1,2-Dibenzamidobenzene inhibitors of human factor Xa

Article abstract of DOI:10.1021/jm990326m

High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound 4, fXa apparent K(ass) = 0.64 x 10⁶ L/mol). SAR studies in this novel structural series of fXa inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tertbutyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced slightly higher levels of activity than other substituents when present in combination with favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds, having fXa apparent K(ass) = 25.5 x 10⁶ L/mol, about 40x more active than the original lead. This lead series does not show potent inhibition of human thrombin. A model for the binding of these ligands to the fXa active site is proposed. The model is consistent with the observed SAR and can serve to guide future SAR studies.

Full text of DOI:10.1021/jm990326m

J . Med. Chem. 2000, 43, 859-872
859
1,2-Diben za m id oben zen e In h ibitor s of Hu m a n F a ctor Xa
David K. Herron,* Theodore Goodson, J r., Michael R. Wiley, Leonard C. Weir,‡ J effrey A. Kyle, Ying K. Yee,
Ann Louise Tebbe, J ennifer M. Tinsley, David Mendel, J ohn J . Masters, J effry B. Franciskovich,
J . Scott Sawyer, Douglas W. Beight, Andrew M. Ratz, Guy Milot,† Steven E. Hall,‡ Valentine J . Klimkowski,
J ames H. Wikel, Brian J . Eastwood, Richard D. Towner, Donetta S. Gifford-Moore, Trelia J . Craft, and
Gerald F. Smith
Lilly Research Laboratories, Eli Lilly & Company, Indianapolis, Indiana 46285, Sphinx Pharmaceuticals, Eli Lilly &
Company, Durham, North Carolina 27707, and Sphinx Pharmaceuticals, Eli Lilly & Company,
Cambridge, Massachusetts 02139
Received J une 24, 1999
High-throughput screening of a combinatorial library of diamidophenols yielded lead compounds
with the ability to inhibit human factor Xa (fXa) at micromolar concentrations (e.g. compound
4, fXa apparent Kass ) 0.64 × 106 L/mol). SAR studies in this novel structural series of fXa
inhibitors showed that the phenolic hydroxyl group was not essential for activity. The best
activity was found in substituted 1,2-dibenzamidobenzenes in which the phenyl group of one
benzoyl group (A-ring) was substituted in the 4-position with relatively small lipophilic or
polarizable groups such as methoxy, vinyl, or chloro and the phenyl group of the other benzoyl
group (B-ring) was substituted in the 4-position with larger lipophilic groups such as tert-
butyl or dimethylamino. The central phenyl ring (C-ring) tolerated a wide variety of
substituents, but methoxy, methanesulfonamido, hydroxyl, and carboxyl substitution produced
slightly higher levels of activity than other substituents when present in combination with
favorable B-ring substitution. Methylation of the amide nitrogen atoms was found to greatly
decrease activity. Compound 12 is the highest affinity fXa inhibitor in this group of compounds,
having fXa apparent Kass ) 25.5 × 106 L/mol, about 40× more active than the original lead.
This lead series does not show potent inhibition of human thrombin. A model for the binding
of these ligands to the fXa active site is proposed. The model is consistent with the observed
SAR and can serve to guide future SAR studies.
In tr od u ction
to be effective in all types of coagulation-based throm-
boembolic processes by means of repression of thrombin
generation. Potential advantages over thrombin inhibi-
tors include the indirect repression of many moles of
generated thrombin per fXa inhibitor molecule, rather
than the 1:1 stoichiometry necessary for direct thrombin
inhibition, and the avoidance of direct interference with
multiple thrombin-mediated activities (platelet aggrega-
tion, protein C activation, factor XIII activation, factors
V and VIII feedback activation).4 Natural and recom-
binant fXa inhibitor proteins and small molecular
weight reversible fXa inhibitors have been shown to be
anticoagulant and antithrombotic in animal models.5
Thromboembolic diseases continue as a leading cause
of morbidity and mortality in developed nations. A
primary medical strategy to treat and to prevent such
diseases has been the use of anticoagulants: heparin
and low-molecular-weight heparins for parenteral short-
term treatments and vitamin K antagonists such as
warfarin for chronic oral therapy. However, there are
serious and well-documented liabilities associated with
the chronic use of warfarin,1 and there has been a
consequent worldwide pharmaceutical discovery effort
to discover safer and more effective anticoagulant
compounds which could be used as chronic oral therapy.
Initially, a large effort focused on the target thrombin
on the basis that direct inhibitors of the fibrinogen-
clotting/platelet-activating protease could produce an-
tithrombotic effects in all types of thrombotic disease.
Thus far, however, an orally effective thrombin inhibitor
has not been successfully developed2 and the discovery
focus to resolve this large unmet medical need has
shifted to the serine protease factor Xa.
The overall development of inhibitors for fXa has to
some extent built upon the effort and success for
R-thrombin. A comparison of their active site binding
regions12,14 reveals that they both have the relatively
deep S1 or “specificity” pocket. The interior of the S1
pocket is fairly hydrophobic except for the presence of
the acidic carboxyl side chain of Asp189 at the base. It
is this acidic group that is usually found forming a
strong salt bridge with a basic amino acid like arginine
or lysine or with other positively charged moieties such
as benzamidine. Both enzymes possess the S3 binding
site made up of Gly216. Additionally, both have a
hydrophobic “distal” S4 region; however the structure
and character of this area differ somewhat between the
two enzymes. In R-thrombin the S4 area is a broad
solvent-exposed hydrophobic region, made up of the side
Factor Xa (fXa) produces thrombin by activating
prothrombin during blood clotting whether coagulation
is triggered by tissue factor or by blood-contact mechan-
isms.3,2a Accordingly, fXa inhibitors have the potential
* To whom correspondence should be addressed. Tel: (317) 276-
4670. Fax: (317) 276-1417. E-mail: dkherron@lilly.com.
‡ Sphinx Pharmaceuticals, North Carolina.
† Sphinx Pharmaceuticals, Massachusetts.
10.1021/jm990326m CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/23/2000

860 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
of the lead compound as shown in Table 1. This finding
allowed us to explore A- and B-ring SAR without having
to include the C-ring hydroxyl group. The A-, B-, and
C-ring designations are defined in Scheme 4 and in
Table 1.
N-Methylation of the monomethylbisamide 10 pro-
vided the dimethylbisamide 11 as shown in Scheme 3.
Compounds with different substitution on the A- and
B-rings were prepared either from the appropriate
diamines as shown in Scheme 4 or from the nitroamines
as shown in Scheme 5.
Molecu la r Mod elin g
Upon confirmation of the lead compound 4 as an
inhibitor of fXa, we sought to place our ligand design
program on a structure-based foundation. At the time,
the only available experimental X-ray structure of
human fXa was the des(1-45) coordinate set of Pad-
manabhan et al.12 Although not complexed with a small
molecule inhibitor, we felt this provided a relatively
accurate description of the active site (2.2 Å resolution)
from which to begin to propose binding modes for the
series lead. An expectation (but not a requirement) for
a competitive enzyme inhibitor is that it occupy many
of the well-defined binding pockets within an active site.
In the case of the current series this consideration would
have the lead compound 4 occupy simultaneously the
fXa S1 and S4 binding pockets. Shown in Figure 2 is
an energy-minimized molecular model of a proposal for
how the lead 4 binds to the active site of fXa. This
binding mode is somewhat based upon the results from
a parallel computational study we performed for various
amidine analogues.13 Details regarding the specific
molecular modeling procedures utilized for the current
study are reported within the Experimental Section.
In the model of 4 bound in the fXa active site (shown
in Figure 2), the two arms of the inhibitor (A- and
B-rings, respectively) lie flat within each of the S1 and
S4 sites. The central phenol ring bridges the two arms
at almost 90° to each other, itself mostly solvent
exposed. This central ring resides near the side of the
opening to the S1 pocket. The p-methoxybenzoyl group
of the A-ring very well complements the shape and the
mostly hydrophobic character of the S1 pocket. The
position of the A-ring buries the methyl of the p-methoxy
group favorably in a small hydrophobic hole found on
the backside of the S1 pocket, formed by the side chains
of Tyr228, Ala190, and Val213. As in R-thrombin,14 this
hole in fXa is normally occupied by a water molecule
which in this case would be expelled. The corresponding
p-methoxybenzoyl group of the B-ring extends down into
the more solvent-exposed hydrophobic S4 region. The
p-methoxy group of this chain is positioned in front of
the side chain of Trp215 and almostly directly between
the aromatic rings of Phe174 and Tyr99. Due to the low
barrier of rotation for this CO single bond (∼2.0 kcal/
mol, CHARMm force field) and the yet available space
within the S4 region, some rotation about this methoxy
group bond was observed during a molecular dynamics
simulation of the active site complex. The energy-
minimized structure shown in Figure 2 has the NH of
the A-ring amide bond simultaneously hydrogen bonded
to the backbone carbonyls of Gly216 and Gly218. During
molecular dynamics simulations of this complex, a more
F igu r e 1. Structures of fXa inhibitors 4, Tenstop (A), and
DX-9065a (B).
chains of Leu99, Trp215, and Ile174. In fXa the S4
region (also known as the “aryl” binding region) is
somewhat more structured, being made up of the
aromatic side chains of residues Tyr99, Trp215, and
Phe174. Finally, fXa does not possess an equivalent to
the small hydrophobic S2 pocket found in R-thrombin.
Several structural types of small molecule fXa inhibi-
tors have been reported. The earliest inhibitors were
peptides, modeled on the conservation of Gly-Arg at the
cleavage site of prothrombin by fXa. An example of one
of these early peptide inhibitors6 is compound A in
Figure 1. In this compound a benzamidine is linked by
a glycine residue to an arylsulfonamide. Far more potent
peptide fXa inhibitors have been made since then with
the aid of combinatorial chemistry.5h The prototype non-
peptide inhibitor is DX-9065a5d,h (compound B in Figure
1). An X-ray crystal structure of fXa with B bound in
its active site7 shows that that the naphthamidine group
binds in the specificity pocket (S1) while the pyrrolidine
imine group binds in the aryl binding site (S4). The
central group connecting the S1 and S4 binding groups
in B is a 4-ethylphenoxy moiety. Recently series have
been reported in which a propyl8 group or an amidopro-
pyl9 group connects the S1 and S4 binding moieties. Still
other fXa inhibitor types have been reviewed.10 The 1,2-
dibenzamidobenzene structures reported here provide
a novel structure on which to build fXa inhibitors.
We discovered, during high-volume screening, the 1,2-
dibenzamidobenzene 4 (Figure 1) which reversibly
inhibited human fXa (apparent Kass ) 0.64 × 106 L/mol).
We have now explored a variety of related structures.
We have modeled some of these structures in the active
site of fXa and we have proposed a mode of binding that
is consistent with the observed SAR. Tests against the
related serine protease thrombin suggest that these
compounds selectively inhibit fXa.
Ch em istr y
The combinatorial library that produced the lead
compound 4 was prepared by solid-phase methods.11
After the lead compound was found, we used the same
methods to prepare additional quantities of 4 and 23
as shown in Scheme 1.
Early in the SAR we prepared compound 2, the
deshydroxy version of the lead compound 4, by the route
shown in Scheme 2. The activity of the deshydroxy
compound was found to be essentially the same as that

