N2-aroylanthranilamide inhibitors of human factor Xa

Article abstract of DOI:10.1021/jm990327e

Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa K(ass) = 0.81 x 10⁶ L/mol) led to a series of human factor Xa (hfXa) inhibitors based on N²-aroylanthranilamide 4. Expansion of the SAR around 4 showed that only smal

Full text of DOI:10.1021/jm990327e

J . Med. Chem. 2000, 43, 873-882
873
N2-Ar oyla n th r a n ila m id e In h ibitor s of Hu m a n F a ctor Xa
Ying K. Yee,* Anne Louise Tebbe, J ared H. Linebarger, Douglas W. Beight, Trelia J . Craft,
Donetta Gifford-Moore, Theodore Goodson, J r., David K. Herron, Valentine J . Klimkowski, J effrey A. Kyle,
J . Scott Sawyer, Gerald F. Smith, J ennifer M. Tinsley, Richard D. Towner, Leonard Weir, and Michael R. Wiley*
Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285
Received J une 24, 1999
Reversal of the A-ring amide link in 1,2-dibenzamidobenzene 1 (fXa Kass ) 0.81 × 106 L/mol)
led to a series of human factor Xa (hfXa) inhibitors based on N2-aroylanthranilamide 4.
Expansion of the SAR around 4 showed that only small planar substituents could be
accommodated in the A-ring for binding to the S1 site of hfXa. Bulky groups such as 4-isopropyl,
4-tert-butyl, and 4-dimethylamino were favored in the B-ring to interact with the S4 site of
hfXa. The central (C) ring containing a 5-methanesulfonamido group yielded greater activity
than carbamoyl groups. Combining the beneficial features from the B- and C-ring SAR,
compound 55 represents the most potent hfXa inhibitor in the N2-aroylanthranilamide 4 series
with hfXa Kass ) 58 × 106 L/mol (Ki ) 11.5 nM).
Sch em e 1a
In tr od u ction
Discovery of selective human factor Xa inhibitors
(hfXa) is of great interest as a treatment for throm-
boembolic disorders.1-3 There are several potential
advantages of a safe and orally effective hfXa inhibitor
over the current treatments, as described in our previ-
ous paper.4 For example, coumadin has a narrow
therapeutic index and its prophylactic treatment carries
a bleeding liability. As a result, patients undergoing
treatment with coumadin require regular monitoring.
These deficiencies provided the impetus to search for
safer and more effective anticoagulant agents.
a
Reagents: (a) 2 equiv p-anisoyl chloride, TEA; (b) 5 N NaOH,
MeOH; (c) p-MeOBnCl, DMF, K2CO3, acetone.
hfXa is upstream from thrombin in the amplification
of the coagulation cascade. Inhibition of hfXa instead
of thrombin could be more effective in attenuating the
coagulation cascade. The previous paper in this series
describes the discovery of 1,2-dibenzamidobenzenes
such as 1-3 (Table 1), which are novel inhibitors of hfXa
with neutral S1 and S4 binding elements.4 The current
goal was to discover more structurally diverse leads
which could serve as starting points for future modifica-
tions to improve oral absorption. In this account, we
report our early efforts to develop inhibitors of hfXa
related to 1 with alternative linkers connecting the
central ring to the S1 and S4 binding elements. These
studies led to the identification of a new series of
anthranilamides (4) which retain high affinity for hfXa.
Sch em e 2a
a
Reagents: (a) 2 equiv p-MeOPhOH, KOtBu; (b) H2 10% Pd/C,
THF; (c) p-anisoyl chloride, pyridine.
elements, p-anisole groups were used for both A- and
B-rings. Preparation of compounds containing an ether
linkage (5 and 6, Table 2) was achieved by a Williamson
synthesis (Schemes 1 and 2). The unsaturated trans-
vinylene linker 7 was constructed by a Heck coupling
reaction (Scheme 3).5 Compound 7 was hydrogenated
to provide the saturated ethylene-linked analogue 8.
The diamide of phthalic acid (10, Table 2) was obtained
from a one-step reaction of the sodium amide of p-
anisidine and diethyl phthalate in DMF.
Two methods were used for the synthesis of anthra-
nilamides 4. The first method started with the coupling
of isatoic anhydride 11 with p-anisole to give amide 12
(Scheme 4).6 Amide 12 was acylated with the appropri-
ate acid chlorides to provide the anthranilamides 4. This
method provided greater flexibility for B-ring variation
Ch em istr y
In our initial studies on alternative linkers for con-
necting the central ring to the S1 and S4 binding
* Corresponding authors. E-mail: yee_ying_k@lilly.com and
wiley_michael_r@lilly.com.
10.1021/jm990327e CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/12/2000

874 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Yee et al.
Sch em e 3a
Sch em e 5a
a
Reagents: (a) tBuPhC(O)Cl, pyr, 0 °C, 50 °C; (b) ClC(O)C(O)Cl,
DMF; (c) H2 NArR3, PhCH3, 80-110 °C; (d) H2, 10% Pd/C, EtOH,
EtOAc; (e) RSO2Cl, pyridine; or succinic anhydride, pyridine.
Sch em e 6a
a
Reagents: (a) 4-vinylanisole, TEA, Pd(OAc)2, 90 °C; (b) p-
anisoyl chloride, pyridine; (c) H2 5% Pd/C, EtOAc.
Sch em e 4a
a
Reagents: (a) 4-AcPhCO2H, SOCl2, 80 °C; or NCPhC(O)Cl,
TEA; (b) NaBH4, MeOH, 0 °C; (c) MeMgBr, 0 °C; (d) Ni(OAc)2,
NaBH4, 0 °C; (e) NH2OH-HCl, NaOAc, EtOH, 80 °C, H2, 5% Pd/
C, EtOH, AcOH; (f) K2CO3, 30% H2O2, DMSO.
method was more versatile for A-ring permutations. The
C-ring was functionalized as described before.
For the more functionalized B-ring moieties (28-31),
anthranilamide 12 was acylated with 4-acetylbenzoyl
chloride to afford 28 (Scheme 6). N-Acylanthranilamide
28 was transformed into 29 and 30 by sodium borohy-
dride reduction and methyl Grignard addition, respec-
tively. Compound 31 was accessed via cyano interme-
diate 31a . N-Acylation of anthranilamide 12 with
4-cyanobenzoyl chloride provided 31a which was hy-
drolyzed with hydrogen peroxide to afford carboxamide
31.
a
Reagents: (a) H2NArR3, PhCH3, 80-110 °C; (b) HO2CArR4,
pyridine, SOCl2; or R4PhC(O)Cl, pyridine; (c) H2 10% Pd/C, EtOH,
EtOAc; (d) RSO2Cl, pyridine; or succinic anhydride, pyridine.
over the second synthetic route. For the introduction of
the C-ring functions, the synthesis started with nitro-
isatoic anhydrides. After the attachment of the A- and
B-rings, functionalization of the C-ring substituent was
achieved by reduction of the nitro group to an amine
followed by its acylation or sulfonylation.
The second method to 4 (R4 ) t-Bu) utilized a
nucleophilic addition of anilines to oxazinone 15 (Scheme
5).7 Formation of the oxazinone 15 was accomplished
in two steps via acylation and cyclization with oxalyl
chloride. This two-step sequence provided more pure
product and greater ease of isolation of 15 over the
previously described one-step procedure.8 Thus, anthra-
nilic acid 13 was N-acylated with a slight excess of
4-tert-butylbenzoyl chloride to give the N-acylated an-
thranilic acid 14 plus a minor amount of the oxazinone
15. Elevated temperature was necessary to drive the
presumed initial mixed anhydride intermediate, derived
from the carboxylate acylation, completely to the N-
acylated product 14. The mixture of N-acylated anthra-
nilic acid and oxazinone was converted to oxazinone 15
with oxalyl chloride and DMF in good overall yield. The
A-rings were readily introduced by heating the corre-
sponding anilines with the oxazinone 15 in toluene at
reflux to produce N2-acylanthranilamides 4. This second
Resu lts a n d Discu ssion
Alternative linkers to the 1,2-diamides in 1 were
explored to determine the conformational and structural
requirements for activity. In theory new linkers could
enhance affinity in two ways: either directly, through
improved interactions with the enzyme, or indirectly,
by facilitating the optimal placement of the A and B aryl
rings in the S1 and S4 regions of the hfXa active site.9
To investigate the importance of preorganization of A-
and B-rings on activity, compounds containing both
flexible and conformationally constrained linkers were
synthesized.9,10 As the data in Table 2 indicate, com-
pounds containing the flexible ether or ethyl linkers (5,
6, and 8) displayed very weak activity or inactivity. The
trans-olefin 7, which enforces geometry similar to the
amide linker, produced a 30-fold decrease in activity
relative to 1. Likewise the phthalamide derivative 10,
in which the orientations of both of the amide linkers
are reversed (retroamides), sustained an 11-fold de-

N2-Aroylanthranilamide Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 875
Ta ble 3. Effect of B-Ring Substituents
Ta ble 1. Activity of 1,2-Dibenzamidobenzenes
compd
R1
R2
4-tBu
4-OMe
4-Me
4-OEt
4-iPr
4-NMe2
4-Cl
4-CF3
3-Me
3-Cl
3-NMe2
3,4-Cl2
C(O)Me
CH(OH)Me
C(OH)Me2
C(O)NH2
fXa Kassa (×106 L/mol)
n
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
OMe
tBu
4.7
0.03
1.8
1.0
7.0
20.1
0.27
0.09
0.03
0.03
0.09
0.01
0.45
0.40
0.48
1.12
5
2
2
2
2
3
2
2
2
2
2
2
1
1
1
3
a
compd
R
fXa Kass (×106 L/mol)
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
1
2
3
OMe
tBu
NMe2
1.3b
7.3b
18.7b
a
Kass is approximately equal to 1/Ki (see explanatory material
b
in Experimental Section). Results reported in ref 4.
