Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-Aryloxy substituents of 2,6-diphenoxypyridines

Article abstract of DOI:10.1016/S0968-0896(01)00338-8

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K_I against factor Xa of 0.12 nM and is greater than 8000- and 2000-fold select

Full text of DOI:10.1016/S0968-0896(01)00338-8

Bioorganic & Medicinal Chemistry 10 (2002) 657–666
Design, Synthesis, and Biological Activity of Novel Factor Xa
Inhibitors: 4-Aryloxy Substituents of 2,6-Diphenoxypyridinesy
Howard P. Ng, Brad O. Buckman,* Keith A. Eagen, William J. Guilford,
Monica J. Kochanny, Raju Mohan, Kenneth J. Shaw, Shung C. Wu, Dao Lentz,
Amy Liang, Lan Trinh, Elena Ho, David Smith, Babu Subramanyam, Ron Vergona,
Janette Walters, Kathy A. White, Mark E. Sullivan, Michael M. Morrissey
and Gary B. Phillips
Pharmaceuticals Research, Berlex Biosciences, 15049 San Pablo Avenue, PO Box 4099, Richmond, CA 94804-0099, USA
Received 31 July 2001; accepted 6 September 2001
Abstract—A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the
coagulation cascade. Inhibitor 5e has a KI against factor Xa of 0.12 nMand is greater than 8000- and 2000-fold selective over two
related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that
tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine
ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investi-
gation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile
of this class of inhibitors is outlined. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
There remains an unmet clinical need for orally active
antithrombotics. To this end, we have been involved in
the search for novel inhibitors of factor Xa (fXa), a
trypsin-like serine protease in the coagulation cascade.2
FXa occupies the strategic juncture of the two arms of
the coagulation cascade, the intrinsic (surface activated)
and extrinsic (vessel injury-tissue factor) pathways.3
Potential clinical indications for an antithrombotic such
as a fXa inhibitor include myocardial infarction, deep
vein thrombosis following orthopedic surgery, compli-
cations following transient ischemic attack, and
unstable angina. Since many of these conditions require
long term administration, an orally available inhibitor
would be preferred. A number of small molecule fXa
inhibitors have been disclosed,4 but clinical results have
not been published.
In an earlier report, we described the discovery and
characterization of a series of pyridine based fXa
inhibitors exemplified by 1.5 Compound 1 is a potent inhi-
bitor of fXa with selectivity over both thrombin and
trypsin, but suffers from pharmacokinetic liabilities.
While we had extensively explored substitution on either
of the phenyl moieties, alternate substitution of the
pyridine had not been investigated for the more
advanced inhibitors. In previous studies, we observed
that amidine-containing 2,6-diphenoxypyridines with a
C4 carboxylic acid moiety had improved although still
low oral availability compared to related inhibitors. For
this reason we concentrated our efforts on inhibitors
with an acidic moiety. The 4-position of the pyridine
is the focus of this study, due in part to the synthesis
and the resulting redundancy of the 2- and 6-positions and
*Corresponding author. Tel.: +1-510-669-4591; fax: +1-510-669-
4310; e-mail: brad_buckman@berlex.com
ySee ref 1.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00338-8

658
H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
the lack of regioisomeric mixtures. Herein, we describe a
novel series of inhibitors with carboxy-substituted aryl-
oxy groups in the 4-position of the pyridine, which
potently and selectively inhibit fXa as well as demonstrate
good in vivo duration of action in rats.
