 129.3, 129.5,
129.6, 130.4, 134.4, 161.1, 164.5, 169.4; HPLC purity (retention
time): 83% (4.02 min); HRMS calcd for C15H12O4N1S1F1 (M+
+ NH4) 339.0809, found 339.0797.
Meth yl [2-Flu or o-3-n itr o-6-(p h en ylth io)p h en yl]a ceta te
(19). Following the same procedure described for 18, fraction
two of column chromatography (30% ethyl acetate-hexane)
afforded 1.2 g (16%) of a yellow oil of product 19; 1H NMR
ppm: 3.78 (s, 3H), 3.99 (d, 2H), 6.80 (dd, 1H), 7.49 (m, 5H),
7.83 (m, 1H); 19F NMR ppm: -119.24 (d, 1F); 13C NMR ppm
(deuteriochloroform): 32.0, 52.8, 123.2, 125.0, 130.0, 130.4,
134.8, 149.6, 152.5, 156.0, 169.5; HPLC purity (retention
time): 83% (4.02 min); HRMS calcd for C15H12O4N1S1F1 (M+
+ NH4) 339.0809, found 339.0793.
Meth yl (3-Am in o-2,6-d iflu or op h en yl)a ceta te (21). Gen-
eral procedure B. Using the nitro compound 16 (6.87 g, 0.029
mmol) and powdered iron (8.29 g, 0.148 mmol) to afford the
crude product. The product was purified by column chroma-
tography (30% ethyl acetate-hexane) to afford 4.07 g (68%)
of a clear oil of product 21; 1H NMR ppm: 3.62 (bs, 2H), 3.71
(s, 2H), 3.74 (s, 3H), 6.70 (m, 2H); 19F NMR ppm: -115.58 (m,
1F), -129.12 (m, 1F); HPLC purity (retention time): >99%
(1.57 min); LRMS for C9H9O2N1F2 (ES, m/z) 202 (M + H).
Ben zyl (1Z)-Am in o(3-{[({3-[(b en zylsu lfon yl)a m in o]-
2,6-d i flu o r o p h e n y l}a c e t y l)a m i n o ]m e t h y l}p h e n y l)-
m eth ylid en eca r ba m a te (27a ). General procedure D afforded
37.7 mg (62%) of a clear oil of product 27a ; 1H NMR ppm: 3.62
(m, 2H), 4.29 (s, 2H), 4.42 (d, 2H), 5.12 (s, 2H), 6.80 (m, 2H),
7.38 (m, 14H), 7.71 (m, 2H); 19F NMR ppm: -117.61 (m, 1F),
-125.19 (m, 1F); HPLC purity (retention time): 69.6% (3.25
min); LRMS for C31H28O5N4S1F2 (ES, m/z) 607 (M + H).
Ben zyl (4-{[({3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu or o-
p h e n yl}a ce t yl)a m in o]m e t h yl}p h e n yl)(im in o)m e t h yl-
ca r ba m a te (27b). General procedure D afforded 49.9 mg
(82%) of a clear oil of product 27b; 1H NMR ppm: 3.58 (m,
2H), 4.27 (s, 2H), 4.32 (d, 2H), 5.18 (s, 2H), 6.74 (m, 2H), 7.14
(d, 2H), 7.37 (m, 12H), 7.68 (d, 2H); 19F NMR ppm: -117.58
(m, 1F), -124.63 (m, 1F); HPLC purity (retention time): 81.7%
(3.14 min); LRMS for C31H28O5N4S1F2 (ES, m/z) 607 (M + H).
ter t-Bu t yl 4-{[({3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu o-
r op h en yl}a cet yl)a m in o]m et h yl}p ip er id in e-1-ca r b oxyl-
a te (27e). General procedure D afforded 67.2 mg (100%) of a
1
Met h yl {3-[Bis(b en zylsu lfon yl)a m in o]-2,6-d iflu or o-
p h en yl}a ceta te (23). Diisopropylethylamine (5.1 mL, 0.029
mol) was added to a solution of the aniline 21 (4.0 g, 0.019
mmol) and R-toluenesulfonyl chloride 6 (8.34 g, 0.042 mol) in
dichloromethane, and the resulting solution stirred at room
temperature for 20 h. The solution was washed with 2 N
clear oil of product 27e; H NMR ppm: 1.14 (m, 2H), 1.47 (s,
9H), 1.65 (m, 3H), 2.69 (m, 2H), 3.18 (m, 2H), 3.62 (s, 2H),
4.10 (m, 2H), 4.38 (s, 2H), 6.01 (m, 1H), 6.90 (m, 2H), 7.35
(m, 6H); 19F NMR ppm: -117.94 (m, 1F), -126.43 (m, 1F);
HPLC purity (retention time): 56.6% (3.57 min); LRMS for
C
26H33O5N3S1F2 (ES, m/z) 560 (M + Na).

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4307
Bis-ter t-b u t yl (1E)-Am in o({5-[({3-[(b en zylsu lfon yl)-
a m in o]-2,6-d iflu or op h en yl}a cetyl)a m in o]p en tyl}a m in o)-
d im eth ylid en eca r ba m a te (27f). General procedure D af-
NMR ppm: 1.04 (m, 2H), 1.32 (m, 3H), 2.35 (m, 3H), 2.86 (m,
2H), 3.06 (s, 2H), 3.78 (s, 2H), 6.26 (m, 1H), 6.74 (m, 6H), 7.57
(bs, 1H), 8.73 (m, 1H), 8.99 (m, 1H); 19F NMR ppm: -118.93
(m, 1F), -123.81 (m, 1F); HPLC purity (retention time): 88.1%
(2.36 min); HRMS calcd for C21H25O3N3S1F2 (M+ + H) 438.1663,
found 438.1642.
1
forded 67.1 mg (100%) of a clear oil of product 27f; H NMR
ppm: 1.57 (m, 18H), 1.58 (m, 6H), 3.26 (m, 2H), 3.40 (m, 2H),
3.60 (s, 2H), 4.35 (s, 2H), 6.10 (m, 1H), 6.86 (m, 1H), 7.37 (m,
8H), 8.31 (m, 1H); 19F NMR ppm: -117.86 (m, 1F), -125.96
(m, 1F); HPLC purity (retention time): 39.2% (3.48 min);
LRMS for C31H43O7N5S1F2 (ES, m/z) 668 (M + H).
N-(5-{[Am in o(im in o)m eth yl]a m in o}p en tyl)-2-{3-[(ben -
zylsu lfon yl)a m in o]-2,6-d iflu or op h en yl}a ceta m id e (29f).
General procedure E afforded 41.2 mg (88%) of a white solid
of product 29f; 1H NMR ppm: 0.83 (m, 6H), 2.71 (m, 2H), 3.05
(s, 2H), 3.16 (m, 2H), 3.68 (s, 2H), 6.25 (m, 1H), 6.74 (m, 8H);
HPLC purity (retention time): >99% (2.66 min); HRMS calcd
for C21H27O3N5S1F2 (M+ + H) 468.1881, found 468.1842.