Ta ble 2. Alternative Amide Linkers
a
Kass is approximately equal to 1/Ki (see explanatory material
in Experimental Section). Each value represents the average of
three separate experiments with a deviation of less than 15%.
pound 17 tests the sensitivity of the B-ring linker to
inversion. The hfXa affinity of 16 and 17 (Table 3)
suggests that inversion of the A-ring linker is well-
tolerated while inversion of the B-ring linker is detri-
mental to affinity for hfXa. In compound 16, which has
the A-ring linker inverted and the tert-butyl group
introduced in the B-ring, the activity increases 4-fold
relative to 9. Compound 16 is also comparable in
enzyme affinity with compound 2, the corresponding 1,2-
dibenzoylaminobenzene derivative. On the other hand,
compound 17, in which the amide connecting the central
ring to the S4 binding element is inverted, sustains a
40-fold loss in activity relative to 9 and is 200-fold less
active than compound 2.
To help rationalize the possible structural details
associated with the observed preference for A-ring
amide inversion relative to B-ring amide inversion,
molecular modeling studies were performed. hfXa active
site complexes of the p-tert-butylphenyl derivatives 2
(1,2-dibenzamidobenzene), 16 (A-chain retroamide), and
17 (B-chain retroamide) were constructed and refined
as described in the Experimental Section. Shown in
Figure 1 is an energy-minimized molecular model for
the A-chain retroamide analogue 16 in the hfXa active
site. Figure 2 provides a comparison of the energy-
minimized models of all three compounds: the A-chain
retroamide 16 (green), the B-chain retroamide 17 (right
side, orange), and the 1,2-dibenzamidobenzene 2 (left
side, purple). From Figures 1 and 2 it is clear that the
proposed overall binding mode for each compound is
generally similar. This binding mode has been described
in detail elsewhere for the 1,2-dibenzamidobenzenes
related to compounds 1-3.4 For the anthranilamides
considered in the current study, the major differences
in the binding mode reside in the arrangement of the
two amide groups. This gives rise to differences in the
possible hydrogen-bonding interactions between the
ligand and the residues in the hfXa active site. Figure
1 suggests that in 16 there is an intramolecular
hydrogen bond between the A-chain carbonyl oxygen
a
fXa Kass
compd
X
Y
(×106 L/mol)
5
6
7
8
9
O
CH2
O
0.01
0.03
0.03
CH2
tCHdCH
CH2
C(O)
CH2
NH
<0.001
1.2b
10
0.07
a
Kass is approximately equal to 1/Ki (see explanatory material
in Experimental Section). Each value represents the average of
three separate experiments with a deviation of less than 15%.
b
Three determinations.
crease in affinity compared to 1. Of the conformationally
constrained analogues 7, 9, and 10, only the anthranil-
amide 9 (Kass of 0.81 × 106 L/mol) showed similar
affinity for hfXa relative to the 1,2-dibenzamidobenzene
1. These results demonstrate that both the rigidity of
the linker and the orientation of hydrogen bond donor
or acceptor groups are important for obtaining optimal
activity.
The relative activity of compounds 1, 9, and 10
illustrates that the position of only one of the two amide
linkers in 1 can be reversed without compromising
enzyme affinity. To determine the relative sensitivity
of each of two amide linkers (A-ring vs B-ring) to
inversion, the tert-butylphenyl derivatives 16 and 17
(Table 3) were synthesized. As reported in our previous
paper, the tert-butylphenyl group nicely complements
the size and shape of the hydrophobic S4 site on hfXa.4
On the other hand, the tert-butylphenyl group is much
too large to fit into the S1 binding site. Therefore,
assuming the p-tert-butylphenyl group occupies the S4
binding site and the p-methoxyphenyl group binds to
the S1 site in each molecule, compound 16 tests the
sensitivity of the A-ring linker to inversion and com-

876 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Yee et al.
Ta ble 4. Effect of A-Ring Substitution
a
compd
R
fXa Kass (×106 L/mol)
n
16
32
33
34
35
36
37
38
39
OMe
H
Me
Et
CF3
OCF3
F
4.7
0.46
3.4
0.001
0.001
<0.001
0.11
5
3
2
3
1
1
1
3
1
Cl
Br
0.49
0.22
a
Kass is approximately equal to 1/Ki (see explanatory material
in Experimental Section). Each value represents the average of
three separate experiments with a deviation of less than 15%.
F igu r e 1. Proposed energy-minimized binding model of the
A-chain retroamide compound 16 complexed with the active
site of fXa. The orientation is such that Asp189 appears in
the upper right-hand corner, Ser195 near the top central, and
Glu97 in the lower left-hand corner. Hydrogen bonds between
16 and the active site for this energy-minimized structure are
shown as dashed lines. All aliphatic hydrogens and waters
have been removed for clarity.
more reasonable conformation for 17 would be similar
to that illustrated for 16, in which an internal hydrogen
bond is formed between the A- and B-chains (7 kcal/
mol lower in energy, CHARMm force field). However,
this more stable conformation for 17 does not allow the
H-bond donors and acceptors on the ligand to be placed
in an orientation to reasonably bind to the protein.
In view of the results from compounds 9 and 16, three
regions of the anthranilamide may be considered for
independent optimization. Results from the B-ring
modification are summarized in Table 3. Consistent
with the 1,2-dibenzamidobenzene series,4 increasing the
size of the 4-substituent on the B-ring (compounds 18-
21) can provide a significant increase in affinity for
hfXa. While the 4-tert-butyl (16) and 4-isopropyl (20)
analogues improve affinity by 6-9-fold, the 4-dimethyl-
amino group (21) provided the greatest enhancement
in activity of 25-fold relative to the methoxy derivative
9. Placing selected electron-withdrawing groups in the
4-position (22 and 23) tends to display a detrimental
effect on hfXa affinity, as does the introduction of
selected substituents at the 3-position of the B-ring (24-
27).
The enhanced affinity observed with compounds 20
and 21 led us to examine B-ring moieties which mimic
the size and shape of the isopropyl group and are also
capable of participating in hydrogen-bonding or ionic
interactions. Compounds 28-31 containing a variety of
hydrogen bond-donating and -accepting groups at the
4-position all have weaker affinity than 20 and 21.
Table 4 shows the results of SAR studies on the S1
binding element and illustrates the sensitivity of this
binding site to structural variation. In this series, the
4-tert-butyl group was used as the B-ring substituent
to ensure that no binding of the B-ring into the S1
pocket could obscure the results. Comparing the results
for compound 32 (R ) H) with compounds 33 (R ) Me)
and 16 (R ) MeO) demonstrates that in this series the
4-methyl and 4-methoxy groups contribute 7-10-fold to
the enzyme affinity. However, even subtle changes in
the size and shape of the S1 binding elements can have
a significant detrimental effect on activity. The 5000-
fold loss in activity suffered by the 4-ethyl derivative
34 relative to 16 demonstrates that 4-substituents must
be able to achieve a conformation coplanar with the
A-ring in order to maintain reasonable affinity. Other
F igu r e 2. Proposed energy-minimized binding models of the
1,2-dibenzamidobenzene 2 (left side, purple) and the B-chain
retroamide 17 (right side, orange) complexed with the active
site of fXa. Included for comparison in each is the ligand from
the equivalently processed complex of the A-chain retroamide
16 (green). The orientation and display features are the same
as in Figure 1.
and the B-chain amide hydrogen, giving the ligand a
stable conformation that incorporates a pseudo-six-
membered ring. As a result, the A-chain amide hydrogen
can only hydrogen bond to the carbonyl oxygen of
Gly218, and the B-chain carbonyl oxygen is at too great
a distance (2.7 Å) to interact with the amide of Gly218.