1,2-Dibenzamidobenzene Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 861
Sch em e 1a
a
P-CO2H ) 4-carboxypolystyrene resin.
Sch em e 2a
Sch em e 3
sphere, not interacting with the protein. One would
expect this group to possibly interact with the guani-
dinium of Arg143. Although this interaction was not
observed during the dynamics simulation of 4, this could
be due to the relatively short simulation time (∼ 50 ps)
never allowing the two groups to come into contact.
However, due to the symmetry of the ligand the two
arms could be interchanged placing the hydroxyl group
in closer proximity of the positively charged Arg143 side
chain. In any case though this interaction would be
drastically modulated due to it being solvent exposed
and therefore not expected to be significant. Finally, no
direct interaction of the ligand with the catalytic triad
is observed in the proposed model of 4 with fXa.
a
(a) Acid chloride, CH2Cl2, aq NaOH; (b) acid chloride, CH2Cl2,
Resu lts a n d Discu ssion
pyridine.
The test results appearing in Table 1 show that the
central ring (C-ring) can tolerate substitution at any
position. The fXa inhibitory activity only varies by a
factor of 5 with the variety of substitutions considered.
This insensitivity to C-ring substitution alone is con-
sistent with the proposed structural model of the lead
4 bound to the fXa active site. Figure 2 illustrates that
generally the C-ring positions 3-6 point into the solvent
and therefore substitutions at these positions need not
realistic hydrogen-bonding arrangement arises in that
the interaction involving the A-ring dynamically ex-
changes between the two protein acceptor carbonyl
oxygens. Although not seen in the minimized structure,
during the simulation the B-ring carbonyl group can
periodically form a hydrogen bond to the backbone NH
of Gly218 (2.5 Å distance in Figure 2). The C-ring
hydroxyl group is shown here extending into the solvent

862 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
Sch em e 4a
Ta ble 1. Effects of Various C-Ring Substituents on fXa
Inhibition by 1,2-Di(4-methoxybenzamido)benzenes
a
a
fXa Kass
fIIa Kass
compd
R1
R2
(×106 L/mol) (×106 L/mol) n (fXa/fIIa)
1
2
3
4
5
7
8
9
4-OMe
H
6-OMe 3-OMe 0.77
4-OH
4-Me
3-OH
4-Cl
H
H
1.25 ( 0.18
-
3
6/3
2
5/4
3/2
1
0.84 ( 0.35
0.02 ( 0.00
0.01
H
H
H
H
H
0.64 ( 0.21
0.45 ( 0.36
0.45
0.42
0.32
0.02 ( 0.01
0.02
0.01
-
0.02
1
2/1
4-CO2H
a
Kass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when n g3 and average when n ) 2. The
precision of single determinations ((std error) is (12.9%. The
precision of mean values is (7.3%. Information about the statisti-
cal analysis that produced these estimates is included in the
Experimental Section. Kass ≈ 1/Ki. A more detailed discussion of
K
ass and its relationship to Ki is given in the Experimental Section.
a
(a) Acid chloride, pyridine, CH3CN; (b) acid chloride, Et3N,
CH3CN; (c) NaOH, water, THF, MeOH.
Sch em e 5a
F igu r e 2. Proposed energy-minimized binding model of the
lead compound 4 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. The molecular surface of the S4 region
is highlighted in small white dots, while the surface for the
B-ring substituent (4-methoxy) is shown in large dots. The
illustrated portion of the ligand surface in contact with the
S4 pocket is colored white, while the exposed portion is colored
orange. Hydrogen bonds between 4 and the active site for this
energy-minimized structure are shown as dashed lines. All
aliphatic hydrogens and waters have been removed for clarity.
D ) Asp, E ) Glu, F ) Phe, G ) Gly, H ) His, Q ) Gln, R )
Arg, S ) Ser, W ) Trp, Y ) Tyr.
a
(a) H2, Pd-C, THF; (b) acid chloride, pyridine; (c) TFA, CH2Cl2;
(d) Bu4NF, THF; (e) MCPBA, CHCl3; (f) MeSO2Cl, CH2Cl2, 2,6-
di-tert-butylpyridine.
Methylation of one or both of the amide nitrogens
greatly decreased fXa affinity as shown in Table 2. As
illustrated by the active site binding model, these NH
groups are involved in at least one hydrogen bond to
the protein, stabilizing the enzyme-inhibitor complex.
Incorporation of methyl groups in either the A- or B-ring
amides would eliminate these interactions. N-Methyl-
ation will also change the conformational preference
have strong interactions with the protein. The similar
observed activity of the original 4-OH lead compound 4
and the C-ring unsubstituted analogue 2 allowed A- and
B-ring SAR to be studied in C-ring unsubstituted
compounds (Tables 2 and 4-6), simplifying the chem-
istry.

1,2-Dibenzamidobenzene Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 863
Ta ble 2. Effects of N-Methylation on fXa Inhibition by
Ta ble 4. Effects of B-Ring Substituents on fXa Inhibition by
1,2-Di(4-methoxybenzamido)benzenes
A-Ring 4-Methoxydibenzamidobenzenes
a
a
fXa Kass
fIIa Kass
a
a
fXa Kass
fIIa Kass
compd
R1
NMe2
CMe2OMe
SMe
O-i-Pr
Ph
O-t-Bu
OMe
SO2NMe2
OEt
SO2Me
OMe
OPr
SOMe
OPh
R2
(×106 L/mol) (×106 L/mol)
16.88 ( 0.07
n
compd R1
R2 (×106 L/mol) (×106 L/mol) n (fXa/fIIa)
33
36
37
38
39
40
41
42
43
44
45
46
47
48
50
51
52
H
0.05 ( 0.01
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
10
11
H
Me
H
H
0.84 ( 0.35
0.02 ( 0.00
6/3
1
1
H
H
H
H
H
Cl
H
H
H
2.35
0.02
0.02
Me Me 0.00
0.00
0.00
1.63
1.19
1.10
0.82
0.71
0.66
0.64
0.53
0.02
0.04
0.00
0.01
0.03
0.00
0.00
0.00
0.02
0.00
0.00
0.00
0.00
0.00
0.00
aKass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when
n g 3. The precision of single
determinations ((std error) is (12.9%. The precision of mean
values is (7.3%. Information about the statistical analysis that
produced these estimates is included in the Experimental Section.
Kass ≈ 1/Ki. A more detailed discussion of Kass and its relationship
to Ki is given in the Experimental Section.
OMe 0.52
H
H
H
H
H
H
0.44
0.43
0.29
0.08
0.01
0.00
Ta ble 3. Effects of B-Ring Substituents and C-Ring
Hydroxylation on fXa Inhibition by A-Ring
4-Methoxydibenzamidobenzenes
OBu
COCF3
OC6H13
a
Kass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when n g 3 and average when n ) 2. The
precision of single determinations ((std error) is (12.9%. The
precision of mean values is (7.3%. Information about the statisti-
cal analysis that produced these estimates is included in the
Experimental Section. Kass ≈ 1/Ki. A more detailed discussion of
K
ass and its relationship to Ki is given in the Experimental Section.
n
a
a
gain in affinity is seen in the analogous deshydroxy
compounds 2 and 33. Similar replacement of 4-methoxy
by tert-butyl gave a 10× increase in affinity in the
hydroxy compounds (4 and 23) and a 9× increase in the
deshydroxy compounds (2 and 22). This general increase
in activity for both of these B-ring 4-position substitu-
tions can be attributed to the increase in ligand surface
area in contact with the hydrophobic S4 binding region.
This difference is evident when comparing Figures 2-4,
where the B-ring substituent contact surface area (large
white dots) and exposed surface area (large orange dots)
are highlighted for analogues 4, 12, and 23, respectively.
These larger substituents may also stabilize the ligand
in the active site, possibly making potential interactions
with the C-ring hydroxyl group more effective. During
a molecular dynamics simulation of the complex, the
tert-butyl group in 23 was observed to undergo slow
rotation within the S4 pocket, while no similar motion
was observed for the N,N-dimethyl group in an equiva-
lent simulation of 12. The further increase in activity
for the N,N-dimethyl substitution (12) compared to tert-
butyl (23) can be attributed to the N,N-dimethyl group
minimizing the solvent-exposed hydrophobic surface
area and much more rigidly positioning the attached
methyl groups directly over the aromatic side chains of
Tyr99 and Phe174.
fXa Kass
fIIa Kass
(fXa/
compd
R1
R2
R3 (×106 L/mol) (×106 L/mol) fIIa)
2
4
12
H
H
H
H
OH
OH
OMe 0.84 ( 0.35 0.02 ( 0.00
OMe 0.64 ( 0.21 0.02 ( 0.01
6/3
5/4
1
1
1
1
2/1
3
1
1
NMe2 25.54
t-Bu 13.00
t-Bu 12.80
0.05
0.13
0.07
0.11
13 MeSO2NH H
16
21
22
23
24
H
H
H
H
H
CO2H
MeSO2NH t-Bu 7.65
H
OH
H
H
H
t-Bu 7.31
0.05
t-Bu 6.57 ( 1.6
0.04 ( 0.02
i-Pr 4.91
t-Bu 3.99
t-Bu 3.56
0.08
0.08
0.05
0.04
27 CO2H
30 OH
32
1
1
H
OH
Et
2.68
a
Kass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when n g 3 and average when n ) 2. The
precision of single determinations ((std error) is (12.9%. The
precision of mean values is (7.3%. Information about the statisti-
cal analysis that produced these estimates is included in the
Experimental Section. Kass ≈ 1/Ki. A more detailed discussion of
K
ass and its relationship to Ki is given in the Experimental Section.
of the bisamides to disfavor the active site binding
conformation and possibly also induce repulsive steric
interactions with the active site.
Tables 3 and 4 show the effects of replacing one of
the 4-methoxy groups of the di(4-methoxybenzoyl)-
benzenes with larger groups. A particularly sharp
increase in fXa affinity was observed when methoxy was
replaced by N,N-dimethyl or tert-butyl. Replacing a
4-methoxy group in compound 4 with N,N-dimethyl in
compound 12 produced a 40× increase in affinity. A 20×
Other B-ring substitutions reported in Tables 3 and
4 exhibit a variety of effects on fXa inhibition. Smaller
alkyl groups such as isopropyl and ethyl (24 and 32,
Table 3) produced smaller increases in affinity over their