(fIIa) for selectivity within the coagulation cascade and
bovine trypsin for general specificity against serine pro-
teases was carried out for the more potent inhibitors
(fXa Ki < 1 mM). The activities of the three serine pro-
teases were determined as the initial rate of the cleavage
of peptide p-nitroanilide by the enzyme. All substrate
concentrations used are equal to their Km values under
the present assay conditions. A modification of the
Morrison equation was used for inhibitors with Ki less
than 3 nMto correct for the proportion of inhibitor
bound to the enzyme relative to the free inhibitor.7
Chemistry
The inhibitors were prepared by sequential addition of
the appropriately substituted phenols to penta-
fluoropyridine (Scheme 1).6 Addition of the first phenol
occurs regioselectively at the C-4 position, allowing the
order of the next two additions to be determined by the
more valuable starting material. Additions were typi-
cally high yield and multiple additions could often be
accomplished in a single reaction vessel. Standard con-
ditions were used for converting the nitrile to the ami-
dine. Hydrolyses of acids were accomplished by heating
esters in acidic solutions. Except for the unsubstituted
guanidine-containing rings, all compounds were pre-
pared by the sequential addition of the fully substituted
aromatic moiety. Guanidine-containing inhibitors were
prepared by reacting aniline intermediate 6 at the nitrile
stage with cyanamide (Scheme 2).
Studies were initiated by examining substituted benzoic
acid derivatives and the intermediate esters that were
isolated (Table 1). The carboxylic acids were approxi-
mately 10-fold more potent than the corresponding
ethyl ester (e.g., 5a vs 5b). Additionally there was a ten-
dency for the methyl ester to be more potent than the
corresponding ethyl ester (5f/5g and 5ff/5gg). There was
no significant difference when varying the regiochem-
istry of the carboxylic acid moiety (5b and 5t), but the
carboxylic acid moiety was important for activity (5kk
vs 5d or 5v, 5nn vs 5j). On the other hand, substitution
of the benzoic acid moiety did not significantly affect
potency (5d, 5e, 5h, 5j, and 5k vs 5b, and 5v, 5x, 5z, and
5aa vs 5t) with the exception of 5dd, which was less
potent than the unsubstituted inhibitor 5t. Extending
the length of the acid chain also had little effect on
activity (5n and 5ii), but when the chain was unsatu-
rated, some decreases in potency were noted (5p, 5q,
and 5hh). Replacement of an acid with an amide gave an
inhibitor intermediate in potency to the acid and ethyl
Results and Discussion
The screening tree to determine the best inhibitors
began with the potency determination against human
fXa. Subsequent screening against human thrombin
Scheme 1.
Scheme 2.

H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
659
Table 1. Activity of compounds 5 against human FXa, human
thrombin and bovine trypsin
more potent than the esters, even though there does not
seem to be direct binding of this moiety with the
enzyme, is that the acids are better able to solvate in the
aqueous environment surrounding the enzyme, resulting
in a non-specific potency increase over the correspond-
ing esters. This is supported by a comparison of the
inhibitors with the non-carboxylate acidic substituents,
5oo and 5pp, with the more lipophilic 5oo being less
potent.
Substitution had little effect on the activity against fIIa
or trypsin. Except for seven inhibitors (5b, 5e, 5k, 5l, 5p,
5dd, and 5mm) the activity against fIIa was within 180–
700 nM. The data for trypsin was even less variable in
that only three inhibitors fell outside of the range of
180–600 nM( 5i, 5l, and 5ll). It is not surprising that
these substitutents have little effect on the activity in
other serine proteases since the groups are outside of the
binding pocket.
Compd
R
Ki (nM)
FXa fIIa trypsin
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
s
t
u
v
w
x
y
z
aa
bb
cc
dd
ee
ff
gg
hh
ii
jj
kk
ll
mm
nn
oo
pp
4-CO2CH2CH3
4-CO2H
2-OCH3-4-CO2CH2CH3
2-OCH3-4-CO2H
2-OH-4-CO2H
2,6-OCH3-4-CO2CH3
2,6-OCH3-4-CO2CH2CH3
2,6-OCH3-4-CO2H
2-Cl-4-CO2CH2CH3
2-Cl-4-CO2H
1.6
0.19
1.4
0.33
0.12
0.89
1.6
0.18
5.8
440
790
340
600
990
350
460
650
380
570
510
340
460
310
280
280
370
180
760
310
270
960
290
250
480
230
210
430
380
240
310
200
300
220
260
180
180
220
340
290
390
520
400
250
290
240
320
140
180
380
560
350
Substitutions were made on the phenyl at the 6-position
of the pyridine (distal position) and were found to be
consistent with our earlier findings (Table 2).8 Structural
findings from crystal structures of related compounds
bound to fXa indicate that this portion of the molecule
is in the S4 pocket.9 Typically, highly basic substituents
are preferred in this pocket and consistent with this, the
compounds with the basic 1-methyl-2-imidazoline were
found to be the most potent inhibitors (5d and 5h).