2-{3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu or op h en yl}-N-[3-
(1H-im id a zol-1-yl)p r op yl]a ceta m id e (29g). General pro-
cedure D afforded 55.5 mg (100%) of a clear oil of product 29g;
1H NMR ppm: 1.97 (m, 2H), 3.24 (m, 2H), 3.58 (s, 2H), 3.94
(m, 2H), 4.34 (s, 2H), 6.61 (m, 1H), 7.71 (m, 2H), 6.89 (m, 3H),
7.32 (m, 7H), 8.02 (s, 1H); 19F NMR ppm: -118.56 (m, 1F),
-125.29 (m, 1F); HPLC purity (retention time): 51.0% (2.28
min); HRMS calcd for C21H22O3N4S1F2 (M+ + H) 449.1459,
found 449.1455.
Ben zyl (4-{[({2,6-Diflu or o-3-[(2-p h en ylet h yl)a m in o]-
p h e n yl}a ce t yl)a m in o]m e t h yl}p h e n yl)(im in o)m e t h yl-
ca r ba m a te (28b). General procedure D. Using 1-hydroxy-
benzotriazole 11 (43.1 mg, 0.31 mmol), acid 26 (0.93 g, 3.19
mmol), PS-carbodiimide (1.00 mmol/g) (9.57 g, 9.57 mmol),
amine (0.96 g, 3.16 mmol), and N-methylmorpholine (2.0 mL,
18.1 mmol) afforded the crude product. The product was
purified by column chromatography (70% ethyl acetate-
hexane) to afford 1.14 g (64%) of a white solid of product 28b;
1H NMR ppm: 2.92 (t, 2H), 3.37 (m, 2H), 3.64 (s, 2H), 3.86
(bs, 1H), 4.39 (d, 2H), 5.23 (s, 2H), 6.41 (m, 1H), 6.57 (m, 1H),
6.79 (m, 1H), 7.34 (m, 14H), 7.72 (d, 2H), 9.43 (bs, 1H);
19F NMR ppm: -131.01 (m, 1F), -136.68 (m, 1F); HPLC
purity (retention time): 83.6% (3.38 min); HRMS calcd for
N-{4-[Am in o(im in o)m et h yl]b en zyl}-2-{2,6-d iflu or o-3-
[(2-p h en yleth yl)a m in o]p h en yl}a ceta m id e (30b). General
Procedure G afforded 0.58 g (82%) of a yellow oil of product
C
32H30O3N4F2 (M+ + H) 557.2364, found 557.2326.
Gen er a l P r oced u r e G. In Sit u TMSI Dep r ot ect ion
1
P r otocol. The benzyloxycarbonyl (Cbz) compound (0.010
mmol), sodium iodide (60.0 mg, 0.040 mmol), and trimethylsilyl
chloride (50.7 uL, 0.040 mmol) were stirred in acetonitrile at
55 °C for one to several hours. Methanol (100 uL) was added
and the solution stirred at room temperature for 1-4 h. The
PS-dimethylbenzylamine resin (3.58 mequiv/g) (0.60 g, 2.1
mmol) was added and the solution stirred at room temperature
for 1-6 h. The reaction mixture was filtered through Celite,
and the solid was rinsed with acetonitrile. The combined
filtrate and washings were evaporated to afford the product.
When necessary, the product was purified by column chroma-
tography.
N-{3-[Am in o(im in o)m eth yl]ben zyl}-2-{3-[(ben zylsu lfo-
n yl)a m in o]-2,6-d iflu or op h en yl}a ceta m id e (29a ). General
Procedure G afforded 89.9 mg (71%) of a white solid of product
29a ; 1H NMR ppm: 3.00 (s, 2H), 3.62 (s, 2H), 3.76 (d, 2H),
6.12 (m, 1H), 6.58-7.32 (m, 13H), 7.88 (m, 1H); 19F NMR ppm:
-120.82 (m, 1F), -124.52 (m, 1F); HPLC purity (retention
time): 66.4% (2.60 min); HRMS calcd for C23H22O3N4S1F2
(M+ + H) 473.1459, found 473.1449.
30b; H NMR ppm: 3.16 (t, 2H), 3.72 (m, 2H), 3.86 (s, 2H),
4.66 (s, 2H), 7.32 (m, 7H), 7.68 (m, 3H), 7.82 (d, 2H), 8.78 (bs,
1H), 9.27 (bs, 1H); 19F NMR ppm: -112.97 (m, 1F), -125.49
(m, 1F); HPLC purity (retention time): 63.3% (2.77 min);
HRMS calcd for C24H24O1N4F2 (M+ + H) 423.1996, found
423.1953.
Meth yl (2-Flu or o-6-h ydr oxy-3-n itr oph en yl)acetate (32).
The nitro compound 16 (18.3 g, 0.079 mol) was added to a
solution of trimethylacetic acid 31 (24.3 g, 0.23 mol) and
potassium carbonate (54.5 g, 0.39 mol) in dimethyl sulfoxide
and the resulting solution stirred at 80 °C for 15 min. The
reaction was cooled to room temperature and diluted with
water and diethyl ether. The resulting solution was acidified
with 2 N hydrochloric acid and extracted with diethyl ether.
The solution was washed with brine, dried over magnesium
sulfate, and filtered. The solvent was removed by evaporation
to afford a mixture of two products. Fraction two of column
chromatography (40% ethyl acetate-hexane) afforded 4.97 g
1
(27%) of a yellow solid of product 32; H NMR ppm: 3.71 (s,
3H), 3.80 (d, 2H, J HF ) 1.7 Hz), 6.95 (dd, 1H, J HH ) 9.2 Hz,
J HF ) 1.1 Hz), 8.05 (m, 1H); 19F NMR ppm: -120.02 (d, 1F,
J HF ) 8.7 Hz); HPLC purity (retention time): >99% (2.25 min);
HRMS calcd for C9H8O5N1F1 (M+ + NH4) 247.0730, found
247.0700.
N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-{3-[(ben zylsu lfo-
n yl)a m in o]-2,6-d iflu or op h en yl}a ceta m id e (29b). General
Procedure G afforded 89.9 mg (71%) of a white solid of product
1
29b; H NMR ppm: 3.21 (s, 2H), 3.94 (s, 2H), 4.06 (m, 2H),
5.94 (bs, 1H), 6.42 (m, 1H), 6.91 (m, 8H), 7.12 (d, 2H), 7.49 (d,
2H), 8.18 (m, 1H); 19F NMR ppm: -119.65 (m, 1F), -124.41
(m, 1F); HPLC purity (retention time): 44.1% (2.57 min);
HRMS calcd for C23H22O3N4S1F2 (M+ + H) 473.1459, found
473.1447.
Meth yl (6-Flu or o-2-h ydr oxy-3-n itr oph en yl)acetate (33).
Following the same procedure described for 32, fraction one
of column chromatography (40% ethyl acetate-hexane) af-
1
forded 12.1 g (67%) of a yellow solid of product 33; H NMR
ppm: 3.71 (s, 3H), 3.81 (s, 2H), 6.80 (m, 1H), 8.18 (m, 1H); 19
F
2-{3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu or op h en yl}-N-[4-
(d im eth yla m in o)ben zyl]a ceta m id e (29c). General proce-
dure D afforded 55.5 mg (100%) of a white solid of product
29c; 1H NMR ppm: 2.50 (s, 6H), 3.26 (s, 2H), 3.89 (s, 2H),
3.93 (d, 2H), 6.27 (bd, 1H), 6.43 (m, 1H), 6.72 (m, 2H), 6.93
(m, 6H), 7.44 (m, 1H), 7.58 (s, 1H), 8.84 (s, 1H); 19F NMR ppm:
-118.49 (m, 1F), -124.01 (m, 1F); HPLC purity (retention
time): >99% (2.57 min); HRMS calcd for C24H25O3N3S1F2 (M+
+ H) 474.1663, found 474.1647.