864 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
Ta ble 5. Effects of A-Ring Substituents on fXa Inhibition by
B-Ring 4-tert-Butyldibenzamidobenzenes
a
a
fXa Kass
fIIa Kass
compd
R1
R2
(×106 L/mol) (×106 L/mol)
n
53
54
55
56
57
60
61
62
63
64
CHCH2
Cl
H
F
H
H
CH3
H
CH2CH3
OCF3
H
H
3.05
2.92
0.56
0.51
0.40
0.38
0.28
0.02
0.01
0.00
0.01
0.05
0.00
0.00
0.00
0.07
0.00
0.00
0.00
0.00
1
1
1
1
1
2
2
1
1
1
CHCH2
H
F
H
H
OCH3
H
H
F igu r e 3. Proposed energy-minimized binding model of
compound 23 complexed with the active site of fXa. The
orientation and display features are the same as in Figure 2.
D ) Asp, E ) Glu, F ) Phe, G ) Gly, H ) His, Q ) Gln, R )
Arg, S ) Ser, W ) Trp, Y ) Tyr.
a
Kass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when n g 3 and average when n ) 2. The
precision of single determinations ((std error) is (12.9%. The
precision of mean values is (7.3%. Information about the statisti-
cal analysis that produced these estimates is included in the
Experimental Section. Kass ≈ 1/Ki. A more detailed discussion of
K
ass and its relationship to Ki is given in the Experimental Section.
Ta ble 6. Effect of 1,3- versus 1,2-Substitution in
Di(4-methoxybenzamido)benzenes
n
a
a
fXa Kass
fIIa Kass
(fXa/
compd
R1
4-anisoylNH
R2
(×106 L/mol) (×106 L/mol) fIIa)
2
65
H
4-anisoylNH 0.84 ( 0.35 0.02 ( 0.00 6/3
F igu r e 4. Proposed energy-minimized binding model of
compound 12 complexed with the active site of fXa. The
orientation and display features are the same as in Figure 2.
D ) Asp, E ) Glu, F ) Phe, G ) Gly, H ) His, Q ) Gln, R )
Arg, S ) Ser, W ) Trp, Y ) Tyr.
H
0.00 0.00
1
a
Kass represents the apparent association constant as measured
by the methods of Smith et al.2k Kass values are expressed as mean
( standard deviation when n g3 and average when n ) 2. The
precision of single determinations ((std error) is (12.9%. The
precision of mean values is (7.3%. Information about the statisti-
cal analysis that produced these estimates is included in the
Experimental Section. Kass ≈ 1/Ki. A more detailed discussion of
methoxy analogues (2 and 4, Table 1). Inserting an
oxygen atom between tert-butyl or isopropyl and the
B-ring decreases affinity, possibly by inducing a less
favorable binding conformation. Compound 40 (Table
4) is 9× less active than 22 (Table 3), and 38 (Table 4)
is 4× less active than 24 (Table 3). The variety of B-ring
4-substituents shown in Tables 3 and 4 produces a
>2500× range of activities, showing the sensitivity of
the SAR to structural variations at this position. Addi-
tion of small substituents at the 2-position of the B-ring
(compounds 41 and 45) had no effect on affinity.
The SAR is sensitive to changes in substitution on
the A-ring, as can be seen by the >300× range of
activities in Table 5. In the proposed binding complex
model this ring resides within the S1 region and
positions the 4-methoxy group into a small hydrophobic
pocket formed by the side chains of Ala90, Val213, and
Tyr228. A similar interaction of aromatic substituents
with a related serine protease S1 pocket was reported
in a series of peptide thrombin inhibitors.15 Only 4-vinyl
(53) and 4-chloro (54) were more active than 4-methoxy
K
ass and its relationship to Ki is given in the Experimental Section.
(2), possibly due in part to less conformational flexibility.
The size constraints on the A-ring 4-substituent appear
to be severe. The ethyl (63) and trifluoromethoxy (64)
compounds are much less active than 2 although their
4-substituents are only slightly larger than the methoxy
group of 2. Moving the 4-vinyl or 4-methoxy group of
53 or 2 to the 3-position in 55 or 62 decreased affinity,
presumably by introducing unfavorable contacts in the
S1 pocket. Compound 65 (Table 6) with 1,3-oriented
benzamido groups was devoid of fXa inhibitory activity.
Members of the new series behave as competitive
inhibitors of fXa. For example compound 2 showed
classical competitive inhibition of fXa by Lineweaver-
Burke analysis with a Ki of 0.68 ( 0.05 µM (n ) 3) from
Dixon plots.
Only two of the compounds presented (13 and 21)
demonstrated apparent Kass values with human throm-

1,2-Dibenzamidobenzene Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 865
bin as high as 0.1 × 10-6 L/mol. The ratio of thrombin
inhibition to fXa inhibition for 13 and 21 is about 1/100.
The remainder of the compounds generally show throm-
bin inhibitory activity e 1/100 of their fXa inhibitory
activities.
this solvated system, the hydrogen positions were initially
energy minimized while all non-hydrogen atoms were fixed
rigidly in space. This was followed by spatially constrained
energy minimization of the entire system by imposing on all
non-hydrogen atoms harmonic constraints that decreased in
stages from 100 to 50 kcal/mol‚Å2 in increments of 10 kcal/
mol‚Å2. The resulting solvated protein system coordinate set
was used as the starting structure for all subsequent protein-
ligand complexes constructed. All energy minimizations were
done using the ABNR algorithm and taken to convergence
(gradient tolerance of 0.01 kcal/mol‚Å). For all energy mini-
mizations and molecular dynamics simulations, the nonbond
interactions were evaluated using a 13 Å nonbond list, updated
by the heuristic testing algorithm. For these calculations a
force shift function for the electrostatics and a shift function
for the van der Waals smoothed the nonbond interactions to
zero at a distance of 12 Å. A constant dielectric of 1.0 was used.
All calculations were performed on Silicon Graphics R4400
workstations.
The 3D structure of compound 4 was constructed in the
ChemNote facility, using default atom types and smoothing
assigned charges over all C and aliphatic H atoms to give a
total sum of 0.0. The overall initial active site conformation
and orientation were taken from that derived in a parallel
study of various related amidine-substituted analogues.13
Compound 4 was positioned by flexibly superimposing it onto
the same bisamide phenyl structure of the related analogue
possessing the derived active site binding mode.
Con clu sion s
The 1,2-bisamidophenyl compounds described above
provide a novel lead structure type for the development
of human fXa inhibitors. This series differs from previ-
ously described small molecule inhibitors of fXa by using
a 1,2-bisamidophenyl group to establish the relative
spacing and orientation between the S1 pocket binding
group (A-ring) and the S4 pocket binding group (B-ring).
In addition to orienting and spacing the A- and B-rings
favorably for S1 and S4 pocket binding, the bisami-
dophenyl unit appears to be able to hydrogen bond to
active site residues to favor specific binding. The
hundred-fold selectivity for fXa inhibition over thrombin
inhibition observed in this series is encouraging, sug-
gesting that fXa inhibitors built on this novel structure
have the potential to discriminate between fXa and
closely related serine proteases. More detailed data on
the specificity of later members of this series are
reported by Wiley et al.13
The structure of 4 was then reinserted into the active site
of the fully solvated fXa protein starting structure, and all
waters having an oxygen within 2.0 Å of any ligand atom were
deleted. The resulting complex was then processed by energy
minimization where the ligand, all protein residues having an
atom within 12 Å, and all waters within 20 Å of the active
site center were unconstrained, while the remainder of the
system was rigidly fixed in place. The final energy-minimized
structure for 4 is shown in Figure 2 where aliphatic hydrogens
and waters have been removed for clarity. A subsequent
molecular dynamics simulation was initiated from this energy-
minimized structure utilizing the same constraint regime. The
simulation utilized a time step of 1 fs and consisted of 3 ps
heating (0-300 °C in increments of 10 °C, reassigning veloci-
ties every 100 steps), 10 ps equilibration (checking the system
temperature every 100 steps and rescaling velocities to cor-
respond to 300 °C if the average deviation is more than (10
°C), and 40 ps production. During the equilibration stage for
each system investigated, velocity rescaling was required only
once or twice and only within the first 2 ps. The final
coordinates from each stage were saved, as well as from every
20th time step during the production dynamics.
All other analogues investigated were constructed from the
energy-minimized fXa complex of 4, using the 3D Editor. Each
of these were also processed by energy minimization and
molecular dynamics in the same manner as described.
Ma ter ia ls. Human fXa and human R-thrombin were pur-
chased 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 fXa) and Bz-Phe-Val-Arg-pNA (for throm-
bin).
Bin d in g Affin ity for fXa (a n d for Th r om bin ). The
binding affinities for human fXa (and for thrombin) were
measured as apparent association constants (Kass) derived from
protease inhibition kinetics as described previously.18,2k The
apparent Kass values were obtained in a high-volume protocol
using automated dilutions of inhibitors (n ) 3 for each of 4-8
inhibitor concentrations) into 96-well plates and chromogenic
substrate hydrolysis rates determined at 405 nm using a
Thermomax plate reader from Molecular Devices (San Fran-
cisco, CA). The assay protocol was: 50 µL buffer (0.06 M tris,
0.3 M NaCl, pH 7.4), 25 µL inhibitor test solution (in MeOH),
25 µL human fXa (32 nM in 0.03 M tris, 0.15 M NaCl, 1 mg/
mL HSA), finally 150 µL substrate (0.3 mM in water) added
within 2 min to start hydrolysis. The final fXa concentration
Exp er im en ta l Section
Or ga n ic Ch em istr y. Preparations of final products are
included in the Experimental Section. Preparations of inter-
mediates are included in the Supporting Information. 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 magnetic
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 tetramethylsilane.
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. Field-
desorption (FD) mass spectra were recorded on a VG Analyti-
cal 70SE instrument, electrospray ionization (ESI) mass
spectra were recorded on a Sciex API 100 instrument, and fast
atom bombardment (FAB) mass spectra were recorded on a
VG Analytical ZAB2-SE instrument. Elemental analyses 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. Analytical HPLC were performed on
a
Hitachi L-6200 instrument over a Vydac C18 analytical column,
eluting with mixtures of CH3CN in water, buffered with 0.1%
TFA.
Molecu la r Mod elin g. The coordinate set for the protein
structure used in this investigation was the human fXa, X-ray
structure of Padmanabhan et al.12 (Brookhaven PDB file 1hcg).
The supplied chymotrypsinogen residue numbering14 was
maintained. All computational construction of ligands, prepa-
ration of the protein and protein-ligand complexes, and
subsequent graphical analysis were performed with QUANTA,
version 96.16 All energy minimization and molecular dynamics
were performed using CHARMm, version 23.2.16,17
The protein was initially prepared by building and regular-
izing coordinates for all atoms in residues 146-151 (undefined
in 1hcg) in an orientation generally pointing away from the
active site. All hydrogens were added to the protein and
crystallographic waters and refined using the HBUILD pro-
cedure. The protein was then completely solvated by centering
a 8 Å sphere of TIPS3P water on each surface-exposed residue.
The ligand binding region was further solvated using a 20 Å
sphere of TIPS3P water, centered at a point central to the
active site, approximately 6 Å beyond the CR of Trp215. For