Additionally, the inhibitors containing less basic groups
(8b, 8d, and 8f) were less active and the acid 8g was
much less active. Contrary to the norm, the non-basic
dimethylamide 8c was comparable to 5d. A series of
guanidines (8h–k) was prepared to investigate the effects
of these highly basic groups. All three substituted gua-
nidines (8i–k) were significantly less potent than the
unsubstituted 8h. As opposed to the cyclic amidine ser-
ies where a significant difference was noted between the
unsubstituted and methyl substituted imidazoline, the
two inhibitors were equipotent in the cyclic guanidine
series (8j vs 8k).
0.30
2,6-CH3-4-CO2H
0.25 1200
2,6-CH3-4-CO2CH2CH3
2-OCH3-4-CH2CO2CH2CH3
2-OCH3-4-CH2CO2H
6.3
0.56
0.14
1000
530
570
510
720
300
340
480
590
300
340
240
250
140
230
660
260
530
130
370
580
520
470
620
320
550
410
140
680
590
300
2,6-OCH3-4-CH¼CHCO2CH2CH3 3.5
2,6-OCH3-4-CH¼CHCO2H
4-CH¼CHCO2H
0.59
0.15
1.1
4-CH¼CHCO2CH3
3-CO2CH2CH3
3-CO2H
1.2
0.11
0.56
0.18
1.0
0.21
0.52
0.22
0.13
0.42
1.1
2-OCH3-5-CO2CH2CH3
2-OCH3-5-CO2H
2,3-OCH3-5-CO2CH2CH3
2,3-OCH3-5-CO2H
3-CONH2-5-CO2CH2CH3
3-CONH2-5-CO2H
3,5-CO2H
3-CO2H-5-CO2CH2CH3
3-CH2CH2CO2CH2CH3
2-OCH2CH2N(CH3)2-5-CO2H
3-CHCHCONH2
0.68
0.93
1.4
3-CHCHCO2CH3
3-CHCHCO2CH2CH3
3-CHCHCO2H
3-CH2CH2CO2H
2.3
0.32
0.19
0.37
0.56
0.36
0.94
0.98
0.69
0.24
Substitution at the distal position had profound effects
on fIIa selectivity. All of the guanidine containing inhi-
bitors had Ki’s>4 mMagainst fIIa and the acid was
essentially inactive against both fIIa and trypsin. The
guanidine 8e is the most selective inhibitor from this
series reported to date. The imidazole 8f and aniline 8b
were more potent against fIIa. Trypsin activity was less
affected by changes in substitution at this position.
2-OCH3-5-(1H-tetrazol-5-yl)
2-OCH3
2-OCH3-5-NHSO2CF3
3-(1-Methyl-1H-imdazolin-2-yl)
2-Cl
2-Cl-4-CONHSO2Ph
2-Cl-4-CONHSO2CH3
ester (5ee vs 5hh and 5gg) in one case, and equipotent to
the acid in two others (5z vs 5aa and 5y vs 5bb). Inhibi-
tors with acidic groups that are not carboxylic acids
such as tetrazole, trifluoromethansulfonylamide, and
phenylsulfonylamide (5jj, 5ll, and 5oo) were less potent
than their carboxylic acid counterparts, but the metha-
nesulfonylamide 5pp was equipotent.
Selected inhibitors were further evaluated for their half-
life and plasma levels after intravenous and oral dosing
in rats (Table 3). Rats were dosed via oral gavage at
10 mg/kg or iv at 1 mg/kg with homogeneous solutions.
Inhibitor plasma concentrations were determined at
various time points by comparing the fXa inhibitory
activity of a plasma sample with the activity of a plasma
sample with exogenously added inhibitor.10 In general,
inhibitors tended to have a short duration when dosed
iv with t1/2 less than 1 h. In rats dosed intravenously,
plasma concentrations tend to be higher with an
increasing number of methoxy groups (5x vs 5v vs
Molecular modeling studies indicate that the sub-
stitutents off of the 4-position of the pyridine are out-
side of the enzyme pocket and extended into the solvent.