However, inversion of the A-chain allows the amide
carbonyl to form a hydrogen bond with the amide of
Gly216. As a result of this interaction, the A-ring in
compound 16 appears to bind slightly deeper into the
S1 pocket when compared to the 1,2-dibenzamidoben-
zene derivative 2 (Figure 2, purple). A comparison of
the A-chain retroamide 16 to the B-chain retroamide
17 (Figure 2, orange) reveals that, for the latter, the
hydrogen bonds involving the A-chain are equivalent
to what is found for the dibenzamidobenzene 2 and the
B-chain carbonyl forms a hydrogen bond with the amide
of Gly216. However, to acquire this binding mode 17
must take on a relatively high-energy conformation that
places the two carbonyl groups in close proximity. A

N2-Aroylanthranilamide Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 877
Ta ble 5. Effect of C-Ring Substituents
a
compd
R1
R2
fXa Kass (×106 L/mol)
n
40
41
42
43
44
45
46
47
48
49
50
51
NO2
NH2
H
H
H
H
H
H
NO2
NH2
0.08
6.3
3.9
0.62
2.2
9.6
1
3
2
2
3
3
4
2
3
3
2
3
NHSO2Me
NHSO2Ph
NHAc
NHC(O)CH2CH2CO2H
H
H
H
H
H
H
2.1
2.5
NHSO2Me
NHSO2Ph
13.5
4.9
NHAc
0.04
0.15
NHC(O)CH2CH2CO2H
a
Kass is approximately equal to 1/Ki (see explanatory material in Experimental Section). Each value represents the average of three
separate experiments with a deviation of less than 15%.
Ta ble 6. Additive Effect of B- and C-Ring Substituents
a
compd
R1
R2
fXa Kass (×106 L/mol)
n
52
53
54
55
56
57
NH2
H
H
NH2
H
22.7
7.0
3
2
2
3
2
2
MeSO2NH
8.0
H
MeSO2NH
57.9b
9.6
HO2CCH2CH2C(O)NH
H
H
HO2CCH2CH2C(O)NH
0.4
a
Kass is approximately equal to 1/Ki (see explanatory material in Experimental Section). Each value represents the average of three
b
separate experiments with a deviation of less than 15%. Ki was determined to be 11.5 ( 2.13 nM (n ) 3) (see Experimental Section).
subtle increases in the size of the 4-substituent also
resulted in significant losses in activity. The 4-trifluo-
romethyl derivative 35 and 4-trifluoromethoxy 36 were
inactive. The 4-halo analogues 37-39 resulted in 10-
44-fold drops in affinity when compared to 16.
which had a positive effect, the 5-succinoylamino (51)
decreased affinity by 31-fold.
Results from incorporation of groups, which were
beneficial in the C-ring SAR, into the p-dimethylamino
B-ring series are shown in Table 6. While the 4-amino
derivative 52 maintained the activity of the unsubsti-
tuted parent compound 21, both the 4-methanesulfa-
moyl (54) and 4-succinoylamino (56) derivatives de-
creased affinity by about one-half. In the 5-substituted
series, the succinoylamino group (57) caused a 50-fold
loss in hfXa affinity. In contrast, the 5-methanesulfa-
moyl group (55) enhanced affinity by 3-fold over 21.
Compound 55 is the most potent analogue within this
series with a Kass for human fXa of 58 × 106 L/mol. In
addition, the Ki for 55 was determined by Dixon plots
and was found to be 11.5 ( 2.13 nM (n ) 3) with
classical competitive inhibition curves. Thus, the C-ring
SAR in the p-dimethylamino B-ring series closely paral-
lels that of the tert-butyl B-ring series.
The SAR of the retroamides 4 directly parallels that
of the 1,2-dibenzamidobenzenes as illustrated by the
relative affinity of 9, 16, and 21 with 1, 2, and 3,
respectively. This correspondence between the two
series strongly suggests that they both bind to the hfXa
in a similar fashion as indicated by the modeling
studies.
The C-ring substituent effects were explored by using
an amino substituent to introduce a variety of functional
groups (Table 5). In the 4-substituted series (40-45),
the amino group (41) has a minimal effect on affinity
compared to 16. While the methanesulfamoyl derivative
(42) was essentially equivalent to 16, the succinoyl-
amino group (45) produced a 2-fold increase in hfXa
affinity. The SAR in the 5-subsituted series diverged
from that of the 4-substituted congeners. In this series,
the 5-methanesulfamoyl derivative (48) was the most
potent C-ring substituent producing nearly a 3-fold
increase in activity. This modest improvement in activ-
ity was expected since modeling studies showed that the
4- and 5-positions of the C-ring lie outside of the hfXa
binding pocket. Unlike the 4-substituted derivative
Con clu sion
Investigation of alternative linking groups relative to
1,2-dibenzamidobenzenes led to the discovery of N2-
aroylanthranilamides 4 with enhanced activity. Molec-
ular modeling studies suggested that N2-aroylanthra-
nilamides 4 having an inverted A-chain bind to hfXa in
a similar mode as the 1,2-dibenzamidobenzenes. On the

878 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Yee et al.
and bound [fXa] were determined from linear standard curves
on the same plate by use of SoftmaxPro software for each
inhibitor concentration and apparent Kass calculated for each
inhibitor concentration which produced hydrolysis inhibition
between 20% and 80% of the control (3.2 nM fXa): apparent
Kass ) [E:I]/[Ef][If] ) [Eb]/[Ef][Io - Ib]. This method allows
affinity determinations for tight binding inhibitors as well as
for weak binding enzyme inhibitors and has generated a large
protease inhibitor SAR database.13 This system of affinity
measurement was designed to (1) validly assess a very large
number of protease inhibitor samples and (2) determine
affinity with tight binding inhibitors where classical Ki meth-
ods fail.17,18 Algebraic solutions for the equation Kass ) [E:I]/
[Ef][If] ) [Eb]/[Ef][Io - Ib] account for the free and bound
inhibitor concentrations. Alternatively, the whole data set can
be graphically analyzed as a linearized solution to the same
Kass equation: Io/(1 - a) ) [1/apparent Kass(a)] + E°, where a
) fraction of free enzyme.13 This equation was also derived
by Henderson17 and Bieth18 for study of tight binding inhibi-
tors, where classical methods fail to account for bound inhibitor
concentrations.
The application of this method has facilitated the accumula-
tion of a large database of apparent Kass values (obtained at a
single appropriate substrate concentration with each protease
of interest) which are self-consistent for each protease.12,13
Apparent Kass values can be corrected by determining the effect
of substrate concentration (apparent Kass ) 1/apparent Ki;
corrected Kass ) 1/Ki).13,17,18 The variability of mean apparent
Kass values determined at the single substrate concentration
was (15%. The assay system Km was measured as 0.347 (
0.031 mM (n ) 4), and Vmax was 13.11 ( 0.76 µM/min. For
classical Ki determinations Dixon plots were generated with
the same protocol using four substrate concentrations (0.112,
0.224, 0.448, 0.896 mM), and the type of inhibition was
confirmed with Lineweaver-Burke graphs.
other hand, anthranilamides having an inverted B-
chain had considerably diminished enzyme affinity.
Exp er im en ta l Section
All reactions were run under an atmosphere of dry nitrogen
unless noted. All solvents and reagents were used as acquired
from commercial sources without purification. Nuclear mag-
netic resonance spectra were recorded at 300 MHz on a GE
QE-300 spectrophotometer in the solvent indicated. Chemical
shifts are reported in parts per million relative to tetrameth-
ylsilane. Infrared spectra were recorded on a Nicolet DX10 FT-
IR spectrometer. Melting points were recorded on a Thomas-
Hoover melting point apparatus and are uncorrected. Mass
spectra were recorded on the following instruments, using the
stated ionization methods: VG Analytical 70SE mass spec-
trometer, field-desorption (FD); VG Analytical ZAB2-SE in-
strument, fast atom bomardment (FAB); Sciex API 100 mass
spectrometer, electrospray ionization (ESI). Elemental analy-
ses were performed by the Physical Chemistry Department
at Lilly Research Laboratories on a Control Equipment Corp.
440 elemental analyzer and are within 0.4% of theory unless
otherwise noted.
Experimental conditions for key compounds 9, 16, 18, 21,
29-32, 36, 37, 40-42, 46-48, and 52-55 are described in
detail below. Preparations for related compounds using analo-
gous procedures are in the Supporting Information.
Ma ter ia ls. hfXa and human thrombin were purchased from
Enzyme Research Laboratories (South Bend, IN). Chromogenic
p-nitroanilide peptide protease substrates were purchased
from Midwest Biotech (Fishers, IN): Bz-Ile-Glu-Gly-Arg-pNA
(for hfXa) and Bz-Phe-Val-Arg-pNA (for thrombin).
Molecu la r Mod elin g. The protein structure coordinate set
used in this investigation was the hfXa X-ray structure of
Padmanabhan et al.14 (Brookhaven PDB file 1hcg). The
computational construction of ligands and protein-ligand
complexes and the graphical analysis of results were performed
with QUANTA, version 96.15 Energy minimization was per-
formed using CHARMm, version 23.2.15,16 Details of the initial
computational preparation of the hfXa X-ray protein structure
as well as the derivation of the bisamide phenyl ligand
template have been described by Herron et al.4 This solvated,
all hydrogen, initial protein system coordinate set was used
as the starting point for all protein-ligand complexes modeled.