866 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
was 3.2 nM. Free [fXa] 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 inhibi-
tors as well as for weak binding enzyme inhibitors21 and has
allowed the generation of a large protease inhibitor SAR
database.19 This system of affinity measurement was designed
to: (1) validly assess a very large number of samples and (2)
determine affinity with tight binding inhibitors where classical
Ki methods fail.20,21 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)] + Eo, where a
) fraction of free enzyme.2k This equation was also derived
by Henderson20 and Beith21 for study of tight binding inhibi-
tors, where classical methods fail to satisfactorily produce Ki
determinations. Application of our method has allowed the
accumulation of a large database of apparent Kass values (the
Kass values obtained at the single appropriate substrate
concentration with each protease of interest) which are self-
consistent for each protease and which can be used to assess
selectivity by directly relating the various Kass values for
respective proteases. Apparent Kass values can be corrected by
determining the effect of substrate concentration.2k,20,21 Cor-
rected Kass values ) 1/Ki. Approximate Ki values ) 1/(apparent
Kass). 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 with 3.2 nM fXa. A single
apparent Kass determination uses 6-8 inhibitor concentrations
(in triplicate) bracketing the IC50 effect concentration, which
allows an estimation regarding the repeatability of the single
apparent Kass result. Statistical treatment of our SAR database
using a random effects analysis of variance22 led to estimates
that the precision (1 standard error) for a single Kass deter-
mination is 12.9% and the precision for a multiple determi-
nation (n ) 3) is 7.3%. The same general method was used for
evaluating protease selectivity of the fXa inhibitors by deter-
mining apparent Kass values with appropriate chromogenic
protease substrates: for human thrombin, 5.9 nM enzyme was
used with 0.2 mM Bz-Phe-Val-Arg-pNA. For classical Ki
determinations Dixon plots were generated with the same
protocol using four substrate concentrations (0.112, 0.224,
0.448, and 0.896 mM), and the type of inhibition was confirmed
with Lineweaver-Burke graphs.
the title compound. A sample was crystallized from CH2Cl2:
mp 205-206 °C; ESMS [M + H]+ 377.37 calcd C22H20N2O4
1
377.41; H NMR (DMSO-d6) δ 3.82 (s, 6H), 7.05 (d, 4H), 7.27
(dd, 2H), 7.63 (dd, 2H), 7.93 (d, 4H), 9.98 (s, 2H). Anal.
(C22H20N2O4) C, H, N.
3,6-Dim et h oxy-N1,N2-b is(4-m et h oxyb en zoyl)-1,2-b en -
zen ed ia m in e (3). To a stirred solution of 3,6-dimethoxy-1,2-
benzenediamine (2.0 mmol) and pyridine (6.1 mL, 75 mmol)
in dichloromethane (30 mL) was added 4-anisoyl chloride (4.9
mmol). After 12 h, the mixture was diluted with dichlo-
romethane and washed with 1 N aq citric acid, saturated aq
NaCl solution, saturated aq NaHCO3 solution, dried (MgSO4),
filtered, and concentrated in vacuo. The resulting solid was
suspended in Et2O, sonicated, filtered and dried in vacuo to
give 555 mg (65%) of the title compound: 1H NMR (DMSO-
d6) δ 3.75 (s, 6H), 3.78 (s, 6H), 6.98 (d, J ) 9.0 Hz, 4H), 6.99
(s, 2H), 7.84 (d, J ) 9.0 Hz, 4H), 9.28 (br s, 2H); MS (FD) m/e
435.9 (M+). Anal. (C24H24N2O6) C, H, N.
N1,N2-Bis(4-m eth oxyben zoyl)-4-h yd r oxy-1,2-ben zen e-
d ia m in e (4). P r oced u r e 1. A mixture of SnCl2‚2H2O (5.0 g,
10.0 mmol) and resin bound nitroamide11 (600 mg, 1.0 mmol
based on a substitution of 2 mequiv) in DMF (20 mL) was
stirred for 5 h. The resin was filtered and washed several times
with DMF, twice with a solution of Et3N/DMF (10%) and
several times with DCM. The resin was dried under vacuum
for a few hours prior to the next step.
To a suspension of the above resin (600 mg, 1.0 mmol) in
dichloromethane (20 mL) were added pyridine (1.3 mL, 15.0
mmoL) and a solution of p-methoxybenzoyl chloride (1.7 g, 10.0
mmol) in dichloromethane (4 mL). The mixture was stirred
for 18 h, drained and the resin washed several times with DMF
followed by several washes with dichloromethane. The resin
was dried under vacuum for a few hours prior to the next step.
Propylamine (3 mL, excess) was added to a suspension of
the resin (∼600 mg, 1.0 mmol) in THF (15 mL). The mixture
stirred for 12 h. The residue obtained after concentration of
the solution was triturated in dichloromethane to afford 250
mg (63%) of pale beige solid. Crystallization of the residue from
dichloromethane/MeOH gave 180 mg of the title product: mp
225-229 °C; 1H NMR (DMSO-d6) δ 3.81 (s, 6H), 6.66 (d, 1H),
7.04 (d, 4), 7.16 (s, 1H), 7.30 (d, 1H), 7.89 (dd, 4H), 9.57 (s,
1H), 9.81 (s, 2H). Anal. (C22H20N2O5) C, H, N.
P r oced u r e 2. Into 25 mL CH2Cl2 was dispersed 0.406 g (1
mmol) 4-methoxy-N1,N2-bis(4-methoxybenzoyl)-1,2-benzene-
diamine. After cooling the solution in an ice bath, 0.284 mL
(3 mmol) BBr3 was added in one portion. After 4 h the reaction
mixture was allowed to warm to room temperature over 1 h,
diluted with CH2Cl2 (75 mL) and washed with water (150 mL)
water. The organic layer was dried (MgSO4) and concentrated.
The product was purified by flash chromatography (silica, 0%
to 20% MeOH in CHCl3), and on cooling in freezer, fractions
crystallized, giving 96 mg (24% yield) of the desired com-
pound: 1H NMR (DMSO-d6) δ 3.80 (s, 6H), 6.66 (d, 1H), 7.05
(d, 4H), 7.18 (s, 1H), 7.30 (d, 1H), 7.89 (d, 2H), 7.95 (d, 2H),
9.57 (br s, 1H), 9.81 (s, 2H); MFD 392 (M+). Anal. (C22H20N2O5)
C, H, N.
4-Meth oxy-N1,N2-bis(4-m eth oxyben zoyl)-1,2-ben zen e-
d ia m in e (1). To a solution of 4-methoxy-1,2-benzenediamine
dihydrochloride (3.60 g, 20 mmol) in CH2Cl2 (400 mL) was
added 0.5 N aq NaOH (168 mL), and the resulting mixture
was cooled in an ice-water bath. p-Anisoyl chloride (7.2 g, 42
mmol) was added slowly with vigorous stirring. The mixture
was allowed to warm slowly to room temperature and stirred
for 18 h. The organic layer was separated and washed with
dil aq HCl, water, and saturated aq NaCl. The title compound,
5.0 g (48%), crystallized from the CH2Cl2 solution: mp 238-
4-Meth yl-N1,N2-bis(4-m eth oxyben zoyl)-1,2-ben zen ed i-
a m in e (5). Following the procedure described for the synthesis
of compound 3, 5 was prepared from 4-methyl-1,2-diaminoben-
1
1
239 °C; H NMR (DMSO-d6) δ 3.78 (s, 3H), 3.82 (s, 6H), 6.93
zene: 83% yield; H NMR (DMSO-d6) δ 2.34 (s, 3H), 3.82 (s,
(dd, J ) 9, 3 Hz, 1H), 7.05 (d, J ) 8 Hz, 4H), 7.31 (d, J ) 3 Hz,
1H), 7.45 (d, J ) 9 Hz, 1H), 7.90 (d, J ) 8 Hz, 2H), 7.93 (d, J
) 8 Hz, 2H), 9.87 (s, 1H), 9.92 (s, 1H). Anal. (C23H22N2O5) C,
H, N.
6H), 7.06 (d, J ) 8.8 Hz, 4H), 7.00-7.10 (m, 1H), 7.45-7.55
(m, 2H), 7.92 (d, J ) 8.8 Hz, 2H), 7.93 (d, J ) 8.8 Hz, 2H),
9.91 (s, 2H); MS (ESI) m/e 391.2 (MH)+. Anal. (C23H22N2O4)
C, H, N.
N1,N2-Bis(4-m eth oxyben zoyl)-1,2-ben zen ed ia m in e (2).
To a solution of o-phenylenediamine (2.16 g, 19.8 mmol) in
CH2Cl2 (200 mL) was added 0.5 N aq NaOH (84 mL), and the
resulting mixture was cooled in an ice-water bath. p-Anisoyl
chloride (6.2 g, 40 mmol) was added slowly with vigorous
stirring. The mixture was allowed to warm slowly to room
temperature and stirred for 18 h. The organic layer was
separated and washed with dil aq NaHCO3 solution, dil aq
HCl, and water. The organic layer was dried (sodium sulfate),
filtered, and concentrated in vacuo to provide 5.2 g (70%) of
2,3-Bis[(4-m eth oxyben zoyl)a m in o]p h en ol (7). A solu-
tion of 2,3-bis[(4-methoxybenzoyl)amino]phenoxy 4-methoxy-
benzoate (7.75 g, 14.7 mmol) in MeOH (100 mL) was cooled to
ice-water bath temperature and 5 N aq NaOH solution (3.0
mL) was added. The reaction mixture was allowed to warm to
room temperature and stirred for 72 h. The reaction mixture
was concentrated in vacuo and the resulting solid was dis-
solved in CH2Cl2 and washed with dil aq HCl, water, and
saturated NaCl solution. The organic layer was dried (sodium
sulfate), filtered, and concentrated in vacuo. Recrystallization