NMR ppm: -99.71 (m, 1F); HPLC purity (retention time):
>99% (2.63 min); HRMS calcd for C9H8O5N1F1 (M+ + NH4)
247.0730, found 247.0700.
Meth yl (6-Flu or o-2-m eth oxy-3-n itr oph en yl)acetate (34).
Iodomethane (20.0 mL, 0.32 mol) was added to a solution of
the phenol 33 (5.0 g, 0.022 mol) and potassium carbonate (9.0
g, 0.065 mol) in tetrahydrofuran. After stirring at 65 °C for
18 h, the solution was diluted with water and extracted with
diethyl ether. The organic layer was washed with brine, dried
over magnesium sulfate, and filtered. The solvent was removed
by evaporation to afford the crude product. The product was
purified by column chromatography (15% ethyl acetate-
hexane) to afford 4.18 g (79%) of a yellow oil of product 34; 1H
NMR ppm: 3.76 (s, 3H), 3.70 (d, 2H, J HF ) 1.5 Hz), 3.93 (s,
3H), 7.00 (m, 1H), 7.94 (m, 1H); 19F NMR ppm: -103.51 (m,
1F); HPLC purity (retention time): >99% (2.60 min); HRMS
calcd for C10H10O5N1F1 (M+ + NH4) 261.0887, found 261.0905.
Met h yl (6-h yd r oxy-2-m et h oxy-3-n it r op h en yl)a cet a t e
(36). Aqueous sodium hydroxide (10%) (25 mL, 0.062 mol) was
added to a solution of the fluoro compound 34 (4.0 g, 0.016
2-{3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu or op h en yl}-N-
(p yr id in -4-ylm eth yl)a ceta m id e (29d ). General procedure
D afforded 51.2 mg (100%) of a white solid of product 29d ;
1H NMR ppm: 3.21 (s, 2H), 3.82 (s, 2H), 3.93 (d, 2H), 6.37
(m, 1H), 6.78 (m, 8H), 7.29 (s, 1H), 7.46 (s, 1H), 8.91 (s, 1H);
19F NMR ppm: -118.95 (m, 1F), -123.95 (m, 1F); HPLC
purity (retention time): 91% (2.34 min); HRMS calcd for
C
21H19O3N3S1F2 (M+ + H) 432.1193, found 432.1164.
2-{3-[(Ben zylsu lfon yl)a m in o]-2,6-d iflu or op h en yl}-N-
(p ip er id in -4-ylm eth yl)a ceta m id e (29e). General procedure
1
E afforded 40.6 mg (93%) of a white solid of product 29e; H

4308 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Parlow et al.
1
mol) in tetrahydrofuran and the resulting solution stirred at
90 °C for 14 h. The solution was acidified with 4 N hydrochloric
acid and extracted with diethyl ether. The solution was washed
with brine, dried over magnesium sulfate, and filtered. The
solvent was removed by evaporation to afford the crude
product 35 in which fluorine NMR revealed no signal. The
product was dissolved into dichloromethane and oxalyl chloride
(7.0 mL, 0.080 mol) was added to the solution followed by a
drop of dimethylformamide. After being stirred at room
temperature for 1.5 h, the solvent was removed by evaporation
to afford the acid chloride. The acid chloride was redissolved
into dichloromethane, and an excess of methanol (50 mL) was
added followed by addition of pyridine (2.6 mL, 0.032 mol).
The solution stirred at room temperature for 5 h. The solution
was washed with water and brine, dried over magnesium
sulfate, and filtered. The solvent was removed by evaporation
to afford a mixture of two products. Fraction two of column
chromatography (25% ethyl acetate-hexane) afforded 1.33 g
oil of product 42a ; H NMR ppm: 3.73 (s, 5H), 7.28 (m, 3H),
7.49 (m, 3H), 8.07 (m, 1H); 19F NMR ppm: -118.75 (m, 1F);
HPLC purity (retention time): >99% (3.81 min); HRMS calcd
for C15H12O4N1F1 (M+ + NH4) 307.1094, found 307.1093.
Meth yl (3′-{[(Ben zyloxy)ca r bon yl]a m in o}-3-flu or o-4-
n itr o-1,1′-bip h en yl-2-yl)a ceta te (42b). General procedure
I. Using triflate 40 (6.5 g, 0.018 mol), the stannane 39b (11.2
mL, 0.022 mol), lithium chloride (2.2 g, 0.051 mol), and
tetrakis(triphenylphosphine)palladium(0) (410 mg, 0.35 mmol)
to afford a mixture of products. Fraction two of column
chromatography (30% ethyl acetate-hexane) afforded 3.8 g
(48%) of an orange oil of product 42b; 1H NMR ppm: 3.71 (m,
5H), 5.21 (s, 2H), 6.86 (bs, 1H), 6.99 (m, 1H), 7.23 (d, 1H), 7.37
(m, 8H), 8.03 (t, 1H); 19F NMR ppm: -118.69 (m, 1F); HPLC
purity (retention time): >99% (4.09 min); HRMS calcd for
C
23H19O6N2F1 (M+ + NH4) 456.1571, found 456.1564.
Met h yl (3-Met h oxy-4-n it r o-1,1′-b ip h en yl-2-yl)a cet a t e
(42c). General procedure I. Using triflate 41 (5.53 g, 14.8
mmol), tri-n-butylphenylstannane 39a (5.8 mL, 17.7 mmol),
lithium chloride (1.9 g, 44.8 mmol), and tetrakis(triphenylphos-
phine)palladium(0) (34.0 mg, 0.29 mmol) to afford the crude
product. The product was purified by column chromatography
(15% ethyl acetate-hexane) to afford 3.32 g (74%) of a white
solid of product 42c; 1H NMR ppm: 3.69 (s, 5H), 3.94 (s, 3H),
7.17 (d, 1H, J ) 8.3 Hz), 7.29 (m, 2H), 7.45 (m, 3H), 7.89 (d,
1H, J ) 8.3 Hz); HPLC purity (retention time): >99% (3.60
min); HRMS calcd for C16H15O5N1 (M+ + NH4) 319.1294, found
319.1312.
Met h yl (4-Am in o-3-flu or o-1,1′-b ip h en yl-2-yl)a cet a t e
(43a ). General procedure B. Using the nitro compound 42a
(0.60 g, 2.0 mmol) and powdered iron (0.60 g, 10.7 mmol) to
afford 0.47 g (91%) of a clear oil of product 43a ; 1H NMR
ppm: 3.64 (d, 2H, J HF ) 2.3 Hz), 3.71 (s, 3H), 6.83 (m, 1H),
7.27 (dd, 1H), 7.37 (m, 5H); 19F NMR ppm: -137.27 (d, 1F,
J HF ) 8.7 Hz); HPLC purity (retention time): 93.2% (2.76 min);
HRMS calcd for C15H14O2N1F1 (M+ + NH4) 277.1352, found
277.1337.