Energy evaluation and minimization were done using the same
nonbond settings, algorithm criteria, and constraint regime
as previously reported.4 The 3D starting structures for ana-
logues 2, 16 (A-chain retroamide), and 17 (B-chain retroamide)
were constructed by modifying the 1,2-dibenzamidobenzene
template. Modifications for each were made using the 3D
Editor, maintaining the original conformation and also position
in space. Default atom types and charges were assigned, the
latter smoothed over all C and nonpolar H atoms to give a
total sum of 0.0. Each analogue was then processed by
reinserting into the active site of the fully solvated hfXa protein
starting structure, deleting waters having an oxygen within
2.0 Å of a ligand atom, and then refining the complex by
constrained energy minimization. The final energy-minimized
structures are shown in Figure 1 (16, green) and Figure 2 (2,
purple; 17, orange). In both figures all aliphatic hydrogens and
waters have been removed for clarity.
Bin d in g Affin ity for h fXa . The binding affinities for hfXa
were measured as apparent association constants (Kass) derived
from protease inhibition kinetics as described previously.12,13
The apparent Kass values were obtained in a high-volume
protocol using automated dilutions of inhibitors (n ) 3 for each
of four to eight inhibitor concentrations) into 96-well plates
and chromogenic substrate hydrolysis rates determined at 405
nm using a Thermomax plate reader from Molecular Devices
(San Francisco, CA). For the assay protocol, 25 µL inhibitor
test solution (in MeOH) was added to 50 µL buffer (0.06 M
tris, 0.3 M NaCl, pH 7.4) followed by 25 µL hfXa (32 nM in
0.03 M tris, 0.15 M NaCl, 1 mg/mL HSA). Finally, 150 µL
substrate (0.3 mM in water) was added within 2 min to start
hydrolysis. The final fXa concentration was 3.2 nM. Free [fXa]
P r ep a r a t ion of N-(4-Met h oxyp h en yl)-2-(4-m et h oxy-
ben za m id o)ben za m id e (9). (a ) Meth yl 2-(4-Meth oxyben -
zoyl)a m in oben zoa te (9a ). To a solution of methyl 2-amino-
benzoate (6.46 mL, 50 mmol) in methylene chloride (150 mL)
at 0 °C was added triethylamine (7.02 mL, 50 mmol) followed
by p-anisoyl chloride (8.55 g, 50 mmol). The reaction mixture
was warmed to room temperature (rt) and stirred for 16 h.
The reaction was shaken with cold dil HCl (200 mL) followed
by satd K2CO3 (200 mL). The organic layer was dried (MgSO4)
and concentrated in vacuo. The residue was crystallized with
hexane to give 9a as a solid (12.0 g, 84%): 1H NMR (DMSO-
d6) δ 3.86 (s, 3H), 3.90 (s, 3H), 7.14 (d, 2H), 7.23 (t, 1H), 7.96
(d, 2H), 8.02 (d, 1H), 8.60 (d, 1H), 11.56 (s, 1H); MS-FD m/e
285 (M+). Anal. (C16H15NO4) C, H, N.
(b) 2-((4-Meth oxyben zoyl)a m in o)ben zoic Acid (9b). To
a mixture of 9a (2.0 g, 7.01 mmol) and MeOH (50 mL) was
added 5 N NaOH (10 mL, 50 mmol) and stirred for 4 h. Most
of the solvent was removed in vacuo and the mixture was
partitioned between water (100 mL) and EtOAc (100 mL). The
aqueous layer was acidified with dil HCl and extracted with
EtOAc (150 mL). The organic layer was dried (MgSO4) and
concentrated in vacuo. The residue was crystallized from
methylene chloride and hexane to give 9b as a solid (1.56 g,
82%): 1H NMR (DMSO-d6) δ 3.85 (s, 3H), 7.12 (d, 2H), 7.19
(t, 1H), 7.65 (t, 1H), 7.92 (d, 2H), 8.05 (d, 1H), 8.70 (d, 1H),
12.13 (s, 1H), 13.76 (s, 1H); MS-FD m/e 271 (M+). Anal. (C15H13
-
NO4) C, H, N.
(c) 2-(4-Meth oxyp h en yl)-4H-3,1-ben zoxa zin -4-on e (9c).
To a 0 °C mixture of 9b (1.36 g, 5.0 mmol), methylene chloride
(75 mL), and DMF (2 drops) was added was oxalyl chloride
(0.50 mL, 5.5 mmol). After 30 m, the reaction was warmed to
rt and stirred for 1 h. The solvent was removed in vacuo at 35
°C to give a solid. The solid was dissolved in methylene chloride
(50 mL) and treated with 0.94 mL (6.66 mmol) triethylamine
at rt. After 16 h the reaction mixture was diluted with 50 mL
methylene chloride and shaken with 200 mL 5 N HCl and 200
mL satd K2CO3. Organic layer was dried (MgSO4) and con-
centrated in vacuo. The product crystallized from CH2Cl2 and
hexanes to give 9c (0.698 g, 55%): 1H NMR (DMSO-d6) δ 3.88

N2-Aroylanthranilamide Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 879
(s, 3H), 7.14 (d, 2H), 7.60 (t, 1H), 7.68 (d, 1H), 7.94 (t, 1H),
8.15 (d, 1H), 8.18 (d, 2H); MS-FD m/e 253 (M+). Anal. for
C15H11NO3 Calcd: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.08;
H, 4.58; N, 5.12.
(d ) N-(4-Met h oxyp h en yl)-2-(4-m et h oxyb en za m id o)-
ben za m id e (9). A mixture of 9c (0.253 g, 1 mmol) ethanol
(25 mL), and p-anisidine (0.123 g, 1 mmol) was refluxed for 4
h. After cooling, the product was crystallized and filtered with
ethanol wash to give 9 as a solid (0.106 g, 28%): 1H NMR
(DMSO-d6) δ 3.73 (s, 3H), 3.81 (S, 3H), 6.93 (d, 2H), 7.09 (d,
2H), 7.23 (t, 1H), 7.23 (t, 1H), 7.57 (t, 1H), 7.58 (d, 2H), 7.85
(d, 2H), 7.90 (d, 1H), 8.52 (d, 1H), 10.42 (s, 1H), 11.81 (s, 1H);
MS-FD m/e 376 (M+). Anal. (C22H20N2O4) C, H, N.
P r ep a r a t ion of 2-(4-ter t-Bu t ylb en zoyla m in o)-N′-(4-
m eth oxyp h en yl)ben za m id e (16). (a ) 2-Am in o-N′-(4-m eth -
oxyp h en yl)ben za m id e (16a ). A mixture of isatoic anhydride
(4.9 g, 30 mmol), p-anisidine (3.7 g, 30 mmol) in toluene (60
mL) was heated to reflux for 5 h.6 After cooling, the superna-
tant was decanted and the solid was suspended in methylene
chloride (500 mL). The resulting suspension was filtered. The
filtrate was combined with the supernatant from above,
partially concentrated, diluted with hexane, and decolorized
with charcoal. The solution was concentrated and crystallized
to yield 16a as a white solid (5.3 g, 73%): mp 116-117 °C; 1H
NMR (300 MHz, DMSO-d6) δ 3.81 (s, 3H), 6.72 (m, 2H), 6.91
(d, J ) 9.0 Hz, 2H), 7.26 (m, 1H), 7.45 (d, J ) 6.8 Hz, 1H),
7.46 (d, J ) 9.0 Hz, 2H); MS-FD m/e 242 (M+). Anal.
(C14H14N2O2) C, H, N.
(b) 2-(4-ter t-Bu tylben zoylam in o)-N′-(4-m eth oxyph en yl)-
ben za m id e (16). To a 0 °C mixture of 16a (728 mg, 3.0 mmol),
pyridine (0.39 mL, 4.5 mmol), and CH2Cl2 (30 mL) was added
tert-butylbenzoyl chloride (0.57 mL, 3.06 mmol). The reaction
was warmed to rt and stirred for 2 h. The reaction was
partially concentrated, stirred vigorously with 1 N HCl (20 mL)
and filtered to yield 11 as a white solid (809 mg, 67%): mp
208-210 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.31 (s, 9H), 3.76
(s, 3H), 6.96 (d, J ) 9.0 Hz, 2H), 7.27 (m, 1H), 7.60 (m, 5H),
7.87 (d, J ) 8.3 Hz, 2H), 7.94 (d, J ) 8.0 Hz, 1H), 8.57 (d, J )
8.0 Hz, 1H), 10.45 (s, 1H), 11.90 (s, 1H); MS-FD m/e 402 (M+).
Anal. (C25H26N2O3) C, H, N.
mL), and washed with water. The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo. The residue was
chromatographed (silica gel, 25% ethyl acetate/75% hexanes
to 50% ethyl acetate/50% hexanes) to give 29 (85 mg, 84%):
1H NMR (CDCl3) δ 1.52 (d, J ) 7.5 Hz, 3H), 3.83 (s, 3H), 4.97
(q, J ) 7.8 Hz, 1H), 6.95 (d, J ) 10.5 Hz, 2H), 7.07 (t, J ) 9.0
Hz, 1H), 7.51 (m, 6H), 7.99 (d, J ) 9.9 Hz, 2H), 8.17 (s, 1H),
8.72 (d, J ) 9.9 Hz, 1H), 11.79 (s, 1H); MS-FD m/e 390 (M+).