1,2-Dibenzamidobenzene Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 867
of the residue from Et2O/hexane gave 2.3 g (40%) of the title
product: mp 208-209 °C; 1H NMR (DMSO-d6) δ 3.82 (s, 3H),
3.84 (s, 3H), 6.79 (d, 1H), 7.04 (dd, 4H), 7.17 (t, 1H), 7.24 (d,
1H), 7.83 (d, 2H), 7.99 (d, 2H), 9.62 (bs, 3H). Anal. (C22H20N2O5)
C, H, N.
1H), 6.73 (d, J ) 8.7, 2H), 7.05 (d, J ) 8.7, 2 H), 7.19 (d, J )
3.0, 1H), 7.28 (d, J ) 8.7, 1H), 7.77 (d, J ) 8.7, 2H), 7.92 (d, J
) 8.7, 2H), 9.49 (s, 1H), 9.61 (s, 1H), 9.85 (s, 1H); IR 1606,
1504, 1292, 1264, 1175 cm-1; MS (ion spray) m/e 406 (M + 1).
Anal. (C23H23N3O4) C, H; N: calcd, 10.36; found, 9.72.
4-Ch lor o-N1,N2-bis(4-m eth oxyben zoyl)-1,2-ben zen ed i-
a m in e (8). Following the procedure described for the synthesis
of compound 3, 8 was prepared from 4-chloro-1,2-diaminoben-
N2-(4-ter t-Bu tylben zoyl)-N5-m eth ylsu lfon yl-N1-(4-m eth -
oxyben zoyl)-1,2,5-ben zen etr ia m in e (13). N2-(4-tert-Butyl-
benzoyl)-N1-(4-methoxybenzoyl)-1,2,5-benzenetriamine triflu-
oroacetate (230 mg, 0.55 mmol) was dissolved in CH2Cl2 (50
mL) and 2,6-di-tert-butylpyridine (247 µL, 1.1 mmol) and
methylsulfonyl chloride (47 µL, 0.61 mmol) were added. The
reaction was stirred at room temperature under nitrogen for
8 h. More methylsulfonyl chloride (47 µL, 0.61 mmol) and 2,6-
di-tert-butylpyridine (247 µL, 1.1 mmol) were added. The
reaction was stirred for an additional 16 h. Additional 2,6-di-
tert-butylpyridine (1 mL, 4.45 mmol) was added and the
reaction was stirred for 2 more h. The reaction was concen-
trated in vacuo and the residue was dissolved in EtOAc and
washed with 1 N HCl (2 × 20 mL). The organic layer was dried
over MgSO4, filtered, and concentrated. The crude residue was
purified by flash column chromatography (1:1 CH2Cl2/EtOAc)
to give the desired product (160 mg, 59%): 1H NMR (DMSO-
d6) δ 1.30 (s, 9H), 3.03 (s, 3H), 3.82 (s, 3H), 7.06(d, J ) 9.0,
2H), 7.11 (dd, J ) 2.6, 8.7, 1H), 7.55 (m, 4H), 7.86 (d, J ) 8.7,
2H), 7.93 (d, J ) 8.7, 2H), 9.83 (s, 1H), 9.94 (s, 1H), 9.99 (s,
1H); IR 3284, 3242, 1657, 1612, 1501, 1388, 1335, 1286, 1257,
1156 cm-1; MS m/e 495.31. Anal. (C26H29N3O5S) C, H, N.
3-[(4-ter t-Bu tylben zoyl)a m in o]-4-[(4-m eth oxyben zoyl)-
a m in o]ben zoic Acid (16). Using the procedure described in
for the preparation of compound 27, methyl 3-[(4-tert-butyl-
benzoyl)amino]-4-[(4-methoxybenzoyl)amino]benzoate (1.00
mmol) yielded, after acidification of the aq layer, 318 mg (71%)
of the title compound as a crystalline solid: 1H NMR (DMSO-
d6) δ 1.28 (s, 9H), 3.80 (s, 3H), 7.05 (d, 2H), 7.52 (d, 2H), 7.81
(s, 1H), 7.89 (d, 1H), 7.94 (d, 1H), 8.19 (s, 1H), 10.11 (s, 1H),
10.19 (s, 1H); IR (KBr) 1645, 1690, 3256 cm-1; MS (FD) m/e
446 (M+). Anal. (C26H26N2O5) C, H, N.
N2-(4-ter t-Bu tylben zoyl)-N4-m eth ylsu lfon yl-N1-(4-m eth -
oxyben zoyl)-1,2,4-ben zen etr ia m in e (21). The N2-(4-tert-
butylbenzoyl)-N1-(4-methoxybenzoyl)-1,2,4-benzenetriamine (230
mg, 0.55 mmol) was dissolved in CH2Cl2 (50 mL) and 2,6-di-
tert-butylpyridine (247 µL, 1.1 mmol) and methylsulfonyl
chloride (47 µL, 0.61 mmol) were added. The reaction was
stirred at room temperature under nitrogen for 8 h. More 2,6-
di-tert-butylpyridine (247 µL, 1.1 mmol) and methylsulfonyl
chloride (47 µL, 0.61 mmol) were added. After stirring for 2
more h, the reaction was then concentrated in vacuo. The
residue was dissolved in EtOAc and washed with 1 N HCl (2
× 20 mL). The organic layer was dried over MgSO4, filtered,
and concentrated. The crude product was purified by flash
column chromatography (1:1 EtOAc/CH2Cl2) to give the pure
product (204 mg, 75%): 1H NMR (DMSO-d6) δ 1.30 (s, 9H),
3.03 (s, 3H), 3.82 (s, 3H), 7.06 (d, J ) 9.0, 2H), 7.11 (dd, J )
2.4, 8.9, 1H), 7.56 (m, 4H), 7.87 (d, J ) 8.7, 2H), 7.93 (d, J )
9.0, 2H), 9.83 (s, 1H), 9.90 (s, 1H), 10.05 (s, 1H); IR 1653, 1608,
1510, 1327, 1255, 1156 cm-1; MS (FD) m/e 495.45. Anal.
(C26H29N3O5S) C, H, N.
1
zene: 86% yield; H NMR (DMSO-d6) δ 3.82 (s, 6H), 7.06 (d,
J ) 9.0 Hz, 4H), 7.32 (dd, J ) 2.6, 8.8 Hz, 1H), 7.65 (d, J )
8.8 Hz, 1H), 7.78 (d, J ) 2.6 Hz, 1H), 7.85-7.95 (m, 4H), 9.97
(s, 1H), 10.00 (s, 1H); MS (ESI) m/e 409.2 (MH)+. Anal. (C22H19
-
ClN2O4) C, H, N.
3,4-Bis[(4-m eth oxyben zoyl)a m in o]ben zoic Acid (9). A
solution of p-anisoyl chloride (5.64 g, 33 mmol) in CH2Cl2 (40
mL) was dropped into pyridine (100 mL) containing 4.56 g (30
mmol) 3,4-diaminobenzoic acid, cooled by an ice bath. Reaction
mixture was allowed to warm gradually to room temperature.
After 72 h, CH2Cl2 (200 mL) was added, forming a precipitate,
which was collected and washed with CH2Cl2. The product was
recrystallized from a concentrated CHCl3-MeOH solution,
giving 148 mg (1.2% yield) of the desired compound: 1H NMR
(DMSO-d6) δ 3.81 (s, 6H), 7.05 (d, 4H), 7.82 (m, 2H), 7.90 (m,
4H), 8.19 (s,1H), 10.07 (s, 1H), 10.08 (s, 1H), 12.9 (br s, 1H);
MS (FD) m/e (420 (M+). Anal. (C23H20N2O6) C, H, N.
N1-Met h yl-N1,N2-b is(4-m et h oxyb en zoyl)-1,2-b en zen e-
d ia m in e (10). Using the procedure described for the prepara-
tion of compound 3, compound 10 was prepared from N1-
1
methyl-1,2-benzenediamine: 95% yield; H NMR (DMSO-d6)
δ 3.25 (s, 3H), 3.70 (s, 3H), 3.85 (s, 3H), 6.72 (br d, J ) 8.0 Hz,
2H), 7.0-7.1 (m, 4H), 7.2-7.3 (m, 3H), 7.52 (d, J ) 8.0 Hz,
1H), 7.90 (d, J ) 9.0 Hz, 2H), 9.68 (s, 1H); MS (FD) m/e 390.0
(M+). Anal. (C23H22N2O4) C, H, N.
N1,N2-Dim eth yl-N1,N2-bis(4-m eth oxyben zoyl)-1,2-ben -
zen ed ia m in e (11). To a stirring solution of compound 10 (0.5
g, 1.28 mmol) in THF (5 mL) at 0 °C was added a 0.5 M
solution of lithium bis(trimethylsilyl)amide in THF (2.4 mL,
1.2 mmol), followed by iodomethane (0.5 mL, 6.4 mmol). The
cold bath was removed and after stirring overnight, the solvent
was removed in vacuo and the residue was partitioned between
EtOAc and water. The organic phase was washed twice with
1 M citric acid, once with brine, twice with saturated aq
NaHCO3, and once again with brine. The organic phase was
then dried with MgSO4, filtered and concentrated in vacuo.
The residue was dissolved in a small amount of CHCl3 and
chromatographed over silica gel, eluting with a gradient of 30%
EtOAc in hexanes through 70% EtOAc in hexanes. The product
containing fractions were combined and concentrated in vacuo
to give 0.5 g (98%) of a white foam: 1H NMR (DMSO-d6) δ
3.72 (br s, 12 H), 6.7-7.4 (br m, 12 H); MS (FD) m/e 404.1
(M+). Anal. (C24H24N2O4) C, H, N.
N2-[4-(Dim eth yla m in o)ben zoyl]-4-h yd r oxy-N1-(4-m eth -
oxyben zoyl)-1,2-ben zen ed ia m in e (12). A solution of 4-(di-
methylamino)benzoic acid and thionyl chloride in CH2Cl2 was
refluxed 4 h. Volatile solvents were removed in vacuo to yield
1.10 g of 4-(dimethylamino)benzoyl chloride. This material was
used in subsequent reactions without purification.
N1-(4-Met h oxyb en zoyl)-N2-(4-ter t-b u t ylb en zoyl)-1,2-
ben zen ed ia m in e (22). Using the procedure described for the
preparation of 3,6-dimethoxy-N1,N2-bis(4-methoxybenzoyl)-1,2-
benzenediamine (3), 4-anisoyl chloride (2.2 mmol) and N1-(4-
tert-butylbenzoyl)-1,2-benzenediamine (1.8 mmol) yielded 441
mg (61%) of the title compound: 1H NMR (DMSO-d6) δ 1.30
(s, 9 H), 3.83 (s, 3 H), 7.07 (d, J ) 9.0 Hz, 2 H), 7.27 (m, 2 H),
7.54 (d, J ) 8.5 Hz, 2 H), 7.64 (m, 2 H), 7.88 (d, J ) 8.5 Hz, 2
H), 7.94 (d, J ) 8.5 Hz, 2 H), 7.88 (d, J ) 8.5 Hz, 2 H), 7.94 (d,
J ) 8.5 Hz, 2 H), 10.00 (br s, 2 H); MS (FD) m/e 402.3 (M+).
Anal. (C25H26N2O3) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-(4-ter t-bu tylben zoyl)-4-h y-
d r oxy-1,2-ben zen ed ia m in e (23). Using 4-tert-butylbenzoyl
chloride and the procedure described for the preparation of
compound 4 yielded 174 mg (40%) of the title compound a
white solid: mp 234-235 °C; 1H NMR (MeOD) δ 1.25 (s, 9H),
3.76 (s, 3H), 6.64 (dd, 1H), 6.90 (d, 2H), 7.09 (d, 1H), 7.24 (d,
To a mixture of 4-tert-butyldimethylsilyloxy-N1-(4-methoxy-
benzoyl)-1,2-benzenediamine (200 mg, 1.20 mmol) and 4-(di-
methylamino)benzoyl chloride (200 mg) in CH2Cl2 (5 mL) were
added excess N-methylmorpholine and a catalytic amount of
4-(dimethylamino)pyridine. The mixture was stirred 16 h at
ambient temperature then was partitioned between EtOAc and
saturated NaHCO3. The organic portion was washed with
brine, dried over MgSO4 and concentrated in vacuo. The
residue was taken up in EtOAc and hexane added until cloudy.
The mixture was sonicated causing a precipitate to form. The
solid silyl ether was collected by filtration then dissolved in 3
mL THF. The solution was treated with 1 mL 5 N HCl and
allowed to stand for 60 h then neutralized with saturated
NaHCO3 solution. Hexane was added and the mixture soni-
cated. The resultant solid was collected by filtration and dried
under vacuum to yield the title bisamide phenol: 1H NMR
(DMSO-d6) δ 2.98 (s, 6H), 3.82 (s, 3H), 6.63 (dd, J ) 2.8, 8.5,