Meth yl (4-Am in o-3′-{[(ben zyloxy)ca r bon yl]a m in o}-3-
flu or o-1,1′-bip h en yl-2-yl)a ceta te (43b). General procedure
I. Using the triflate 40 (1.31 g, 3.6 mmol), stannane 39b (2.17
mL, 4.2 mmol), lithium chloride (440 mg, 10.0 mmol), and
tetrakis(triphenylphosphine)palladium(0) (80.8 mg, 0.070 mmol)
in 18 mL of anhydrous dioxane. The solution was heated to
85 °C for 21 h. The organic layer was washed with brine, dried
over magnesium sulfate, and filtered. The solvent was removed
by evaporation to afford the crude product. The product was
purified by column chromatography (40% ethyl acetate-
hexane) to afford 0.56 g (38%) of a yellow oil of product 43b;
1H NMR ppm: 3.64 (d, 2H), 3.70 (s, 3H), 5.23 (s, 2H), 6.90
(m, 4H), 7.40 (m, 8H); 19F NMR ppm: -136.68 (d, 1F); HPLC
purity (retention time): >99% (3.50 min); HRMS calcd for
1
(34%) of a white solid of product 36; H NMR ppm: 3.84 (s,
3H), 3.85 (s, 2H), 3.94 (s, 3H), 6.78 (d, 1H, J ) 8.9 Hz), 7.90
(d, 1H, J ) 8.9 Hz), 8.19 (s, 1H); HPLC purity (retention
time): >99% (2.14 min); HRMS calcd for C11H13O6N1 (M+
NH4) 259.0930, found 259.0921.
+
Gen er a l P r oced u r e H. Tr ifla te F or m a tion . Triethyl-
amine (1.2 mmol) was added to a solution of the phenol (1.0
mmol) and triflic anhydride (1.1 mmol) in dichloromethane at
-10 °C. After being stirred at room temperature for 1-18 h,
the solution was acidified with hydrochloric acid 2 N and
extracted with dichloromethane. The organic layer was washed
with brine, dried over magnesium sulfate, and filtered. The
solvent was removed by evaporation to afford the product.
When necessary, the product was purified by column chroma-
tography.
Meth yl (2-Flu or o-3-n itr o-6-{[(tr iflu or om eth yl)su lfon yl]-
oxy}p h en yl)a ceta te (40). General procedure H. Using tri-
ethylamine (729 uL, 5.2 mmol), phenol 32 (1.0 g, 4.36 mmol),
and triflic anhydride (807 uL, 4.7 mmol) to afford the crude
product. The product was purified by column chromatography
(20% ethyl acetate-hexane) to afford 1.0 g (68%) of a clear oil
1
of product 40; H NMR ppm: 3.79 (s, 3H), 3.90 (d, 2H, J HF
)
2.0 Hz), 7.38 (dd, 1H,, J HH ) 7.6 Hz, J HF ) 1.8 Hz), 8.18 (m,
1H); 19F NMR ppm: -73.61 (s, 3F), -113.78 (m, 1F); HPLC
purity (retention time): 94.6% (3.69 min); LRMS for C9H8O5N1F1
(ES, m/z) 3.62 (M + H).
Meth yl (2-Meth oxy-3-n itr o-6-{[(tr iflu or om eth yl)su lfo-
n yl]oxy}p h en yl)a ceta te (41). General procedure H. Using
triethylamine (910 uL, 6.5 mmol), phenol 36 (1.3 g, 5.5 mmol),
and triflic anhydride (1.0 mL, 5.9 mmol) to afford the crude
product. The product was purified by column chromatography
(15% ethyl acetate-hexane) to afford 1.7 g (83%) of a clear oil
1
of product 41; H NMR ppm: 3.78 (s, 3H), 3.87 (d, 2H), 3.96
C
23H21O4N2F1 (M+ + NH4) 426.1829, found 426.1846.
Meth yl (4-Am in o-3-m eth oxy-1,1′-bip h en yl-2-yl)a ceta te
(43c). General procedure B. Using the nitro compound 42c
(1.0 g, 3.3 mmol) and powdered iron (0.92 g, 16.4 mmol) to
afford the product 43c. The product was not purified and
carried on to the next step. HPLC purity (retention time):
>99% (2.48 min); HRMS calcd for C16H17O3N1 (M+ + H)
272.1278, found 272.1325.
(s, 3H), 7.27 (d, 1H, J ) 9.2 Hz), 7.95 (d, 1H, J ) 9.2 Hz); 19
F
NMR ppm: -73.89 (s, 3F); HPLC purity (retention time):
>99% (3.62 min); HRMS calcd for C11H10O8N1F3 (M+ + NH4)
391.0423, found 391.0396.
Gen er a l P r oced u r e I. Stille Cou p lin g. The triflate
compound (1.0 mmol) was added to a solution of stannane (1.1
mmol), lithium chloride (3.0 mmol), and tetrakis(triphenylphos-
phine)palladium(0) (0.02 mmol) in anhydrous dioxane (0.2
mM). The solution was heated to 85 °C for one to several hours.
The solution was allowed to cool to room temperature followed
by addition of diethyl ether and water. The organic layer was
washed with brine, dried over magnesium sulfate, and filtered.
The solvent was removed by evaporation to afford the product.
When necessary, the product was purified by column chroma-
tography.
Met h yl
{3-F lu or o-4-[(2-p h en ylet h yl)a m in o]-1,1′-b i-
p h en yl-2-yl}a ceta te (47a ). General procedure F. Using
sodium triacetoxyborohydride (1.7 g, 8.0 mmol), aniline 43a
(0.051 g, 2.0 mmol), and phenylacetaldehyde 45 (281 uL, 0.015
1
mol) to afford 0.58 g (80%) of a yellow oil of product 47a ; H
NMR ppm: 3.02 (t, 2H), 3.51 (m, 2H), 3.64 (d, 2H, J HF ) 2.9
Hz), 3.70 (s, 3H), 6.80 (m, 1H), 7.02 (dd, 1H), 7.31 (m, 10H);
19F NMR ppm: -138.50 (d, 1F); HPLC purity (retention
time): 83.2% (4.69 min); HRMS calcd for C23H22O2N1F1 (M+
+ H) 364.1713, found 364.1703.
Met h yl (4-(Ben zyla m in o)-3′-{[(b en zyloxy)ca r b on yl]-
a m in o}-3-flu or o-1,1′-bip h en yl-2-yl)a ceta te (47b). General
procedure F. Using sodium triacetoxyborohydride (1.12 g, 5.28
mmol), aniline 43b (0.54 g, 1.32 mmol), and benzaldehyde 44
(147.8 uL, 1.45 mmol) to afford the crude product. The product
Meth yl (3-Flu or o-4-n itr o-1,1′-biph en yl-2-yl)acetate (42a).