Anal. (C23H22N2O4‚0.25H2O) C, H, N.
2-[4-[2-(2-Hyd r oxyp r op yl)]ben zoyla m in o]-N′-(4-m eth -
oxyp h en yl)ben za m id e (30). To a solution of 28 (161 mg,
0.410 mmol) in tetrahydrofuran (10 mL) cooled to 0 oC was
added 3 M methylmagnesium bromide in diethyl ether (0.2
mL, 0.6 mmol). After 1 h, the reaction mixture was quenched
with saturated aqueous ammonium chloride solution (2 mL),
diluted with ether, and washed with water. The organic layer
was dried (MgSO4), filtered, and concentrated in vacuo. The
residue was chromatographed (silica gel, 15% ethyl acetate/
85% hexanes to 35% ethyl acetate/65% hexanes) to give 30
(24 mg, 14%): 1H NMR (CDCl3) δ 1.64 (d, J ) 9.3 Hz, 6H),
3.88 (s, 3H), 6.99 (d, J ) 3.7 Hz, 2H), 7.13 (t, J ) 8.4 Hz, 1H),
7.55-7.67 (m, 6H), 8.02 (d, J ) 8.4 Hz, 2H), 8.17 (s, 1H), 8.78
(d, J ) 8.4 Hz, 1H); MS-FD m/e 404 (M+); HRMS (C24H24N2O4-
Na) theoretical 427.1634, found 427.1634 (M + Na).
P r ep a r a tion of N-(4-Meth oxyp h en yl)-2-[4-(a m in oca r -
b on yl)b en za m id o]b en za m id e (31). (a ) N-(4-Met h oxy-
p h en yl)-2-(4-cya n oben za m id o)ben za m id e (31a ). Using a
procedure described in 16b, N-(4-methoxyphenyl)-2-aminobenz-
amide and 4-cyanobenzoyl chloride yielded 31a (76%): 1H
NMR (DMSO-d6) δ 3.71 (s, 3H), 6.91 (d, 2H), 7.28 (t, 1H), 7.56
(d, 2H), 7.59 ((t, 1H), 7.90 (d, 2H), 8.02 (s, 4H), 8.39 (d, 1H),
10.43 (s, 1H), 11.91 (s, 1H); MS-ES m/e 372 (M + H). Anal.
(C22H17N3O3) C, H, N.
(b ) N-(4-Met h oxyp h en yl)-2-[4-(a m in oca r b on yl)b en z-
a m id o]ben za m id e (31). To a mixture of 31a (0.5 g, 1.35
mmol), DMSO (10 mL), and K2CO3 (0.5 g, 3.62 mmol) was
added 30% H2O2 (1 mL, 8.82 mmol). After stirring for 1 h, the
reaction mixture was diluted with water (100 mL) and the
precipitate was filtered and dried to give 31 (0.45 g, 86%): 1H
NMR (DMSO-d6) δ 3.75 (s, 3H), 6.91 (d, 2H), 7.26 (t, 1H), 7.58
(d, 2H), 7.59 (t, 1H), 7.91 (d, 1H), 7.94 (d, 2H), 7.99 (d, 1H),
8.03 (s, 1H), 8.09 (s, 1H), 8.50 (d, 1H), 10.43 (s, 1H), 11.90 (s,
1H); MS-ES m/e 390 (M + H). Anal. (C22H19N3O4) C, H, N.
P r ep a r a t ion of 2-[(4-ter t-Bu t ylb en zoyl)a m in o]-N-
p h en ylben za m id e (32). (a ) 2-(4-ter t-Bu tylp h en yl)-4H-3,1-
ben zoxa zin -4-on e (32a ). To a stirred solution of anthranilic
acid (34.3 g, 250 mmol) in pyridine (400 mL) was added 4-tert-
butylbenzoyl chloride (94 mL, 502 mmol) dropwise. The
reaction was for 12 h and poured onto ice and 2 N hydrochloric
acid (100 mL). After partitioning, the organic layer was washed
with 2 N hydrochloric acid, satd sodium chloride, satd sodium
bicarbonate, water, dried (MgSO4), and concentrated in vacuo.
The residue was crystallized from ether/hexanes to give 32a
(32.1 g, 46%): 1H NMR (DMSO-d6) δ 8.26 (m, 3 H), 7.83 (t, J
) 8.7 Hz, 1 H), 7.70 (d, J ) 8.7 Hz, 1 H), 7.52 (m, 3 H), 1.39
(s, 9 H); MS-FIA 280.2 (M+). Anal. (C18H17NO2) C, H, N.
(b) 1-N-P h en yl-2-N-(4-ter t-bu tylben zoyl)an th r an ilam ide
(32). A mixture of 32a (1.0 g, 3.6 mmol), aniline (0.33 g, 3.6
mmol) and toluene (15 mL) was refluxed for 8 h. After cooling,
the reaction was added diethyl ether and filtered to 32 (120
mg, 9%): 1H NMR (300 MHz, DMSO-d6) δ 1.30 (s, 9H), 7.14 (t,
J ) 7.5 Hz, 1H), 7.27 (dt, J ) 0.8, 7.9 Hz, 1H), 7.37 (t, J ) 7.9
Hz, 2H), 7.59 (d, J ) 8.7 Hz, 2H), 7.61 (m, 1H), 7.71 (d, J )
7.9 Hz, 2H), 7.84 (d, J ) 8.7 Hz, 2H), 7.93 (dd, J ) 1.1, 7.5 Hz,
1H), 8.51 (d, J ) 7.9 Hz, 1H), 10.53 (s, 1H), 11.70 (s, 1H); MS-
FD m/e 372. Anal. (C24H24N2O2) C, H, N.
2-(4-Meth ylben zoyla m in o)-N′-(4-m eth oxyp h en yl)ben z-
a m id e (18). Using a procedure described for 16, 4-methyl-
benzoyl chloride yielded 18 (64%): 1H NMR (300 MHz, DMSO-
d6) δ 2.38 (s, 3H), 3.75 (s, 3H), 6.95 (d, J ) 9.2 Hz, 2H), 7.26
(m, 1H), 7.37 (d, J ) 8.1 Hz, 2H), 7.60 (m, 3H), 7.81 (d, J )
8.1 Hz, 2H), 7.93 (d, J ) 7.7 Hz, 1H), 8.54 (d, J ) 8.4 Hz, 1H),
10.43 (s, 1H), 11.86 (s, 1H); FIA-MS m/e 359.2 (M - 1). Anal.
(C22H20N2O3) C, H, N.
2-(4-Dim eth ylam in oben zoylam in o)-4-am in o-N-(4-m eth -
oxyp h en yl)ben za m id e (21). To a mixture of 4-dimethyl-
aminobenzoic acid (10 g, 60.5 mmol) and CH2Cl2 (400 mL) was
added thionyl chloride (6.6 mL, 90.5 mmol). The reaction was
refluxed for 3 h and concentrated in vacuo to give 4-dimethyl-
aminobenzoyl chloride (11.11 g, quant). 4-Dimethylaminoben-
zoyl chloride (377 mg, 2.05 mmol) was added to a solution of
2-amino-N-(4-methoxyphenyl)benzamide (304 mg, 1.25 mmol),
pyridine (0.15 mL, 1.85 mmol), DMAP (159 mg, 1.30 mmol)
and CH2Cl2 (15 mL). After stirring for 14 h, the reaction was
diluted with CH2Cl2 (75 mL) and washed with satd Na2CO3
(2 × 10 mL). The organic layer was dried (Na2SO4) and
concentrated. The residue was chromatographed on (CH2Cl2
to 5% EtOAc/CH2Cl2) to yield 21 as a white solid (444 mg,
91%): 1H NMR (300 MHz, DMSO-d6) δ 2.96 (s, 6H), 3.72 (s,
3H), 6.76 (d, J ) 9.0 Hz, 2H), 6.92 (d, J ) 8.7 Hz, 2H), 7.17
(m, 1H), 7.59-7.54 (m, 3H), 7.72 (d, J ) 9.0 Hz, 2H), 7.88 (d,
J ) 7.5 Hz, 1H), 8.57 (d, J ) 8.1 Hz, 1H), 10.39 (s, 1H), 11.74
(s, 1H); MS-IS m/e 390.2 (M + H). Anal. (C23H23N3O3) C, H,
N.