868 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
1H), 7.44 (d, 2H), 7.73-7.83 (m, 5H). Anal. (C25H26N2O4‚1/
3H2O) C, H, N.
Hz), 7.92 (d, 2 H, J ) 8.7 Hz), 9.53 (s, 1 H), 9.80 (s, 1 H), 9.84
(s, 1 H); MS (FAB) 391.1 (M + 1). Anal. (C23H22N2O4) C, H, N.
N1-(4-Met h oxyb en zoyl)-N2-(4-isop r op ylb en zoyl)-1,2-
ben zen ed ia m in e (24). To a mixture of 4-isopropylbenzoic
acid (1.65 mmol) and pyridine (2.1 mmol) in toluene (15 mL)
was added thionyl chloride (2.1 mmol). After heating at 80 °C
for 3 h, the reaction mixture was cooled and concentrated in
vacuo to give 4-isopropylbenzoyl chloride. A solution of this
material (1.65 mmol) in CH2Cl2 (15 mL) was added to a
mixture of N1-(4-methoxybenzoyl)-1,2-benzenediamine (399
mg, 1.65 mmol) and pyridine (1.65 mmol) in CH2Cl2 (15 mL)
cooled to 0 °C. The reaction mixture was warmed to room
temperature and stirred for 1 h. The reaction was partitioned
between CH2Cl2 (25 mL) and 1 N HCl (10 mL). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo
to give 523 mg (82%) of the title compound as a white solid:
1H NMR (CDCl3) δ 1.30 (d, 6H, J ) 6.9 Hz), 3.00 (m, 1H), 6.94-
6.92 (m, 2H), 7.00 (d, 2H, J ) 8.7 Hz), 7.36 (d, 2H, J ) 8.1
Hz), 7.46-7.40 (m, 2H), 7.93 (d, 2H, J ) 8.4 Hz), 7.99 (d, 2H,
J ) 9.0 Hz), 9.21 (s, 1H), 9.23 (s, 1H); MS (FD) m/e 388 (M+);
IR (CHCl3) 1256, 1507, 1608, 1646 cm-1. Anal. (C24H24N2O3)
C, H, N.
3-[(4-Meth oxyben zoyl)a m in o]-4-[(4-ter t-bu tylben zoyl)-
a m in o]ben zoic Acid (27). To a solution of methyl 3-[(4-
methoxybenzoyl)amino]-4-[(4-tert-butylbenzoyl)amino]-ben-
zoate (0.487 g, 1.00 mmol) in THF (32 mL) and MeOH (8 mL)
was added 5 N aq NaOH (0.6 mL). The resulting mixture was
stirred for 16 h, a second portion of 5 N aq NaOH (0.6 mL)
added, and the mixture stirred for an additional 16 h. The
solvent was concentrated in vacuo and the crude product
acidified with dil aq HCl and diluted with EtOAc. The mixture
was extracted with saturated aq K2CO3 solution. The aq layer
was acidified and extracted with EtOAc. The organic layer was
dried (MgSO4), filtered, and concentrated in vacuo. Crystal-
lization of the residue from CH2Cl2/hexane provided 376 mg
(100%) of the title product: 1H NMR (DMSO-d6) δ 1.28 (s, 9H),
3.81 (s, 3H), 7.05 (d, 2H), 7.53 (d, 2H), 7.82 (d, 1H), 7.86 (d,
2H), 7.87 (d, 1H), 8.18 (s, 1H), 10.08 (s, 1H), 10.12 (s, 1H); MS
(FD) m/e 446 (M+); IR (KBr) 1608, 1659, 1687, 2963 cm-1.;
Anal. (C26H26N2O5) H, N; C: calcd, 69.94; found, 70.90.
N1-(4-Meth oxyben zoyl)-N2-(4-ter t-bu tylben zoyl)-5-h y-
d r oxy-1,2-ben zen ed ia m in e (30). The N1-(4-methoxyben-
zoyl)-N2-(4-tert-butylbenzoyl)-5-tert-butyldimethylsilylhydroxy-
1,2-benzenediamine (220 mg, 0.41 mmol) was dissolved in THF
(20 mL) and the solution was cooled to 0 °C. A 1 M THF
solution of tetra-n-butylammonium fluoride (0.82 mL, 0.82
mmol) was added and the reaction turned yellow. After stirring
for 15 min at 0 °C, the reaction was quenched with water (5
mL). The THF was removed under reduced pressure and the
residue was partitioned between EtOAc and brine. The organic
layer was dried over MgSO4, filtered, and concentrated in
vacuo. The crude product was purified using flash column
chromatography (10% EtOAc/CH2Cl2-50% EtOAc/CH2Cl2) to
give the desired compound (140 mg, 81%): 1H NMR (DMSO-
d6) δ 1.29 (s, 9H), 3.81 (s, 3H), 6.65 (dd, J ) 2.4, 8.5, 1H), 7.04
(d, J ) 8.7, 2H), 7.17 (d, J ) 2.3, 1H), 7.31 (d, J ) 9.0, 1H),
7.51 (d, J ) 8.3, 2H), 7.87 (t, J ) 7.3, 4H), 9.53 (s, 1H), 9.79 (s,
1H), 9.84 (s, 1H); MS m/e 418.03. Anal. (C25H26N2O4) C, H; N:
calcd, 6.69; found, 5.98.
N1-(4-Meth oxyben zoyl)-N2-(4-eth ylben zoyl)-4-h yd r oxy-
1,2-ben zen ed ia m in e (32). To a solution of N1-(4-methoxy-
benzoyl)-N2-(4-propylbenzoyl)-4-(tert-butyldimethylsilyloxy)-
1,2-benzenediamine (300 mg, 0.59 mmol) in THF (20 mL)
cooled to 0 °C was added a 1 M solution of tetra-n-butylam-
monium fluoride in THF (1.4 mL, 1.4 mmol). After 15 min,
the reaction mixture was diluted with water and partitioned
with EtOAc. The organic layer was dried (MgSO4), filtered,
and concentrated in vacuo. The residue was chromatographed
(silica gel, 10% EtOAc/90% CH2Cl2 to 40% EtOAc/60% CH2-
Cl2) to give 200 mg (87%) of the title compound as a white
solid: 1H NMR (DMSO-d6) δ 1.18 (t, 3 H, J ) 7.5 Hz), 2.65 (q,
2 H, J ) 7.5 Hz), 3.81 (s, 3 H), 6.65 (dd, 1 H, J ) 8.7, 2.6 Hz),
7.04 (d, 2 H, J ) 8.7 Hz), 7.21 (d, 1 H, J ) 2.6 Hz), 7.29 (d, 1
H, J ) 9.0 Hz), 7.33 (d, 2 H, J ) 7.9 Hz), 7.81 (d, 2 H, J ) 8.3
N1-[4-(Dim eth ylam in o)ben zoyl]-N2-(4-m eth oxyben zoyl)-
1,2-ben zen ed ia m in e (33). A mixture of N1-(4-methoxyben-
zoyl)-1,2-benzenediamine (242 mg, 1.00 mmol), 4-(dimethy-
lamino)benzoicacid(200mg,1.24mmol),1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (2040 mg, 1.26 mmol), and
1-hydroxybenzotriazole (135 mg, 1.00 mmol) in 3 mL CH2Cl2
was stirred 48 h at ambient temperature. The mixture was
partitioned between EtOAc and 10% citric acid. The organic
portion was washed with water and saturated NaHCO3 then
dried over MgSO4, filtered and concentrated in vacuo. The
residue was purified on silica gel with 40% EtOAc in hexane
to yield the title compound (77 mg, 20%) as a white powder:
1H NMR (CDCl3) δ 3.07 (s, 6H), 3.87 (s, 3H), 6.86 (d, J ) 8.7,
2H), 6.97 (d, J ) 8.7, 2H), 7.06 (m, 2H), 7.43 (m, 1H), 7.56 (m,
1H), 7.91 (d, J ) 8.7, 2H), 7.96 (d, J ) 8.7, 2H), 8.87 (s, 1H),
9.22 (s, 1H); MS (FD) m/e 389 (M+). Anal. (C23H23N3O3) C, H,
N.
N1-(4-Meth oxyben zoyl)-N2-[4-(1-m eth oxy-1-m eth yleth -
yl)ben zoyl]-1,2-ben zen ed ia m in e (36). Using the procedure
described for the preparation of compound 24, N1-(4-methoxy-
benzoyl)-1,2-benzenediamine (125 mg, 0.52 mmol) was allowed
to react with 4-[2-(2-methoxypropyl)]benzoic acid (93 mg, 0.48
mmol) to yield 107 mg (49%) of the title compound as a white
solid: 1H NMR (CDCl3) δ 1.58 (s, 6H), 3.13 (s, 3H), 3.90 (s,
3H), 6.92-6.97 (m, 2H), 7.01 (d, 2H, J ) 9.0 Hz), 7.46-7.42
(m, 2H), 7.55 (d, 2H, J ) 8.4 Hz), 7.97-8.02 (m, 4H), 9.20 (s,
1H), 9.36 (s, 1H); MS (FD) m/e 418 (M+). Anal. (C25H26 N2O4)
C, H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(m eth ylth io)ben zoyl]-1,2-
ben zen ed ia m in e (37). Using the procedure described for the
preparation of compound 24, N1-(4-methoxybenzoyl)-1,2-ben-
zenediamine (772 mg, 3.19 mmol) was allowed to react with
4-(methylthio)benzoic acid (772 mg, 4.59 mmol) to yield 1.13
g (91%) of the title compound as a white solid: 1H NMR
(CDCl3) δ 9.37 (s, 1H), 9.18 (s, 1H), 7.95 (d, 2H, J ) 10.5 Hz),
7.90 (d, 2H, J ) 10.2 Hz), 7.40-7.33 (m, 2H), 7.30 (d, 2H, J )
10.8 Hz), 6.97 (d, 2H, J ) 10.5 Hz), 6.85 (m, 2H), 3.87 (s, 3H),
2.52 (s, 3H); MS (FD) 392 (M+); IR (CHCl3) 1256, 1508, 1599,
1644 cm-1. Anal. (C22H20N2O3S) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(1-m eth yleth oxy)ben zoyl]-
1,2-ben zen ed ia m in e (38). Using the procedure described for
the preparation of compound 43, 4-(1-methylethoxy)benzoic
acid (2.00 mmol) yielded, after recrystallization from CH2Cl2/
hexane, 272 mg (34%) of the title compound: 1H NMR (DMSO-
d6) δ 1.24 (d, 6H), 3.78 (s, 3H), 4.68 (septet, 1H), 6.99 (d, 2H),
7.02 (d, 2H), 7.2 (m, 2H), 7.6 (m, 2H), 7.86 (d, 2H), 7.91 (d,
2H), 9.96 (s, 2H); IR (KBr) 1607, 1648, 3300 cm-1; MS (FD)
m/e 404 (M+). Anal. (C24H24N2O4) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-(4-p h en ylben zoyl)-1,2-ben -
zen ed ia m in e (39). Using a procedure similar to that de-
scribed for the preparation of compound 3, 4-phenylbenzoic
acid (200 mg, 1.01 mmol) yielded 75 mg (18%) of the title
compound: 1H NMR (DMSO-d6) δ 3.83 (s, 3H), 7.08 (d, J )
9.0, 2H), 7.31 (m, 2H), 7.43 (m, 1H), 7.52 (t, J ) 7.3, 2H), 7.68
(m, 2H), 7.76 (d, J ) 7.2, 2H), 7.85 (d, J ) 8.3, 2H), 7.97 (d, J
) 9.0, 2H), 8.05 (d, J ) 8.3, 2H); MS (FD) m/e 422 (M+). Anal.
(C27H22N2O3) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-(4-ter t-bu toxyben zoyl)-1,2-
ben zen ed ia m in e (40). The title compound was prepared from
4-tert-butoxybenzoyl chloride using the methods described for
the synthesis of compound 43 to give 0.231 g (28%) crystallized
from CH2Cl2-hexane: 1H NMR (DMSO-d6) δ 1.35 (s, 9H), 3.79
(s, 3H), 7.03 (d, 2H), 7.09 (d, 2H), 7.15 (m, 2H), 7.6 (m, 2H),
7.86 (d, 2H), 9.90 (d, 2H), 9.93 (s, 1H), 9.99 (s, 1H); MS (FD)
m/e 418 (M+). Anal. (C25H26N2O4) C, H, N.
N1-(2-Ch lor o-4-m et h oxyb en zoyl)-N2-(4-m et h oxyb en -
zoyl)-1,2-ben zen ed ia m in e (41). Using the procedure de-
scribed for compound 45, 2-chloro-4-methoxybenzoyl chloride
(410 mg, 2.42 mmol) yielded 770 mg (77%) of the title
compound: 1H NMR δ (DMSO-d6) 3.81 (s, 3H), 3.82 (s, 3H),