General procedure I. Using triflate 40 (1.0 g, 2.76 mmol), tri-
n-butylphenylstannane 39a (1.0 mL, 3.0 mmol), lithium
chloride (351 mg, 8.28 mmol), and tetrakis(triphenylphos-
phine)palladium(0) (63.9 mg, 0.055 mmol) to afford the crude
product. The product was purified by column chromatography
(20% ethyl acetate-hexane) to afford 0.62 g (78%) of a yellow

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4309
was purified by column chromatography (20% ethyl acetate-
hexane) to afford 0.44 g (67%) of a yellow oil of product 47b;
1H NMR ppm: 3.65 (d, 2H), 3.71 (s, 3H), 4.44 (s, 2H), 5.23 (s,
2H), 6.72 (m, 2H), 6.97 (m, 2H), 7.36 (m, 8H); 19F NMR ppm:
-138.06 (d, 1F); HPLC purity (retention time): >99% (4.58
min); HRMS calcd for C30H27O4N2F1 (M+ + H) 499.2033, found
499.2018.
Meth yl [3′-{[(Ben zyloxy)ca r bon yl]a m in o}-3-flu or o-4-
(isop r op yla m in o)-1,1′-bip h en yl-2-yl]a ceta te (47c). Gen-
eral procedure F. Using sodium triacetoxyborohydride (5.6 g,
26.4 mmol), aniline 43b (2.69 g, 6.6 mmol), and acetone 46
(8.0 mL, excess) to afford the crude product. The product was
purified by column chromatography (30% ethyl acetate-
hexane) to afford 1.34 g (45%) of a white solid of product 47c;
1H NMR ppm: 1.30 (d, 6H), 3.62 (d, 2H), 3.68 (s, 3H), 4.13 (m,
1H), 5.21 (s, 2H), 6.70 (m, 2H), 6.99 (m, 2H), 7.36 (m, 9H);
HPLC purity (retention time): >99% (3.84 min); HRMS calcd
for C26H27O4N2F1 (M+ + H) 451.2033, found 451.2023.
(retention time): >99% (2.84 min); LRMS for C25H25O4N2F1
(ES, m/z) 437 (M + H).
{3-Meth oxy-4-[(2-p h en yleth yl)a m in o]-1,1′-bip h en yl-2-
yl}a cetic Acid (48d ). General procedure C. Using sodium
hydroxide (10%) (2.9 mL, 7.2 mmol) and methyl ester 47d (0.69
g, 1.8 mmol) to afford 0.66 g (100%) of an orange foam of
product 48d ; HPLC purity (retention time): 80% (3.83 min).
[4-(Isop r op yla m in o)-3-m eth oxy-1,1′-bip h en yl-2-yl]a ce-
tic Acid (48e). General procedure C. Using sodium hydroxide
(10%) (7.6 mL, 19.0 mmol) and methyl ester 47e (1.51 g, 4.81
mmol) to afford the crude product. The product was purified
by column chromatography (40% ethyl acetate-hexane) to
1
afford 0.44 g (31%) of a white solid of product 48e; H NMR
ppm: 1.25 (d, 6H), 3.62 (m, 3H), 3.75 (s, 3H), 6.50 (d, 1H, J )
8.3 Hz), 6.94 (d, 1H, J ) 8.3 Hz), 7.27 (m, 5H); HPLC purity
(retention time): >99% (2.11 min); HRMS calcd for C18H21O3N1
(M+ + H) 300.1600, found 300.1587.
Ben zyl (4-{[({3-F lu or o-4-[(2-p h en yleth yl)a m in o]-1,1′-
b ip h e n yl-2-yl}a ce t yl)a m in o]m e t h yl}p h e n yl)(im in o)-
m eth ylca r ba m a te (49a ). General procedure D. Using 1-hy-
droxybenzotriazole 11 (180 mg, 1.3 mmol), acid 48a (0.47 g,
1.34 mmol), PS-carbodiimide (1.00 mmol/g) (2.6 g, 2.6 mmol),
amine (0.51 g, 1.6 mmol), and N-methylmorpholine (295 uL,
2.6 mmol) to afford the product. The product was purified by
column chromatography (60% ethyl acetate-hexane) to afford
0.79 g (96%) of a white solid of product 49a ; 1H NMR ppm:
2.92 (m, 2H), 3.19 (m, 2H), 3.77 (d, 2H), 4.67 (d, 2H), 5.40 (s,
2H), 5.60 (bm, 1H), 7.08 (m, 1H), 7.15 (dd, 1H), 7.54 (m, 16H),
8.28 (m, 3H), 8.59 (bt, 1H), 9.40 (bs, 1H), 9.65 (bs, 1H); 19F
NMR ppm: 138.15 (d, 1F); HPLC purity (retention time):
>99% (4.00 min); HRMS calcd for C22H20O2N1F1 (M+ + H)
615.2771, found 615.2760.
Met h yl {3-m et h oxy-4-[(2-p h en ylet h yl)a m in o]-1,1′-b i-
p h en yl-2-yl}a ceta te (47d ). General procedure F. Using
sodium triacetoxyborohydride (2.8 g, 13.2 mmol), aniline 43c
(0.89 g, 3.3 mmol), and phenylacetaldehyde 45 (540 uL, 39.2
mmol) to afford the crude product. The product was purified
by column chromatography (25% ethyl acetate-hexane) to
afford 0.69 g (56%) of a yellow oil of product 47d ; 1H NMR
ppm: 3.04 (t, 2H), 3.50 (t, 2H), 3.69 (s, 3H), 3.67 (s, 2H), 3.69
(s, 3H), 6.75 (d, 1H, J ) 8.3 Hz), 7.02 (d, 1H, J ) 8.3 Hz), 7.35
(m, 10H); HPLC purity (retention time): >99% (4.37 min);
HRMS calcd for
376.1872.
C
24H25O3N1 (M+ + H) 376.1913, found
Meth yl [4-(isop r op yla m in o)-3-m eth oxy-1,1′-bip h en yl-
2-yl]a ceta te (47e). General procedure F. Using sodium tri-
acetoxyborohydride (9.3 g, 43.0 mmol), aniline 43c (2.99 g, 11.0
mmol), and acetone 46 (1.0 mL, 13.6 mmol) to afford the crude
product. The product was purified by column chromatography
(15% ethyl acetate-hexane) to afford 3.55 g (100%) of a yellow
Ben zyl [4-({[(4-(Ben zyla m in o)-3′-{[(ben zyloxy)ca r bo-
n yl]a m in o}-3-flu or o-1,1′-b ip h en yl-2-yl)a cet yl]a m in o}-
m eth yl)p h en yl](im in o)m eth ylca r ba m a te (49b). General
procedure D. Using PS-carbodiimide (1.00 mmol/g) (1.4 g, 1.4
mmol), acid 48b (0.29 g, 0.71 mmol), 1-hydroxybenzotriazole
11 (95.9 mg, 0.71 mmol), amine (0.27 g, 0.84 mmol), and
N-methylmorpholine (624 uL, 5.6 mmol) to afford a the crude
product. The product was purified by reverse-phase chroma-
tography to afford 180 mg (34%) of an orange oil of product
1
oil of product 47e; H NMR ppm: 1.24 (d, 6H), 3.59 (s, 3H),
3.62 (m, 2H), 3.72 (s, 3H), 4.09 (m, 1H), 6.62 (d, 1H, J ) 8.3
Hz), 6.92 (d, 1H, J ) 8.3 Hz), 7.28 (m, 5H); HPLC purity
(retention time): 97.5% (2.57 min); HRMS calcd for C19H23O3N1
(M+ + H) 314.1756, found 314.1758.