P r ep a r a tion of 2-(4-ter t-Bu tylben zoyl)a m in o-N-(4-tr i-
flu or om eth oxyp h en yl)ben za m id e (36). (a ) 2-Am in o-N-(4-
tr iflu or om eth oxyp h en yl)ben za m id e (36a ). To a stirring
solution of 4-(trifluoromethoxy)aniline (0.34 mL, 2.5 mmol) in
dichloromethane (15 mL) was added pyridine (0.6 mL, 7.5
mmol), followed by 2-nitrobenzoyl chloride (0.36 mL, 2.7
mmol). After 1 h, the solvent was removed in vacuo and the
residue was partitioned between ethyl acetate and water. The
2-[4-(1-H yd r oxyet h yl)b en zoyla m in o]-N′-(4-m et h oxy-
p h en yl)ben za m id e (29). To a solution of 28 (101 mg, 0.260
o
mmol) in methanol (5 mL) cooled to 0 C was added sodium
borohydride (17 mg, 0.46 mmol). After 20 min, the reaction
mixture was quenched with saturated aqueous ammonium
chloride solution (1 mL), diluted with methylene chloride (30

880 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Yee et al.
organic phase was separated and washed twice with 1 M citric
acid, once with brine, twice with satd aq NaHCO3, and again
with brine. The organic phase was then dried with MgSO4,
filtered and the filtrate was concentrated. The resulting solid
was then suspended in ether, sonicated, filtered and dried to
give 0.67 g of a white solid.
2-(4-ter t-Bu t ylb en zoyla m in o)-4-a m in o-N′-(4-m et h ox-
yp h en yl)ben za m id e (41). Using a procedure analogous to
6b, 40 was hydrogenated in a mixture of ethyl acetate, ethanol,
1
and glacial acetic acid to yield 41 (61%): mp 137-140 °C; H
NMR (300 MHz, DMSO-d6) δ 1.31 (s, 9H), 3.75 (s, 3H), 5.98
(br s, 2H), 6.34 (dd, J ) 2.3, 9.0 Hz, 1H), 6.92 (d, J ) 9.0 Hz,
2H), 7.54 (d, J ) 9.0 Hz, 2H), 7.58 (d, J ) 8.3 Hz, 2H), 7.73 (d,
J ) 9.0 Hz, 1H), 7.84 (d, J ) 8.3 Hz, 2H), 8.00 (d, J ) 2.3 Hz,
1H), 9.92 (s, 1H), 12.78 (s, 1H); MS-FD m/e 377 (M+). Anal.
(C25H27N3O3) C, H, N.
The solid was then dissolved in THF (60 mL) and to this
stirring solution (under nitrogen) was added 10% Pd/C (0.3
g). The reaction was hydrogenated overnight at atmospheric
pressure and filtered. The filtrate was concentrated and the
residue was dissolved in ether. The organic phase was washed
with water and brine, dried (MgSO4), and concentrated to give
of 36a as a white solid (0.43 g, 77%): 1H NMR (300 MHz,
DMSO-d6) δ 6.32 (br s, 2H), 6.58 (t, J ) 7.2 Hz, 1H), 6.75 (d,
J ) 8.3 Hz, 1H), 7.20 (t, J ) 7.2 Hz, 1H), 7.33 (d, J ) 8.7 Hz,
2H), 7.62 (d, J ) 7.9 Hz, 1H), 7.82 (d, J ) 8.7 Hz, 2H), 10.14
(s, 1H); MS-FD m/e 296.1. Anal. (C14H11N2O2F3) C, H, N.
2-(4-ter t-Bu tylben zoyla m in o)-4-m eth ylsu lfon yla m in o-
N′-(4-m eth oxyp h en yl)ben za m id e (42). Using the procedure
described for 48, 41 yielded 42 (68%): 1H NMR (DMSO-d6) δ
12.27 (s, 1H), 10.31 (s, 1H), 8.57 (s, 1H), 10.30 (s, 1H), 7.92 (d,
1H, J ) 8.7 Hz), 7.83 (d, 2H, J ) 8.4 Hz), 7.01 (d, 1H, J ) 8.7
Hz), 7.60-7.54 (m, 4 H), 6.93 (d, 2H, J ) 9.0 Hz), 3.73 (s, 3H),
1.29 (s, 9 H), 3.11 (s, 3H); MS-FD m/e 495. Anal. Calcd for
C
26H29N3O5S: C, 63.09; H, 5.90; N, 8.48. Found: C, 66.12*;
(b) 2-(4-ter t-Bu tylben zoyl)am in o-N-(4-tr iflu or om eth ox-
yp h en yl)ben za m id e (36). Using a procedure described in
16b, 36a yielded 36 as a white solid (0.207 g, 89%): 1H NMR
(300 MHz, DMSO-d6) δ 1.30 (s, 9H), 7.29 (dt, J ) 1.1, 7.5 Hz,
1H), 7.38 (d, J ) 9.0 Hz, 2H), 7.58 (d, J ) 8.3 Hz, 2H), 7.62
(dt, J ) 1.1, 7.9 Hz, 1H), 7.82 (d, J ) 9.0 Hz, 2H), 7.83 (d, J )
8.3 Hz, 2H), 8.47 (d, J ) 8.3 Hz, 1H), 10.68 (s, 1H), 11.54 (s,
1H); MS-FD m/e 456.2. Anal. (C25H23N2O3F3) C, H, N.
H, 6.08; N, 9.68*.
P r ep a r a tion of 2-(4-ter t-Bu tylben zoyla m in o)-5-n itr o-
N′-(4-m eth oxyp h en yl)ben za m id e (46). (a ) 2-(4-ter t-Bu tyl-
ben zoyla m in o)-5-n itr oben zoic Acid (46a ). To a mixture of
5-nitroanthranilic acid (24.6 g, 135 mmol) and pyridine (14.2
mL, 175 mmol) in N,N-dimethylformamide (140 mL) cooled
to 0 °C was added tert-butylbenzoyl chloride (31.6 mL, 162
mmol). After stirring for 1 h, the reaction mixture was heated
at 75 °C for 4 h, cooled, and poured into an ice/water mixture.
The resulting solid was filtered, washed with water and a
mixture of 1:2 diethyl ether:hexanes, and dried in vacuo at
150 °C for 2 h to give a mixture 9:1 of 2-(4-tert-butylbenzoyl-
amino)-5-nitrobenzoic acid:6-nitro-2-[4-tert-butylphenyl]-4H-
3,1-benzoxazin-4-one as a light brown solid (37.1 g, 80%): mp
245-249 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.32 (s, 9H), 7.63
(d, J ) 8.7 Hz, 2H), 7.91 (d, J ) 8.7 Hz, 2H), 8.50 (dd, J ) 2.6,
9.0 Hz, 1H), 8.78 (d, J ) 2.6 Hz, 1H), 8.94 (d, J ) 9.0 Hz, 1H),
12.55 (s, 1H); MS-FD m/e 342. Anal. (C18H18N2O5) C, H, N.
(b ) 5-Nit r o-2-[4-ter t-b u t ylp h en yl]-4H -3,1-b en zoxa zin -
4-on e (46b). To a suspension of 2-(4-tert-butylbenzoylamino)-
5-nitrobenzoic acid, 5-nitro-2-[4-tert-butylphenyl]-4H-3,1-benz-
oxazin-4-one, and N,N-dimethylformamide (0.4 mL, 5.4 mmol)
in methylene chloride (200 mL) was added oxalyl chloride (10.4
mL, 119 mmol) in a dropwise manner. After stirring for 2 h,
the mixture was filtered. The filtrate was concentrated in
vacuo to give 46b as a light brown solid (32.9 g, 94%): mp
159-161 °C; 1H NMR (300 MHz, DMSO-d6) δ 1.32 (s, 9H), 7.65
(d, 2H, J ) 8.7 Hz), 7.89 (d, 1H, J ) 8.7 Hz), 8.16 (d, 2H, J )
8.7 Hz), 8.35 (dd, 1H, J ) 8.7, 2.7 Hz), 8.75 (d, 1H, J ) 2.7
Hz); MS-FD m/e 324. Anal. (C18H16N2O4) C, H, N.
P r ep a r a t ion of 2-(4-ter t-Bu t ylb en zoyl)a m in o-N-(4-
flu or op h en yl)ben za m id e (37). (a ) 1-N-(4-F lu or op h en yl)-
2-n itr oben za m id e (37a ). Using a procedure described in 36a ,
4-fluoroaniline yielded 37a (79%): 1H NMR (300 MHz, DMSO-
d6) δ 7.21 (t, J ) 9.0 Hz, 2H), 7.67(dd, J ) 5.3, 9.0 Hz, 2H),
7.70-7.80 (m, 3H), 7.87 (t, J ) 7.5 Hz, 1H), 8.15 (d, J ) 8.3
Hz, 1H), 10.71 (s, 1H); MS-FD m/e 260 (M+). Anal. (C13H9N2O3F)
C, H, N.
(b) 2-Am in o-N-(4-flu or op h en yl)ben za m id e (37b). To a
stirred mixture of 37a (4.0 g, 15.4 mmol), methanol (220 mL),
tetrahydrofuran (110 mL) and nickel acetate tetrahydrate (7.7
g, 31 mmol) was added sodium borohydride (2.3 g, 62 mmol)
was added in small portions. After gas evolution had ceased,
the solvent was removed in vacuo. The residue was partitioned
between ethyl acetate and concentrated ammonium hydroxide.