A possible explanation for the fact that the acids are

660
H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
Table 2. Activity of compounds 8 against human FXa, human thrombin and bovine trypsin
Compd
R
R0
Ki (nM)
FXa
fIIa
Trypsin
5d
8a
8b
8c
8d
8e
8f
8g
5h
8h
8i
1-Methyl-1H-imidazolin-2-yl
C(NH)NH2
N(CH3)2
CON(CH3)2
CH2N(CH3)2
NHC(NH)NH2
1-methyl-1H-imidazol-2-yl
COOH
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
0.23
0.47
1.2
0.66
3.6
0.13
3.2
27
0.18
0.17
35
6.8
6.6
460
1900
140
240
520
570
870
580
560
880
790
2600
4200
50
>5000
650
4100
4300
4000
3800
>5000
180
550
1200
650
1-methyl-1H-imidazolin-2-yl
NHC(NH)NH2
(pyrrolidin-1-yl)(imino)methylamino
(1H-imidazolin-2-yl)amino
(1-methyl-1H-imidazolin-2-yl)amino
8j
8k
1200
Table 3. Inhibitor plasma concentrations after iv or po dosing in
ratsa
Compd
iv
po
30 min
60 min
30 min
60 min
90 min
5b
5d
5e
5h
5j
5n
5p
5t
5v
5x
8a
8b
8d
8e
2.4
8.4
1.5
11
1.7
1.1
12
2.1
9.1
13
7.9
0.8
1.3
12
1.0
4.7
1.0
7.7
1.4
0.5
9.4
0.9
6.4
11
0.01
<0.9
nd
2.2
nd
2.4
0.03
0.3
>0.06
0.04
0.2
0.8
0.05
0.06
0.04
<0.02
0.04
0.05
<0.02
0.08
0.1
Figure 1. Plasma concentrations of 5d after oral and iv administration.
5.0
0.3
0.4
7.5
aReported values are the plasma levels in mM.
life after oral dosing was >1.5 h and the maximum
plasma concentration was 0.6 mM. As was typically seen
with these inhibitors, after intravenous administration
the maximum plasma concentrations are much higher.
The calculated oral availability of 5d is 6% in dogs and
was not considered adequate for further development of
this inhibitor or series.
5t and 5d vs 5b). Although structurally similar to other
compounds tested, when dosed orally, only 5d showed
significant plasma concentrations.
The pharmacokinetic parameters of inhibitor 5d looked
promising in the rat and it was further evaluated in dogs
(Fig. 1). Conscious beagles were dosed via oral gavage
at 10 mg/kg or intravenously at 1 mg/kg with homo-
geneous aqueous solutions of 5d. Inhibitor concentra-
tions were determined at various time points with the
same methods as those determined in the rat. The half-
Conclusion
A novel series of potent inhibitors of human fXa
have been designed and prepared with subnanomolar
potencies and greater than 30,000-fold selectivity over

H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
661
human fIIa and greater than 2000-fold selectivity over
bovine trypsin, two related serine proteases. Substitu-
tion of the distal phenyl ring with guanidine containing
substituents significantly improved fIIa selectivity.
Although the inhibitors demonstrated poor oral avail-
ability, the four position of the pyridine has been iden-
tified as a site for substitution that does not negatively
affect either the fXa potency or selectivity. A future
publication will further explore the 4-position of the
pyridine and the effect of those substitutents on
potency, selectivity, and oral availability.
layer was separated, dried (MgSO4), and the solvent was
removed in vacuo. Purification by chromatography on
silica with hexane/CH2Cl2 (1/4) gave 3.21 g (86%) of a
white amorphous solid. NMR (CDCl3) d 7.66 (d, 1H),
7.63 (dd, 1H), 7.54 (dd, 1H), 7.49 (d, 1H), 7.34 (m, 3H),
7.2 (m, 2H), 7.08 (d, 1H), 6.92 (d, 1H), 5.1 (s, 2H), 4.4
(q, 2H), 3.85 (s, 3H), 1.4 (t, 3H).