{3-Flu or o-4-[(2-p h en yleth yl)a m in o]-1,1′-bip h en yl-2-yl}-
a cetic Acid (48a ). General procedure C. Using sodium
hydroxide (10%) (2.5 mL, 6.2 mmol) and methyl ester 47a (0.58
g, 1.6 mmol) to afford 0.47 g (84%) of an orange glass of product
48a ; 1H NMR ppm: 3.02 (t, 2H), 3.51 (m, 2H), 3.68 (d, 2H,
J HF ) 2.6 Hz), 6.84 (m, 1H), 7.04 (dd, 1H), 7.31 (m, 10H); 19F
NMR ppm: -137.47 (d, 1F); HPLC purity (retention time):
>99% (4.08 min); HRMS calcd for C22H20O2N1F1 (M+ + H)
350.1556, found 350.1532.
(4-(Ben zyla m in o)-3′-{[(ben zyloxy)ca r bon yl]a m in o}-3-
flu or o-1,1′-bip h en yl-2-yl)a cetic Acid (48b). General pro-
cedure C. Using sodium hydroxide (10%) (1.4 mL, 3.5 mmol)
and methyl ester 47b (0.44 g, 0.88 mmol) to afford 0.29 g
(100%) of a white solid of two products. Partial exchange of
benzyloxy with methoxy of the Cbz group occurred resulting
in a mixture of two products. The mixture of products were
not separated and carried on to the next.
[3′-{[(Ben zyloxy)ca r b on yl]a m in o}-3-flu or o-4-(isop r o-
p yla m in o)-1,1′-bip h en yl-2-yl]a cetic Acid (48c). Aqueous
sodium hydroxide (10%) (4.3 mL, 10.0 mmol) was added to a
solution of the methyl ester 47c (1.21 g, 2.6 mmol) in
tetrahydrofuran and the resulting solution stirred at 60 °C
for 48 h. Partial Cbz deprotection occurred so, benzyl chloro-
formate (382 uL, 2.6 mmol) was added to the solution and the
resulting solution stirred at room temperature for 1 h. The
solution was acidified with 2 N hydrochloric acid and extracted
with diethyl ether. The solution was washed with brine, dried
over magnesium sulfate, and filtered. The solvent was removed
by evaporation to afford the crude product. The product was
purified by column chromatography (50% ethyl acetate-
hexane) to afford 0.24 g (21%) of a brown oil of product 47c;
1H NMR ppm: 1.28 (d, 6H), 3.65 (d, 2H), 4.12 (m, 1H), 5.19 (s,
2H), 6.69 (m, 1H), 6.97 (m, 2H), 7.36 (m, 9H); HPLC purity
1
49b; H NMR ppm: 3.68 (m, 4H), 4.34 (s, 2H), 5.08 (s, 2H),
5.26 (s, 2H), 6.68-7.60 (m, 28H); 19F NMR ppm: -136.67 (bs,
1F); HPLC purity (retention time): 61% (4.15 min); HRMS
calcd for C45H40O5N5F1 (M+ + H) 672.2622, found 672.2604.
Ben zyl {4-[({[3′-{[(Ben zyloxy)car bon yl]am in o}-3-flu or o-
4-(isop r op yla m in o)-1,1′-b ip h e n yl-2-yl]a ce t yl}a m in o)-
m eth yl]p h en yl}(im in o)m eth ylca r ba m a te (49c). General
procedure D. Using PS-carbodiimide (1.00 mmol/g) (0.59 g, 0.59
mmol), acid 48c (0.13 g, 0.29 mmol), 1-hydroxybenzotriazole
11 (40.2 mg, 0.29 mmol), amine (0.11 g, 0.34 mmol), and
N-methylmorpholine (163 uL, 1.4 mmol) to afford the crude
product. The product was carried on to the next step. HPLC
purity (retention time): 79% (2.77 min); HRMS calcd for
C
41H40O5N5F1 (M+ + H) 702.3092, found 702.3114.
Ben zyl Im in o(4-{[({3-m eth oxy-4-[(2-ph en yleth yl)am in o]-
1,1′-b ip h en yl-2-yl}a cet yl)a m in o]m et h yl}p h en yl)m et h -
ylca r ba m a te (49d ). General procedure D. Using PS-carbo-
diimide (1.00 mmol/g) (3.6 g, 3.6 mmol), acid 48d (0.66 g, 1.83
mmol), 1-hydroxybenzotriazole 11 (247 mg, 1.8 mmol), amine
(0.70 g, 2.2 mmol), and N-methylmorpholine (1.0 mL, 9.0
mmol) to afford the product. The product was purified by
column chromatography (60% ethyl acetate-hexane) to afford
1
0.52 g (45%) of a white solid of product 49d ; H NMR ppm:
3.17 (m, 2H), 3.65 (m, 2H), 3.77 (s, 2H), 3.86 (s, 3H), 4.64 (d,
2H), 5.27 (bt, 1H), 4.40 (s, 2H), 6.96 (d, 1H), 7.11 (d, 1H), 7.56
(m, 16H), 8.23 (m, 3H), 8.41 (bt, 1H), 9.40 (bs, 1H), 9.75 (bs,
1H); HPLC purity (retention time): >99% (3.80 min); HRMS
calcd for C39H38O4N4 (M+ + H) 627.2971, found 627.2918.
Ben zyl Im in o{4-[({[4-(isop r op yla m in o)-3-m eth oxy-1,1′-
b ip h e n yl-2-yl]a c e t yl}a m in o)m e t h yl]p h e n yl}m e t h yl-
ca r ba m a te (49e). General procedure D. Using PS-carbodi-
imide (1.00 mmol/g) (2.8 g, 2.8 mmol), acid 48e (0.42 g, 1.4
mmol), 1-hydroxybenzotriazole 11 (0.19 mg, 1.4 mmol), amine

4310 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Parlow et al.
(0.54 g, 1.6 mmol), and N-methylmorpholine (620 uL, 5.6
mmol) to afford the crude product. The product was purified
by column chromatography (60% ethyl acetate-hexane) but
stuck to the column and was washed off with 100% ethyl
acetate to afford 0.73 g (92%) of a white solid of product 49e;
1H NMR ppm: 1.25 (d, 6H), 3.62 (m, 3H), 3.71 (s, 3H), 4.33 (d,
2H), 5.19 (s, 2H), 6.05 (bt, 1H), 6.63 (d, 2H, J ) 8.3 Hz), 6.93
(d, 2H, J ) 8.3 Hz), 7.29 (m, 13H), 7.77 (d, 2H); HPLC purity
(retention time): >99% (2.55 min); HRMS calcd for C34H36O4N4
(M+ + H) 565.2815, found 565.2839.
chloride (162 uL, 1.2 mmol) to afford a mixture of two products.
Fraction one of reverse-phase chromatography afforded 4.5 mg
(4%) of a white solid of product 51; 1H NMR ppm: 3.12 (t, 2H),
3.66 (m, 4H), 4.51 (s, 2H), 6.96 (d, 1H), 7.36 (m, 12H), 7.55 (d,
2H), 7.80 (d, 2H); HPLC purity (retention time): 92% (2.53
min); HRMS calcd for C30H30O2N4 (M+ + H) 479.2447, found
479.2446.