The organic layer was washed with concentrated ammonium
hydroxide and satd sodium chloride, dried (MgSO4), filtered,
and concentrated to give 37b (2.86 g, 81%): 1H NMR (300
MHz, DMSO-d6) δ 6.31 (br s, 2H), 6.58 (dt, J ) 1.1, 7.9 Hz,
1H), 6.75 (d, J ) 7.9 Hz, 1H), 7.16 (d, J ) 8.7 Hz, 2H), 7.15-
7.25 (m, 1H), 7.60 (d, J ) 7.9 Hz, 1H), 7.71 (dd, J ) 5.3, 9.0
Hz, 2H), 10.02 (s, 1H); MS-FD m/e 230.2 (M+). Anal. (C13H11N2-
OF) C, H, N.
(c) 2-(4-ter t-Bu tylben zoyla m in o)-5-n itr o-N′-(4-m eth ox-
yp h en yl)ben za m id e (46). A mixture of 6-nitro-2-[4-tert-
butylphenyl]-4H-3,1-benzoxazin-4-one (1.21 g, 3.73 mmol) and
p-anisidine (551 mg, 4.47 mmol) in N,N-dimethylformamide
(5 mL) was heated at 80 °C for 2.5 h. After cooling to room
temperature, the reaction mixture was poured into an ice/
water mixture and extracted twice with methylene chloride.
The combined organic layers were washed with water, dried
(Na2SO4), and filtered. The solution was concentrated and
crystallized to give 46. The mother liquor was chromato-
graphed (silica gel, 20% diethyl ether/80% hexanes to 40%
diethyl ether/60% hexanes) to yield a combined product of 46
as a light brown solid (948 mg, 56%): mp 210-211 °C; 1H NMR
δ 1.31 (s, 9H), 3.30 (s, 3H), 6.97 (d, J ) 9.0 Hz, 2H), 7.60 (d, J
) 9.0 Hz, 2H), 7.62 (d, J ) 8.7 Hz, 2H), 7.87 (d, J ) 8.7 Hz,
2H), 8.48 (dd, J ) 2.7, 9.0 Hz, 1H), 8.84 (m, 2H), 12.31 (s, 1H);
MS-FD m/e 447. Anal. (C25H25N3O5) C, H, N.
2-(4-ter t-Bu t ylb en zoyla m in o)-5-a m in o-N′-(4-m et h ox-
yp h en yl)ben za m id e (47). A mixture of 2-(4-tert-butylben-
zoylamino)-5-nitro-N′-(4-methoxyphenyl)benzamide (895 mg,
2.00 mmol), 10% palladium-on-carbon (90 mg) and ethyl
acetate (5 mL) in ethanol (5 mL) was hydrogenated at one
atmospheric pressure for 5 h. The reaction was degassed and
a suspension of 10% palladium-on-carbon (45 mg) and ethyl
acetate (3 mL) was added. This mixture was hydrogenated for
2 more days. The reaction was filtered through diatomaceous
(c) 2-(4-ter t-Bu tylben zoyl)a m in o-N-(4-flu or op h en yl)-
ben za m id e (37). Using the procedure described in 16b, 37b
yielded 37 (1.03 g, 64%): 1H NMR (300 MHz, DMSO-d6) δ 1.31
(s, 9H), 7.22 (t, J ) 9.0 Hz, 2H), 7.28 (t, J ) 7.5 Hz, 1H), 7.55-
7.65 (m, 3H), 7.73 (dd, J ) 5.3, 9.0 Hz, 2H), 7.85 (d, J ) 8.3
Hz, 2H), 7.93 (d, J ) 7.5 Hz, 1H), 8.51 (d, J ) 8.3 Hz, 1H),
10.57 (s, 1H), 11.69 (s, 1H); MS-FD m/e 230.2 (M+). Anal.
(C24H23N2O2F) C, H, N.
P r ep a r a tion of 2-(4-ter t-Bu tylben zoyla m in o)-4-n itr o-
N′-(4-m eth oxyp h en yl)ben za m id e (40). (a ) 2-Am in o-4-n i-
tr o-N′-(4-m eth oxyp h en yl)ben za m id e (40a ). Using a pro-
cedure described in 16a, 4-nitroisatoic anhydride and p-anisidine
yielded 40a as a greenish yellow solid (95%): mp 193-197 °C;
1H NMR (DMSO-d6) δ 3.72 (s, 3H), 6.23 (br s, 2H), 6.90 (d,
2H, J ) 9.3 Hz), 7.33 (dd, 1H, J ) 2.1, 8.7 Hz), 7.59 (d, J )
9.3 Hz, 1H), 7.59 (d, J ) 2.1 Hz, 1H), 7.77 (d, J ) 8.7 Hz, 2H),
10.19 (s, 1H); MS-FD m/e 287. Anal. (C14H13N3O4) C, H, N.
(b) 2-(4-ter t-Bu tylben zoyla m in o)-4-n itr o-N′-(4-m eth ox-
yp h en yl)b en za m id e (40). Using a procedure described in
1
16b, 40a yielded 40 as a yellow solid (77%): mp 221 °C; H
NMR (300 MHz, DMSO-d6) δ 1.36 (s, 9H), 3.84 (s, 3H), 6.97
(d, J ) 9.0 Hz, 2H), 7.55 (m, 4H), 7.76 (d, J ) 8.7 Hz, 1H),
7.87 (dd, J ) 2.3, 8.7 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 2H), 8.66
(br s, 1H), 9.65 (d, J ) 2.3 Hz, 1H), 11.77 (br s, 1H); MS-FD
m/e 447 (M+). Anal. (C25H25N3O5) C, H, N.

N2-Aroylanthranilamide Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 881
J ) 8.8 Hz, 2H), 8.05 (d, J ) 8.8 Hz, 1H), 10.29 (s, 1H), 10.83
earth with ethyl acetate/ethanol washes. The filtrate was
concentrated in vacuo and chromatographed (silica gel, 10%
ethyl acetate/90% methylene chloride to 35% ethyl acetate/
65% methylene chloride) to give 47 as a white solid (487 mg,
58%): mp 212-214.5 °C; 1H NMR δ 1.29 (s, 9H), 3.73 (s, 3H),
5.21 (br s, 2H), 6.76 (dd, J ) 2.6, 9.0 Hz, 1H), 6.91 (d, J ) 9.0
Hz, 2H), 6.98 (d, J ) 2.6 Hz, 1H), 7.53 (d, J ) 8.3 Hz, 2H),
7.59 (d, J ) 9.0 Hz, 2H), 7.77 (d, J ) 8.3 Hz, 2H), 8.01 (d, J )
9.0 Hz, 1H), 10.28 (s, 1H), 10.95 (s, 1H); MS-FD m/e 417. Anal.
(C25H27N3O3) Calcd: C, 71.92; H, 6.52; N, 9.80. Found: C,
71.38*; H, 6.56; N, 9.80.
.
(s, 1H); FIA-MS m/e 405.4 (M + H). Anal. (C23H24N4O3 0.25H2O)
C, H, N.
2-(4-(Dim eth yla m in o)ben zoyla m in o)-4-m eth a n esu lfo-
n yla m in o-N′-(4-m eth oxyp h en yl)ben za m id e (54). Using
procedure described in 48, 52 yielded 54 (27%): 1H NMR (300
MHz, DMSO-d6) δ 3.00 (s, 6H), 3.76 (s, 3H), 3.12 (s, 3H), 6.81
(d, J ) 8.8 Hz, 2H), 6.98 (m, 3H), 7.58 (d, J ) 8.8 Hz, 2H),
7.76 (d, J ) 8.8 Hz, 2H), 7.91 (d, J ) 8.8 Hz, 1H), 8.61 (d, J )
1.8 Hz, 1H), 10.24 (s, 1H), 10.29 (s, 1H), 12.12 (s, 1H); FIA-
MS m/e 483.3 (M + H). Anal. (C24H26N4O5S) C, H, N.
2-(4-(Dim eth yla m in o)ben zoyla m in o)-5-m eth a n esu lfo-
n yla m in o-N′-(4-m eth oxyp h en yl)ben za m id e (55). Using a
procedure described for 48, 53 yielded 55 (18%): 1H NMR (300
MHz, DMSO-d6) δ 2.99 (s, 6H), 3.75 (s, 3H), 3.04 (s, 3H), 6.78
(d, J ) 9.2 Hz, 2H), 6.96 (d, J ) 9.2 Hz, 2H), 7.39 (dd, J ) 2.2,
9.2 Hz, 1H), 7.58 (d, J ) 9.2 Hz, 1H), 7.64 (d, J ) 2.2 Hz, 2H),
7.73 (d, J ) 8.8 Hz, 1H), 8.41 (d, J ) 8.8 Hz, 2H), 9.75 (s, 1H),
10.47 (s, 1H), 11.18 (s, 1H); FIA-MS m/e 483.3 (M + H). Anal.
(C24H26N4O5S) C, H, N.