1,2-Dibenzamidobenzene Inhibitors of fXa
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5 869
6.98-7.15 (m, 4H), 7.22-7.31 (m, 2H), 7.60-7.70 (m, 3H), 7.95
(d, 2H), 9.80 (bs, 1H), 10.02 (bs, 1H). Anal. (C22H19ClN2O4) C,
H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(m eth ylsu lfin yl)ben zoyl]-
1,2-ben zen ed ia m in e (47). To a solution of N1-(4-methoxy-
benzoyl)-N2-[4-(methylthio)benzoyl]-1,2-benzenediamine (417
mg, 0.60 mmol) in CHCl3 (20 mL), cooled to 0 °C was added
m-chloroperoxybenzoic acid (346 mg, 1.16 mmol). After 30 min,
the reaction mixture was warmed to room temperature and
calcium hydroxide (123 mg, 1.66 mmol) was added. After 15
min, the reaction mixture was filtered and the filtrate con-
centrated in vacuo. The residue was chromatographed (silica
gel, 50% EtOAc/50% hexanes to 80% EtOAc/20% hexanes) to
give 360 mg (83%) of the title compound as a white solid: 1H
NMR (CDCl3) δ 2.77 (s, 3H) 3.89 (s, 3H), 6.91 (m, 2H), 7.00 (d,
2H, J ) 8.7 Hz), 7.45 (m, 1H), 7.32 (m, 1H), 7.75 (d, 2H, J )
8.7 Hz), 7.97 (d, 2H, J ) 8.7 Hz), 8.13 (d, 2H, J ) 8.7 Hz),
9.12 (s, 1H), 9.79 (s, 1H); MS (FD) 408 (M+); IR (CHCl3) 1257,
1508, 1607, 1651, 3008 cm-1. Anal. (C22H20N2O4) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(d im eth yla m in osu lfon yl-
)ben zoyl]-1,2-b en zen ed ia m in e (42). Using the procedure
described for the preparation of compound 24, N1-(4-methoxy-
benzoyl)-1,2-benzenediamine (534 mg, 2.21 mmol) was allowed
to react with 4-(dimethylaminosulfonyl)benzoic acid (534 mg,
2.33 mmol) to yield 349 mg (35%) of the title compound: 1H
NMR (CDCl3) δ 2.76 (s, 6H), 3.90 (s, 3H), 6.97 (m, 2H), 7.02
(d, 2H, J ) 9.0 Hz), 7.32 (d, 1H, J ) 9.6 Hz), 7.53 (d, 1H, J )
9.3 Hz), 7.89 (d, 2H, J ) 8.4 Hz), 7.96 (d, 2H, J ) 8.7 Hz),
8.15 (d, 2H, J ) 8.4 Hz), 8.90 (s, 1H), 9.79 (s, 1H); MS (FD)
m/e 453 (M+); IR (CHCl3) 1166, 1257, 1508, 1607, 1652 cm-1
.
Anal. (C23H23N3O5S) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-(4-eth oxyben zoyl)-1,2-ben -
zen ed ia m in e (43). To a mixture of 4-ethoxybenzoic acid
(0.332 g, 2.00 mmol) and a few drops of N,N-dimethylforma-
mide in CH2Cl2 (50 mL) cooled to 0 °C was added oxalyl
chloride (0.21 mL, 2.2 mmol). After 30 min reaction the
mixture was warmed to room temperature and was stirred for
an additional 10 min. The mixture was concentrated in vacuo,
and the residue was dissolved in CH2Cl2 (10 mL). The resulting
solution was added in two portions to a mixture of N1-(4-
methoxybenzoyl)-1,2-benzenediamine (0.455 g, 2.00 mmol) and
Et3N (0.281 mL, 2.00 mmol) in CH2Cl2 (40 mL) cooled to 0 °C.
After 4 h, the mixture was allowed to warm to room temper-
ature and was stirred for an additional 12 h. The reaction was
quenched with cold dil aq HCl (50 mL), diluted with hexane,
and shaken in a separatory funnel. The organic layer was
washed with cold dil aq HCl and saturated aq NaHCO3
solution. The solution was dried (MgSO4), filtered, and con-
centrated in vacuo. Recrystallization of the residue from CH2-
Cl2/hexane provided 317 mg (41%) of title compound: 1H NMR
(DMSO-d6) δ 1.37 (t, 3H), 3.82 (s, 3H), 4.10 (q, 2H), 7.05 (d,
2H), 7.08 (d, 2H), 7.3 (m, 2H), 7.6 (m, 2H), 7.86 (d, 4H), 9.97
(s, 2H); IR (KBr) 1606, 1646, 3259 cm-1; MS (FD) m/e 390 (M+).
Anal. (C23H23N2O4) H, N; C: calcd, 70.75; found, 66.03.
N1-(4-Meth oxyben zoyl)-N2-(4-m eth ylsu lfon ylben zoyl)-
1,2-ben zen ed ia m in e (44). Using the procedure described for
the preparation of compound 24, N1-(4-methoxybenzoyl)-1,2-
benzenediamine (399 mg, 1.65 mmol) and 4-methylsulfonyl-
benzoic acid (463 mg, 2.31 mmol) yielded 591 mg (84%) of the
title compound as a white solid: 1H NMR (CDCl3) δ 3.08 (s,
3H), 3.88 (s, 3H), 7.00 (d, 2H, J ) 8.7 Hz), 7.32-7.12 (m, 3H),
7.71 (d, 1H, J ) 8.4 Hz), 7.91 (d, 2H, J ) 8.7 Hz), 8.04 (d, 2H,
J ) 8.4 Hz), 8.15 (d, 2H, J ) 8.4 Hz), 8.51 (s, 1H), 9.75 (s,
1H); MS (FD) m/e 424 (M+); IR (KBr) 757, 1155, 1437, 1509,
1659, 3300 cm-1. Anal. (C22H20N2O5S) C, H, N.
N1-(2,4-Dim eth oxyben zoyl)-N2-(4-m eth oxyben zoyl)-1,2-
ben zen ed ia m in e (45). To a solution N1-(4-methoxybenzoyl)-
1,2-benzenediamine hydrochloride (558 mg, 2.08 mmol) in
CH2Cl2 (100 mL) was added 0.5 N aq NaOH (8 mL), and the
resulting mixture was cooled in an ice-water bath. 2,4-
Dimethoxybenzoyl chloride (0.40 g, 2.0 mmol) was added
slowly with vigorous stirring. The mixture was allowed to
warm slowly to room temperature and stirred for 18 h. The
organic layer was separated and washed with dil aq NaHCO3
solution, dil aq HCl, and water. The organic layer was dried
(sodium sulfate), filtered, and concentrated in vacuo to provide
0.52 g (64%) of the title compound: 1H NMR δ (DMSO-d6) 3.58
(s, 3H), 3.82 (s, 3H), 3.88 (s, 3H), 6.61 (d, 1H), 6.68 (dd, 1H),
7.0-7.4 (m, 5H), 7.95-8.20 (m, 4H), 9.92 (s, 1H), 10.12 (s, 1H).
Anal. (C23H22N2O5) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(p r op yloxy)ben zoyl]-1,2-
ben zen ed ia m in e (46). The title compound was prepared from
4-propyloxybenzoyl chloride using the methods described for
the synthesis of compound 43 to give 0.224 g (28%) of a
crystalline title compound from CH2Cl2-hexane: 1H NMR
(DMSO-d6) δ 0.96 (t, 3H), 1.74 (hextet, 2H), 3.83 (s, 3H), 3.99
(t, 2H), 3.99 (t, 2H), 7.05 (d, 4H), 7.27 (m, 2H), 7.65 (m, 2H),
7.94 (d, 4H), 9.59 (s, 2H); MS (FD) m/e 404 (M+). Anal.
(C24H24N2O4) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-(4-ph en oxyben zoyl)-1,2-ben -
zen ed ia m in e (48). The N1-(4-methoxybenzoyl)-1,2-benzene-
diamine (200 mg, 0.826 mmol), 4-phenoxybenzoic acid (177 mg,
0.826 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (316 mg, 1.65 mmol), and DMAP (10 mg, 0.083
mmol) were combined in CH2Cl2 (2 mL) and stirred for 18 h.
The mixture was then diluted with additional CH2Cl2, washed
with 1 N NaOH, and with 1 N HCl. The organic layer was
dried over sodium sulfate, filtered, and concentrated in vacuo
to provide the desired product (294 mg, 0.67 mmol, 81%) as a
foam which solidified: 1H NMR (CDCl3) δ 3.89 (s, 3H), 6.75
(m, 2H), 7.05 (m, 6H), 7.21 (m, 1H), 7.32 (m, 2H), 7.41 (t, J )
7.8, 2H), 8.00 (d, J ) 7.2, 4H), 9.50 (s, 1H), 9.61 (s, 1H); IR
1646, 1607, 1505, 1245 cm-1; MS m/e 438.70. Anal. (C27H22N2O4)
H, N; C: calcd, 73.96; found, 72.22.
N1-(4-Bu toxyben zoyl)-N2-(4-m eth oxyben zoyl)-1,2-ben -
zen ed ia m in e (50). The title compound was prepared from
4-butoxybenzoyl chloride using the methods described for the
synthesis of compound 43 to give 0.277 g of crystalline product
(33%) from CH2Cl2-hexane: 1H NMR (DMSO-d6) δ 0.982 (s,
3H), 1.44 (hextet, 2H), 1.71 (quintet, 2H), 3.82 (s, 3H), 4.03
(q, 2H), 7.04 (d, 2H), 7.06 (d, 2H), 7.25 (t, 1H), 7.28 (t, 1H),
7.54 (m, 2H), 7.91 (d, 2H), 7.93 (d, 2H), 8.89 (s, 2H); MS (FD)
m/e 418 (M+). Anal. (C25H26N2O4) C, H, N.
N1-(4-Meth oxyben zoyl)-N2-[4-(tr iflu or oacetyl)ben zoyl]-
1,2-ben zen ed ia m in e (51). The title compound was prepared
from 4-trifluoroacetylbenzoyl chloride using the methods
described for the synthesis of compound 43 to give 0.511 g
(58%) crystallized from CH2Cl2-hexane: 1H NMR (DMSO-d6)
δ 3.80 (s, 3H), 7.03 (d, 2H), 7.28 (m, 2H), 7.64 (m, 2H), 7.92 (d,
2H), 8.15 (s, 4H), 9.89 (s, 1H), 10.32 (s, 1H); MS (FD) m/e 442
(M+). Anal. (C23H17F3N2O4) C, H, N.
N1-[4-(Hexyloxy)ben zoyl]-N2-(4-m eth oxyben zoyl)-1,2-
ben zen ed ia m in e (52). The title compound was prepared from
4-hexyloxybenzoyl chloride using the methods described for
the synthesis of compound 43 to give 0.415 g (46%) of the title
compound as crystals from CH2Cl2-hexane: 1H NMR (DMSO-
d6) δ 0.84 (t, 3H), 1.30 (m, 4H), 1.41 (quintet, 2H), 1.73 (quintet,
2H), 3.82 (s, 3H), 4.03 (t, 2H), 7.05 (d, 2H), 7.07 (d, 2H), 7.74
(m, 2H), 7.78 (d, 2H), 7.79 (d, 2H), 9.59 (s, 2H); MS (FD) m/e
446 (M+). Anal. (C27H30N2O4) C, H, N.
N2-(4-ter t-Bu t ylb en zoyl)-N1-(4-vin ylb en zoyl)-1,2-b en -
zen ed ia m in e (53). Using the procedure described for the
preparation of compound 44, 4-vinylbenzoic acid (0.67 mmol)
yielded 210 g (79%) of the title compound as a white amor-
phous solid: 1H NMR (DMSO-d6) δ 1.30 (s, 9 H), 5.40 (d, 1 H,
J ) 13.5 Hz), 5.99 (d, 1 H, J ) 21.0 Hz), 6.81 (dd, 1 H, J )
21.3, 13.2 hz), 7.31-7.27 (m, 2 H), 7.54 (d, 2 H, J ) 10.5 Hz),
7.88 (d, 2 H, J ) 10.2 Hz), 7.94 (d, 2 H, J ) 10.2 Hz), 10.01 (s,
1 H), 10.07 (s, 1 H); MS (FD) m/e 398 (M+). Anal. (C26H26N2O2)
C, H, N.
N1-(4-ter t-Bu tylben zoyl)-N2-(4-ch lor oben zoyl)-1,2-ben -
zen ed ia m in e (54). To a solution of N1-(4-tert-butylbenzoyl)-
1,2-benzenediamine (100 mg, 0.37 mmol) in 3 mL CH2Cl2 was
added 4-chlorobenzoyl chloride (95 µL, 0.74 mmol) and excess
K2CO3. The mixture was stirred 30 min then a 1:1 solution of
THF and 5 N NaOH was added. The resultant mixture was
stirred an additional 20 min and diluted with Et2O. The