4-P h en yl-7-[(2-ph en yleth yl)am in o]-1-ben zofu r an -2(3H)-
on e (52). Following the same procedure described for 51,
fraction two of reverse-phase chromatography afforded 17.7
1
N-{4-[Am in o(im in o)m et h yl]b en zyl}-2-{3-flu or o-4-[(2-
p h en yleth yl)a m in o]-1,1′-bip h en yl-2-yl}a ceta m id e (50a ).
A catalytic amount of palladium on carbon (10%) in dioxane
was added to a methanol-4 N hydrochloric acid/dioxane (3:1)
solution of the Cbz compound 49a (200 mg, 0.32 mmol) and
the mixture was stirred under a balloon of hydrogen at room
temperature for 12 h. The mixture was filtered through Celite,
and the solvent was evaporated to afford the product. The
product was purified by reverse-phase chromatography to
afford 142 mg (92%) of a white solid of product 50a ; 1H NMR
ppm: 2.97 (t, 2H), 3.48 (t, 2H), 3.61 (d, 2H), 4.64 (d, 2H), 6.80
(m, 1H), 6.97 (dd, 1H), 7.33 (m, 11H), 7.47 (d, 2H), 7.79 (d,
2H); 19F NMR ppm: -77.60 (s, 6F), -139.78 (d, 1F); HPLC
purity (retention time): >99% (3.22 min); HRMS calcd for
mg (32%) of a white solid of product 52; H NMR ppm: 3.06
(t, 2H), 3.63 (t, 2H), 3.88 (s, 2H), 7.09 (d, 1H), 7.39 (m, 11H);
HPLC purity (retention time): >99% (4.33 min); HRMS calcd
for C22H22O2N2 (M+ + H) 330.1494, found 330.1492.
N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-[3-h ydr oxy-4-(iso-
p r op yla m in o)-1,1′-bip h en yl-2-yl]a ceta m id e (53). Boron
tribromide 1 M (2.7 mL, 2.7 mmol) was added to a solution of
the methyl ether 49e (0.50 g, 0.88 mmol) in dichloromethane
at -10 °C. The solution stirred at -10 °C for 1.5 h. The solution
was quenched with water and evaporated to a minimal amount
of solvent. The crude product mixture was purified directly
by reverse-phase chromatography to afford a mixture of two
products. Fraction one of reverse-phase chromatography af-
forded 0.22 mg (60%) of a purple-white solid of product 53; 1H
NMR ppm: 1.41 (d, 6H), 3.68 (s, 2H), 3.89 (m, 1H), 4.28 (s,
2H), 6.97 (m, 1H), 7.25-7.92 (m, 11H); HPLC purity (retention
C
30H29O1N4F1 (M+ + H) 481.2398, found 481.2413.
2-[3′-Am in o-4-(ben zyla m in o)-3-flu or o-1,1′-bip h en yl-2-
yl]-N-{4-[a m in o(im in o)m eth yl]ben zyl}a ceta m id e (50b).
Hydrogen bromide in acetic acid (30 wt %) was added to the
carbamate 49b and the solution stirred at room temperature
for 16 h. The solution was evaporated to afford a mixture of
two products. Fraction one of reverse-phase chromatography
afforded 32 mg (52%) of an orange solid of product 50b; 1H
NMR ppm: 3.64 (s, 3H), 4.36 (d, 2H), 4.45 (s, 2H), 6.64 (m,
1H), 6.84 (dd, 1H), 7.37 (m, 13H), 7.67 (d, 2H), 7.90 (bs, 2H),
8.97 (bs, 2H); 19F NMR ppm: -76.96 (s, 9F), -138.72 (d, 1F);
HPLC purity (retention time): >99% (2.62 min); HRMS calcd
for C29H28O1N5F1 (M+ + H) 482.2356, found 482.2332.
Gen er al P r ocedu r e J . Hydr ogen ation . A catalytic amount
of palladium on carbon (5%) in methanol was added to a
methanol solution of the Cbz compound and the mixture was
stirred under a balloon of hydrogen at room temperature for
one to several hours. The mixture was filtered through Celite,
and the solvent was evaporated to afford the product. When
necessary, the product was purified by column chromatogra-
phy.
time): >99% (1.42 min); HRMS calcd for C25H28O2N4 (M+
H) 417.2291, found 417.2295.
+
N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-[5-(isopr opylam i-
n o)-3,6-d ioxo-2-p h e n ylcycloh e xa -1,4-d ie n -1-yl]a ce t a -
m id e (54). A solution of potassium nitrosodisulfonate (Fremy’s
salt) (0.15 g, 0.56 mol) in 0.5 mL of water was added to a
solution of the phenol 53 (76.9 mg, 0.18 mmol) in 4 mL of a
mixture of tetrahydrofuran and water (1:1). The solution
stirred at room temperature for 2 h (turning deep red-brown).
The solution was diluted with a saturated solution of sodium
bicarbonate and extracted with diethyl ether. The organic layer
was washed with brine, dried over magnesium sulfate, and
filtered. The solvent was removed by evaporation to afford the
crude product. The product was purified by reverse-phase
chromatography to afford 4.2 mg (5%) of a red-brown solid of
product 54; 1H NMR ppm: 1.25 (d, 6H), 3.69 (s, 2H), 3.71 (m,
1H), 4.38 (s, 2H), 7.28 (m, 3H), 7.38 (m, 5H), 7.75 (d, 2H), 7.89
(bs, 1H), 7.99 (bs, 1H); HPLC purity (retention time): >99%
(1.52 min); HRMS calcd for C25H26O3N4 (M+ + H) 431.2083,
found 431.2102.
2-[3′-Am in o-3-flu or o-4-(isop r op yla m in o)-1,1′-bip h en yl-
2-yl]-N-{4-[a m in o(im in o)m eth yl]ben zyl}a ceta m id e (50c).
General procedure J afforded 122.3 mg (95%) of a white
solid of product 50c; 1H NMR ppm: 1.68 (d, 6H), 4.04 (d, 2H),
4.15 (sept, 1H), 4.85 (d, 2H), 7.27 (m, 6H), 7.65 (t, 1H), 7.83
(d, 2H), 8.07 (bt, 1H), 8.24 (d, 2H), 9.20 (bs, 1H), 10.38 (bs,
1H); 19F NMR ppm: -76.41 (s, 9F), -138.02 (d, 1F); HPLC
purity (retention time): >99% (1.37 min); HRMS calcd for
Assa ys for Biologica l Activity. Recombinant soluble TF,
consisting of amino acids 1-219 of the mature protein se-
quence was expressed in E. coli and purified using a Mono Q
Sepharose FPLC. Recombinant human VIIa was purchased
from American Diagnostica, Greenwich CT and chromogenic
substrate N-methylsulfonyl-D-phe-gly arg-p-nitroaniline was
prepared by American Peptide Company, Inc., Sunnyvale, CA.
Factor Xa was obtained from Enzyme Research Laboratories,
South Bend, IN, thrombin from Calbiochem, La J olla, CA, and
trypsin and L-BAPNA from Sigma, St. Louis, MO. The
chromogenic substrates S-2765 and S-2238 were purchased
from Chromogenix, Sweden.
C
25H29O1N5F1 (M+ + H) 434.2356, found 434.2360.
N-{4-[Am in o(im in o)m et h yl]b en zyl}-2-{3-m et h oxy-4-
[(2-ph en yleth yl)am in o]-1,1′-biph en yl-2-yl}acetam ide (50d).