2-(4-ter t-Bu tylben zoyla m in o)-5-m eth ylsu lfon yla m in o-
N′-(4-m eth oxyp h en yl)ben za m id e (48). To a solution of 47
(150 mg, 0.36 mmol) in methylene chloride (5 mL) at 0 C was
o
added pyridine (32 mL, 0.40 mmol) followed by methanesulfo-
nyl chloride (31 µL, 0.40 mmol). After 5 min, the reaction
mixture was allowed to warm to rt and stirred for 30 min. The
reaction mixture was diluted with methylene chloride and
washed twice with water. The organic layer was dried (Mg-
SO4), filtered, and concentrated in vacuo. The residue was
chromatographed (silica gel, 30% ethyl acetate/70% methylene
chloride) to give 48 (130 mg, 73%): 1H NMR (DMSO-d6) δ 1.29
(s, 9H), 3.04 (s, 3H), 3.73 (s, 3H), 6.93 (d, 2H, J ) 8.7 Hz),
7.39 (d, 1H, J ) 8.7 Hz), 7.56 (d, 4H, J ) 7.8 Hz), 7.61 (s, 1H),
7.80 (d, 2H, J ) 7,8 Hz), 8.31 (d, 1H, J ) 9.00 Hz), 9.81 (s,
1H), 10.47 (s, 1H), 11.24 (s, 1H); MS-FD m/e 495. Anal.
(C26H29N3O5S‚0.50H2O) C, H; N: calcd, 8.33; found, 7.89.
Su p p or tin g In for m a tion Ava ila ble: Procedures for com-
pounds not included in the Experimental Section. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
P r ep a r a tion of 2-(4-(Dim eth yla m in o)ben zoyla m in o)-
4-a m in o-N′-(4-m eth oxyp h en yl)ben za m id e (52). (a ) 2-(4-
(Dim et h yla m in o)b en zoyla m in o)-4-n it r o-N′-(4-m et h ox-
yp h en yl)ben za m id e (52a ). Using a procedure described for
21, 40a and 4-dimethylaminobenzoic acid yielded 52a (88%):
1H NMR (300 MHz, DMSO-d6) δ 3.01 (s, 6H), 3.76 (s, 3H), 6.81
(d, J ) 9.15 Hz, 2H), 6.98 (d, J ) 9.15 Hz, 2H), 7.63 (d, J )
9.15 Hz, 2H), 7.77 (d, J ) 9.15 Hz, 2H), 8.02 (dd, J ) 2.2, 8.8
Hz, 1H), 8.15 (d, J ) 8.8 Hz, 1H), 9.45 (d, J ) 2.20 Hz, 1H),
10.71 (s, 1H), 11.76 (s, 1H); FIA-MS m/e 433.4 (M - 1). Anal.
(C23H22N4O5) C, H, N.
(1) The principle that inhibitors of fXa can produce anticoagulant
effects in blood and can produce preclinical antithrombotic effects
has been demostrated from studies of potent natural fXa
inhibitors from the leech and the tick. (a) Vlasuk, G. P.
Structural and Functional Characterization of Tick Anticoagu-
lant Peptide (TAP): A Potent and Selective Inhibitor of Blood
Coagulation Factor Xa. Thromb. Haemostasis 1993, 70, 212-
216. (b) Vlasuk, G. P.; Ramjit, D.; Fujita, T.; Dunwiddie, C. T.;
Nutt, E. M.; Smith D. E.; Shebuski, R. J . Comparison of the In
Vivo Anticoagulant Properties of Standard Heparin and the
Highly Selective Factor Xa Inhibitors Antistatin and Tick
Anticoagulant Peptide (TAP) in
a Rabbit Model of Venous
Thrombosis. Thromb. Haemostasis 1991, 65, 257-262. (c) Schaf-
fer, L. W.; Davidson, J . T.; Vlasuk, G. P.; Dunwiddie, C. T.; Siegl,
P. K. S. Selective Factor Xa Inhibition by Recombinant Anti-
stasin Prevents Vascular Graft Thrombosis in Baboons. Arte-
rioscl. Thromb. 1992, 12, 879-885.
(b) 2-(4-(Dim eth yla m in o)ben zoyla m in o)-4-a m in o-N′-(4-
m eth oxyp h en yl)ben za m id e (52). Using procedure described
in 6b, 52a was reduced with 10% Pd/C in 20% EtOH/EtOAc
to give 52 (66%): 1H NMR (300 MHz, DMSO-d6) δ 2.99 (s, 6H),
3.75 (s, 3H), 5.92 (s, 2H), 6.31 (dd, J ) 2.20, J ) 8.78 Hz, 1H),
6.79 (d, J ) 8.78 Hz, 2H), 6.93 (d, J ) 8.78 Hz, 2H), 7.55 (d,
J ) 9.15 Hz, 2H), 7.70 (d, J ) 8.78 Hz, 1H), 7.76 (d, J ) 9.15
Hz, 2H), 8.01 (d, J ) 1.83 Hz, 1H), 9.89 (s, 1H), 12.55 (s, 1H);
FIA-MS m/e 405.6 (M + H). Anal. (C23H24N4O3‚0.25H2O) C,
H, N.
(2) Small organic molecules which reversibly inhibit fXa have also
been shown to produce anticoagulant activity in plasma and
antithrombotic activity in animal models. (a) Hara, T.; Yokoya-
ma, A.; Ishihara, H.; Yokoyama, Y.; Nagahara, T.; Iwamoto, M.
DX-9065a, A New Synthetic, Potent Anticoagulant And Selective
Inhibitor Of Factor Xa. Thromb. Haemostasis 1994, 71, 314-
319. (b) Herbert, J . M.; Bernat, A.; Dol, F.; Herault, J . P.; Crepon,
B.; Lormeau, J . C. DX-9065a, A Novel, Synthetic Selective And
Orally Active Inhibitor Of Factor Xa: In-Vitro And In-Vivo
Studies. J . Pharmacol. Exp. Ther. 1996, 276, 1030-1038. (c)
Taniuchi, Y.; Sakai, Y.; Hisamichi, H.; Kayama, M.; Mano, Y.;
Sato, K.; Hirayama, F.; Koshio, H.; Matsumoto, Y.; Kawasaki,
T. Biochemical and Pharmacological Characterization of YM-
60828, a Newly Synthesized and Orally Active Inhibitor of
Human Factor Xa. Thromb. Haemostasis 1998, 79, 543-548.
(3) Reviews: (a) Vacca, J . P. Thrombosis and Coagulation. Annu.
Rep. Med. Chem. 1996, 33, 81-90. (b) Al-Obeidi, F.; Ostrem, J .
A. Factor Xa Inhibitors by Classical and Combinatorial Chem-
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Masters, J . J .; Franciskovich, J . B.; Sawyer, J . S.; Beight, D.
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P r ep a r a tion of 2-(4-(Dim eth yla m in o)ben zoyla m in o)-
5-am in o-N′-(4-m eth oxyph en yl)ben zam ide (53).(a)2-Am in o-
5-n itr o-N′-(4-m eth oxyp h en yl)ben za m id e (53a ). Using a
procedure described in 16a , 5-nitroisatoic anhydride and
p-anisidine yielded 53a (95%): 1H NMR (300 MHz, DMSO-d6)
δ 3.75(s, 3H), 6.84(d, J ) 9.2 Hz, 1H), 6.93(d, J ) 9.2 Hz, 2H),
7.64(s, 2H), 7.59(d, J ) 9.2 Hz, 2H), 8.59(d, J ) 2.6 Hz, 1H),
8.06(dd, J ) 2.56, 9.2 Hz, 1H), 10.31 (s, 1H); FIA-MS m/e 288.0
(M + H). Anal. (C14H13N3O4) C, H, N.
(b) 2-(4-(Dim eth yla m in o)ben zoyla m in o)-5-n itr o-N′-(4-
m eth oxyp h en yl)ben za m id e (53b). Using procedure de-
scribed in 21, 53a and 4-dimethylaminobenzoic acid yielded
53b (84%): 1H NMR (300 MHz, DMSO-d6) δ 3.77 (s, 3H), 3.01
(s, 6H), 6.82 (d, J ) 8.8 Hz, 2H), 6.99 (d, J ) 9.2 Hz, 2H), 7.62
(d, J ) 8.8 Hz, 2H), 7.79 (d, J ) 9.2 Hz, 2H), 8.45 (dd, J ) 2.6
Hz, J ) 9.2 Hz, 1H), 8.82 (d, J ) 9.2 Hz, 1H), 8.84 (d, J ) 2.6
Hz, 1H), 10.81 (s, 1H), 12.24 (s, 1H); FIA-MS m/e 433.4 (M -
H). Anal. (C23H22N4O5) C, H, N.
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3.74 (s, 3H), 5.15 (s, 2H), 6.76 (m, 3H), 6.92 (d, J ) 8.8 Hz,
2H), 6.99 (d, J ) 2.2 Hz, 2H), 7.60 (d, J ) 8.8 Hz, 2H), 7.70 (d,
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