870 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 5
Herron et al.
mixture was washed twice with water, dried over MgSO4,
filtered and concentrated in vacuo. The residue was sonicated
with hexane causing a precipitate to form which was collected
by filtration and dried under vacuum to yield 100 mg (66%) of
the title compound: 1H NMR (CDCl3) δ 1.37 (s, 9H), 6.95 (m,
2H), 7.33 (d, J ) 9.4, 1H), 7.50 (d, J ) 8.7, 3H), 7.53 (d, J )
8.3, 2H), 7.91 (d, J ) 8.7, 2H), 7.95 (d, J ) 8.7, 2H), 9.01 (s,
g, 2.75 mmol). After 12 h, the solvent was removed in vacuo.
The residue was dissolved in EtOAc and washed twice with 1
M aq citric acid, once with water, twice with saturated aq
NaHCO3 solution, once with water, and once with saturated
aq NaCl solution. The organic phase was dried (MgSO4),
filtered, and concentrated in vacuo. The residue was triturated
with Et2O, filtered, and dried in vacuo to give 610 mg (84%)
of the title compound: 1H NMR (DMSO-d6) δ 1.30 (s, 9H), 3.78
(s, 3H), 7.15 (dd, J ) 1.5, 8.3 Hz, 1H), 7.25-7.35 (m, 2H), 7.44
(t, J ) 8.0 Hz, 1H), 7.48-7.52 (m, 2H), 7.55 (d, J ) 8.5 Hz,
2H), 7.66-7.77 (m, 2H), 7.92 (d, J ) 8.3 Hz, 2H), 10.05 (s,
2H); MS (FD) m/e 402.2 (M+). Anal. for (C25H26N2O3) C, H, N.
N1-(4-ter t-Bu t ylb en zoyl)-N2-(4-et h ylb en zoyl)-1,2-b en -
zen ed ia m in e (63). Following the procedure used in the
preparation of compound 3, 63 was prepared from 4-ethylben-
zoyl chloride. The product was purified by silica gel chroma-
tography, eluting with a gradient of 10% through 50% EtOAc
in hexanes: 54% yield; 1H NMR (DMSO-d6) δ 1.20 (t, J ) 7.5
Hz, 3H), 1.30 (s, 9H), 2.67 (q, J ) 7.5 Hz, 2H), 7.25-7.35 (m,
2H), 7.36 (d, J ) 8.3 Hz, 2H), 7.55 (d, J ) 8.3 Hz, 2H), 7.60-
7.70 (m, 2H), 7.87 (d, J ) 8.3 Hz, 2H), 7.89 (d, J ) 8.3 Hz,
2H), 10.0 (s, 2H); MS (FD) m/e 400.4 (M+). Anal. (C26H28N2O2)
C, H, N.
N1-(4-ter t-Bu t ylb en zoyl)-N2-(4-t r iflu or om et h oxyb en -
zoyl)-1,2-ben zen ed ia m in e (64). Following the procedure
described for the preparation of compound 62, 64 was prepared
from 4-trifluoromethoxybenzoic acid: 64% yield; 1H NMR
(DMSO-d6) δ 1.29 (s, 9H), 7.25-7.30 (m, 2H), 7.52 (d, J ) 8.3
Hz, 4H), 7.60-7.70 (m, 2H), 7.87 (d, J ) 8.3 Hz, 2H), 8.07 (d,
J ) 8.3 Hz, 2H), 9.93 (s, 1H), 10.15 (s, 1H); MS (FD) m/e 456
(M+). Anal. (C25H23N2O3F3) C, H, N.
N1,N3-Bis(4-m eth oxyben zoyl)-1,3-ben zen edia m in e (65).
Into 100 mL CH2Cl2 was dissolved 1.08 g (10 mmol) m-
phenylenediamine. The solution was cooled in an ice bath, and
2.81 mL (20 mmol) Et3N was added in one portion, followed
by 3.42 g (20 mmol) p-anisoyl chloride dropwise. Reaction
mixture was allowed to warm gradually to room temperature
and after 16 h was quenched with cold dil HCl (100 mL). A
precipitate formed, which was collected, washed with cold dil
HCl, and dried at 50 °C, giving 3.58 g (95% yield) of the desired
compound: 1H NMR (DMSO-d6) δ 3.73 (s, 3H), 7.06 (d, 4H),
7.30 (t, 1H), 7.50 (d, 2H), 8.00 (d, 4H), 8.33 (s, 1H), 10.15 (s,
2H); MS (FD) m/e 376 (M+). Anal. (C22H20N2O4) C, H, N.
1H), 9.48 (s, 1H); IR 1651, 1600, 1505, 1319, 1271, 1092 cm-1
;
MS (FD) m/e 406 (M+). Anal. (C24H23ClN2O2) C, H, N.
N2-(4-ter t-Bu t ylb en zoyl)-N1-(3-vin ylb en zoyl)-1,2-b en -
zen ed ia m in e (55). To a solution of 3-vinylbenzoic acid (200
mg, 1.35 mmol) in THF (10 mL) was added thionyl chloride
(241 mg, 2.02 mmol) and pyridine (214 mg, 2.7 mmol). The
reaction was heated at 80 °C for 2 h and cooled to room
temperature. N1-(4-tert-Butylbenzoyl)-1,2-benzenediamine (362
mg, 1.35 mmol) was added. After stirring for 2 h, the reaction
mixture was diluted with CH2Cl2 and washed once with
saturated aq copper sulfate solution, once with saturated aq
NaCl solution, dried (MgSO4), filtered, and concentrated in
vacuo. Chromatography (silica gel, 10% EtOAc/90% CH2Cl2)
provided 200 mg (37%) of the title compound as a white solid:
1H NMR (DMSO-d6) δ 1.32 (s, 9 H), 5.34 (d, 1 H, J ) 10.9 Hz),
5.94 (d, 1 H, J ) 17.7 Hz), 6.79 (dd, 1 H, J ) 10.9, 17.7 Hz),
7.52-7.56 (m, 2 H), 7.29-7.33 (m, 2 H), 7.84 (d, 1 H, J ) 7.8
Hz), 7.67-7.70 (m, 2 H), 7.91 (d, 2 H, J ) 8.4 Hz), 8.01 (s, 1
H), 10.01 (s, 1 H), 10.09 (s, 1 H); MS (FAB) 399 (M + 1). Anal.
(C26H26N2O2) C, H, N.
N2-(4-ter t-Bu tylben zoyl)-N2-(4-flu or oben zoyl)-1,2-ben -
zen ed ia m in e (56). Using the procedure described for the
preparation of compound 55, 4-fluorobenzoic acid (0.71 mmol)
yielded 260 mg (94%) of the title compound as a white
amorphous solid: 1H NMR (DMSO-d6) δ 1.29 (s, 9 H), 7.30-
7.26 (m, 2 H), 7.36 (t, 2 H, J ) 8.3 Hz), 7.52 (d, 2 H, J ) 8.7
Hz), 7.68-7.61 (m, 2 H), 7.87 (d, 2 H, J ) 8.3 Hz), 8.00-8.04
(m, 2 H), 9.95 (s, 1 H), 10.08 (s, 1 H); MS (FD) m/e 390. Anal.
(C24H23FN2O2) C, H, N.
N1-(3-F lu or oben zoyl)- N2-(4-ter t-bu tylben zoyl)-1,2-ben -
zen ed ia m in e (57). The N2-(4-tert-butylbenzoyl)-1,2-benzene-
diamine (200 mg, 0.75 mmol) was dissolved in THF (10 mL).
Pyridine (121 µL, 1.50 mmol) and 3-fluorobenzoyl chloride (91
µL, 0.75 mmol) were added. After 24 h, the reaction was
quenched with saturated aq NH4Cl. The mixture was poured
into a separatory funnel and diluted with CH2Cl2. The mixture
was washed with saturated aqueous NH4Cl and the organic
layer was dried over MgSO4, filtered, and concentrated. The
crude product was purified by flash column chromatrography
(10% EtOAc/CH2Cl2) to give the desired product (280 mg,
96%): 1H NMR (DMSO-d6) δ 1.20 (s, 9H), 7.29 (m, 2H), 7.44
(m, 1H), 7.53 (d, J ) 8.3, 2H), 7.67 (m, 5H), 7.88 (d, J ) 8.3,
2H), 9.95 (s, 1H); IR 1654, 1588, 1510, 1474, 1444, 1322, 1270
cm-1; MS m/e 390.35. Anal. (C24H23FN2O2) C, H, N.
N 1-Be n zoyl-N 2-(4-t er t -b u t ylb e n zoyl)-1,2-b e n ze n e d i-
a m in e (60). Using the procedure described for the preparation
of compound 3, benzoyl chloride (0.80 mmol) and N1-(4-tert-
butylbenzoyl)-1,2-benzenediamine (0.75 mmol) yielded 150 mg
(54%) of the title compound: 1H NMR (DMSO-d6) δ 1.30 (s,
9H), 7.25-7.35 (m, 2H), 7.49-7.60 (m, 5H), 7.66 (dd, J ) 4.0,
5.8 Hz, 2H), 7.88 (d, J ) 8.3 Hz, 2H), 7.95 (d, J ) 6.8 Hz, 2H),
10.01 (s, 1H), 10.08 (s, 1H); MS (FD) m/e 372.1 (M+). Anal.
(C24H24N2O2) C, H, N.
N1-(4-Meth ylp h en yl)-N2-(4-ter t-bu tylben zoyl)-1,2-ben -
zen ed ia m in e (61). Using the procedure described for the
synthesis of compound 3, 4-toluoyl chloride (1.1 mmol) and N1-
(4-tert-butylbenzoyl)-1,2-benzenediamine (0.93 mmol) yielded
360 mg (100%) of the title compound: 1H NMR (DMSO-d6) δ
1.30 (s, 9 H), 2.37 (s, 3 H), 7.28 (m, 2 H), 7.33 (d, J ) 8.0 Hz,
2 H), 7.54 (d, J ) 8.5 Hz, 2 H), 7.66 (m, 2 H), 7.85 (d, J ) 8.0
Hz, 2 H), 7.89 (d, J ) 8.3 Hz, 2 H), 10.02 (br s, 2 H); MS (FD)
m/e 386.3 (M+). Anal. (C25H26N2O2) C, H, N.
Ack n ow led gm en t. We thank the Physical Chem-
istry Department of the Lilly Research Laboratories for
their help in characterizing the compounds reported
here.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the preparation of compounds 6, 14, 15, 17-20,
25, 26, 28, 29, 31, 34, 35, 49, 58, and 59. This information is
available free of charge via the Internet at http://pubs.acs.org.
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