4-[2-[5-Cyano-2-(phenylmethoxy)phenoxy]-3,5-difluoro-6-
[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phenoxy]-3-
methoxybenzoic acid, ethyl ester (4, R=2-OCH3, 4-
COOCH2CH3). 4-[2-[5-Cyano-2-(phenylmethoxy)phen-
oxy]-3,5,6-trifluoropyridin-4-yl]-3-methoxybenzoic acid,
ethyl ester (1.0 g, 1.8 mmol) was dissolved in DMSO
(20 mL) and 4,5-dihydro-3-(hydroxyphenyl)-1-methyl-
imidazole (0.47 g, 1.8 mmol) and cesium carbonate (1.5 g,
4.5 mmol) were added. After stirring for 15 h, starting
material remained. After heating at 35 ꢁC for 2 h, the
mixture was partitioned with EtOAc and 0.1 N aq
KOH. The organic layer was separated, washed with
0.5 N aq KOH and brine, dried (MgSO4), and the sol-
vent was removed in vacuo to give 1.18 g (84%) of a
white amorphous solid that was used without further
purification. NMR (CDCl3) d 7.7 (s, 1H), 7.65 (d, 1H),
7.1–7.4 (m, 10H), 6.9 (m, 3H), 5.0 (s, 2H), 4.4 (q, 2H),
3.9 (s, 3H), 3.9 (m, 2H), 3.45 (t, 2H ), 2.7 (s, 3H), 1.4 (t,
3H).
Experimental
All starting materials not described below were pur-
chased from commercial sources. All reagents and sol-
vents were used as received from commercial sources
without additional purification. Elemental analyses were
performed by Robertson Microlit Laboratories; Madi-
son, NJ and results were within ꢀ0.4% of the calcu-
lated values. NMR spectra were recorded with a Varian
XL-300 spectrometer and were consistent with the
assigned structures. HPLC were performed with a
Rainin SD-1 Dynamax system and a C-18 reverse-phase
Dynamax 60A column using a gradient of acetonitrile
(0.1% TFA) in water (0.1% TFA).
Human fXa and human fIIa were from Enzyme
Research Lab., South Bend, IN, USA and bovine tryp-
sin was from Boehringer Mannheim Corp., Indianapo-
lis, IN, USA. All peptide-p-nitroanilide substrates were
purchased from Pharmacia Hepar Inc., Franklin, OH,
USA. Tris–HCl, NaCl and CaCl2 were from J. T. Baker
Inc., Jackson, TN, USA and polyethylene glycol 6000
was from BDH Laboratory Supplies, Poole, UK.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-methoxybenzoic acid, ethyl ester,
trifluoroacetic acid salt (5c). 4-[2-[5-Cyano-2-(phenyl-
methoxy)phenoxy]-3,5-difluoro-6-[3-(4,5-dihydro-1-methyl-
1H-imidazol-2-yl)phenoxy]-3-methoxybenzoic
acid,
ethyl ester (1.1 g, 1.4 mmol) was dissolved in absolute
EtOH (6 mL) and cooled in a dry ice/acetone bath. HCl
(g) was bubbled into the solution to saturate and the
reaction was sealed. After stirring for 15 h at ambient
temperature, the solvent was removed in vacuo to give
1.55 g of a white foam. The material was dissolved in
EtOH (6 mL) and cooled in a dry ice/acetone bath.
Ammonia was condensed in the tube and the tube was
sealed. After heating at 75 ꢁC for 1.5 h, the solvent was
removed in vacuo. The residue was redissolved in EtOH
(20 mL) and Pd/C (10%, 250 mg) was added. After
shaking under 55 psi of H2 for 3 h, the solid was
removed by filtration and the solvent removed in vacuo.