General procedure J afforded 14.6 mg (81%) of a white solid
1
of product 50d ; H NMR ppm: 3.00 (t, 2H), 3.50 (t, 2H), 3.63
(s, 2H), 3.67 (s, 3H), 4.41 (s, 2H), 6.86 (m, 1H), 6.96 (m, 1H),
7.35 (m, 10H), 7.46 (d, 2H), 7.77 (d, 2H), 8.21 (bs, 1H); HPLC
purity (retention time): >99% (3.29 min); HRMS calcd for
TF /VIIa Assa y. 100 nM Recombinant soluble tissue factor
and 2 nM recombinant human Factor VIIa were added to a
96-well assay plate containing 0.4 mM of the substrate,
N-methylsulfonyl-D-phe-gly-arg-p-nitroaniline and either in-
hibitor or buffer (5 mM CaCl2, 50 mM Tris-HCl, pH 8.0, 100
mM NaCl, 0.1% BSA). The reaction, in a final volume of 100
µL, was measured immediately at 405 nm to determine
background absorbance. The plate was incubated at room
temperature for 60 min, at which time the rate of hydrolysis
of the substrate was measured by monitoring the reaction at
405 nm for the release of p-nitroaniline. All compounds were
assayed in duplicate at seven concentrations. Percent inhibi-
tion at each concentration was calculated from OD405nm value
from the experimental and control sample. IC50 values were
calculated from a four-parameter logistic regression equation.
C
31H32O2N4 (M+ + H) 493.2604, found 493.2626.
N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-[4-(isopr opylam i-
n o)-3-m eth oxy-1,1′-bip h en yl-2-yl]a ceta m id e (50e). Gen-
eral procedure J afforded 120 mg (79%) of a purple-white solid
of product 50e; 1H NMR ppm: 1.41 (m, 6H), 3.65 (s, 2H), 3.82
(m, 1H), 3.95 (s, 3H), 4.39 (s, 2H), 6.98 (d, 1H), 7.19 (d, 1H),
7.42 (m, 7H), 7.78 (d, 2H); HPLC purity (retention time):
>99% (1.50 min); HRMS calcd for C26H30O2N4 (M+ + H)
431.2447, found 431.2447.
N-{4-[Am in o(im in o)m eth yl]ben zyl}-2-{3-h yd r oxy-4-[(2-
p h en ylet h yl)a m in o]-1,1′-b ip h en yl-2-yl}a cet a m id e (51).
General procedure G. Using compound 49d (0.10 g, 0.16
mmol), sodium iodide (0.19 g, 1.2 mmol), and trimethylsilyl

Tissue Factor VIIa Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4311
For each compound the individual IC50 values were within 10%
of each other. The reported IC50 represents an average of the
duplicates.
Access Team (IMCA-CAT) at the Advanced Photon
Source. IMCA-CAT facilities are supported by the
corporate members of the IMCA and through a contract
with Illinois Institute of Technology (IIT), executed
through the IIT's Center for Synchrotron Radiation
Research and Instrumentation. Use of the Advanced
Photon Source was supported by the U.S. Department
of Energy, Basic Energy Sciences, Office of Science,
under Contract No. W-31-109-Eng-38.
F a ctor Xa Assa y. 0.3 nM Human Factor Xa and 0.15 mM
N-R-benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitro-
aniline dihydrochloride (S-2765) were added to a 96-well assay
plate containing either inhibitor or buffer (50 mM Tris-HCl,
pH 8.0, 100 mM NaCl, 0.1% BSA). The reaction, in a final
volume of 100 µL, was measured immediately at 405 nm to
determine background absorbance. The plate was incubated
at room temperature for 60 min, at which time the rate of
hydrolysis of the substrate was measured by monitoring the
reaction at 405 nm for the release of p-nitroaniline. All
compounds were assayed in duplicate at seven concentrations.
Percent inhibition at each concentration was calculated from
OD405nm value from the experimental and control sample. IC50
values were calculated from a four-parameter logistic regres-
sion equation. For each compound the individual IC50 values
were within 10% of each other. The reported IC50 represents
an average of the duplicates.
Su p p or tin g In for m a tion Ava ila ble: Analytical and spec-
tral characterization data for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Braunwald E.; Califf R. M.; Cannon C. P.; Fox K. A.; Fuster V.;
Gibler W. B.; Harrington R. A.; King S. B.; Kleiman N. S.;
Theroux P.; Topol E. J .; Van de Werf F.; White H. D.; and
Willerson J . T. Redefining medical treatment in the management
of unstable angina. Am. J . Med. 2000, 108, 41-53.
Th r om bin Assa y. Human thrombin (0.28 nM) and H-D-
phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline dihydrochlo-
ride (0.06 mM) were added to a 96-well assay plate containing
either inhibitor or buffer (50 mM Tris-HCl, pH 8.0, 100 mM
NaCl, 0.1% BSA). The reaction, in a final volume of 100 µL,
was measured immediately at 405 nm to determine back-
ground absorbance. The plate was incubated at room temper-
ature for 60 min, at which time the rate of hydrolysis of the
substrate was measured by monitoring the reaction at 405 nm
for the release of p-nitroaniline. All compounds were assayed
in duplicate at seven concentrations. Percent inhibition at each
concentration was calculated from OD405nm value from the
experimental and control sample. IC50 values were calculated
from a four-parameter logistic regression equation. For each
compound the individual IC50 values were within 10% of each
other. The reported IC50 represents an average of the dupli-
cates.
Cr ysta l Str u ctu r e. Tissue factor was expressed in E. coli
and was refolded from inclusion bodies.13 Factor VII was
expressed in mammalian cells (BHK cells) and purified using
a Ca2+-dependent monoclonal antibody directed against the
GLA domain of factor VII. It was then eluted with a buffer
that contains 20 mM EDTA, followed by further purification
using a Mono Q column. Factor VII was subsequently activated
to factor VIIa using recombinant factor Xa. The protein was
dialyzed into a 9% NaCl solution at pH 6.0 and stored at -80
°C.
Prior to crystallization experiments, Factor VIIa was buffer-
exchanged to a storage solution of 50 mM Tris pH 7.5, 50 mM
NaCl, and 2 mM CaCl2 and concentrated to 6 mg/mL. Crystals
of the TF-VIIa complex were obtained by sitting drop vapor
diffusion experiments using a reservoir solution of 16-24%
PEG 4K, 50-150 mM MgCl2, and 50 mM citrate pH 7.5.
The crystals are orthorhombic, space group P212121 with
lattice lengths a ) 69.71 Å, b ) 81.02 Å and c ) 125.90 Å for
the fluorobenzene complex and a ) 69.8 Å, b ) 81.25 Å, and
c ) 126.12 Å for the benzoquinone complex. Complete diffrac-
tion data were measured from crystals of the two complexes
at the 17ID IMCA beamline at the Advanced Photon Source,
Argonne National Laboratories. The overall Rsymm was 10.8%
for the fluorobenzene (50c) complex to 2.4 Å resolution and
9.1% for the benzoquinone (54) complex to 2.22 Å resolution.
The structure of the two complexes was solved by difference
Fourier methods using the structure of the covalent complex
of TF-VIIa with a peptidomimetic inhibitor.7a
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