A portion of the residue was purified by HPLC using a
Dynamax column and a gradient of CH3CN in H2O
with 0.1% TFA. The resulting fractions were combined
and the solvent was removed by lyophylization to give
530 mg of 5c. NMR (DMSO-d6) d 11.25 (s, 1H), 10.3 (s,
1H), 9.1 (s, 2H), 9.05 (s, 2H), 7.6 (m, 5H), 7.4 (m, 4H),
7.0 (d, 1H), 4.35 (q, 2H), 4.1 (m, 2H), 3.95 (s, 3H), 3.95
(m, 2H), 3.0 (s, 3H), 1.35 (t, 3H). Anal.
4 -(2,3,5,6 - Tetrafluoropyridin - 4 - yl) - 3 - methoxybenzoic
acid, ethyl ester (2, R=2-OCH3, 4-COOCH2CH3).
Pentafluoropyridine (0.75 mL, 6.8 mmol) was added to
an ice cooled solution of 2-methoxy-4-hydroxybenzoic
acid (1.34 g, 6.8 mmol), and cesium carbonate (2.9 g,
8.8 mmol) in acetonitrile (60 mL). The solution was
warmed to ambient temperature. After stirring for 12 h
the mixture was partitioned with water and EtOAc. The
organic layer was separated, dried (MgSO4), and the
solvent was removed in vacuo to give 2.38 g (100%) of
an orange solid that was used without further purifica-
tion. NMR (CDCl3) d 7.7 (d, 1H), 7.7 (s, 1H), 7.2 (d,
1H), 4.4 (q, 2H), 3.9 (s, 3H), 1.4 (t, 3H).
4-[2-[5-Cyano-2-(phenylmethoxy)phenoxy]-3,5,6-trifluoro-
pyridin-4-yl]-3-methoxybenzoic acid, ethyl ester (3,
.
R=2-OCH3,
4-COOCH2CH3).
4-(2,3,5,6-Tetra-
(C32H29F2N5O7 2.4 TFA) C, H, N.
fluoropyridin-4-yl)-3-methoxybenzoic acid, ethyl ester
(2.35 g, 6.8 mmol) was dissolved in CH3CN (60 mL) and
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-methoxybenzoic acid, trifluoroacetic
acid salt (5d). A portion of the residue prior to purifica-
tion from the procedure for 5c was dissolved in 6 N aq
HCl (30 mL). After heating at 85 ꢁC for 4 h, the solvent
3-hydroxy-4-(phenylmethoxy)benzonitrile
(1.53
g,
6.8 mmol) and cesium carbonate (2.9 g, 8.8 mmol) were
added. After stirring for 15 h starting material
remained. After heating at 35 ꢁC for 5 h, the mixture
was partitioned with EtOAc and 1 N KOH. The organic

662
H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
was removed in vacuo. The residue was purified by
HPLC using a Dynamax column and a gradient of
CH3CN in H2O with 0.1% TFA. The resulting fractions
were combined and the solvent was removed by lyo-
phylization to give 170 mg of 5c as a white solid. NMR
(DMSO-d6) d 11.25 (s, 1H), 10.3 (s, 1H), 9.1 (s, 2H), 9.0
(s, 2H), 7.6 (m, 5H), 7.4 (m, 4H), 7.05 (d, 1H), 4.1 (m,
2H), 3.95 (s, 3H), 3.95 (m, 2H), 3.0 (s, 3H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-chlorobenzoic acid, ethyl ester,
trifluoroacetic acid salt (5i). NMR (DMSO-d6) d 11.25
(s, 1H), 10.3 (s, 1H), 9.1 (s, 2H), 8.95 (s, 2H), 8.2 (s, 1H),
8.0 (d, 1H), 7.4–7.7 (m, 7H), 7.05 (d, 1H), 4.35 (q, 2H),
4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H), 1.35 (t, 3H). Anal.
.
(C31H26F2N5O6 2.5 TFA) C, H, N.
.
(C30H25F2N5O7 2.5 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-chlorobenzoic acid, trifluoroacetic
acid salt (5j). NMR (DMSO-d6) d 11.25 (s, 1H), 10.3 (s,
1H), 9.1 (s, 2H), 8.95 (s, 2H), 8.2 (s, 1H), 7.95 (d, 1H),
7.4–7.7 (m, 7H), 7.05 (d, 1H), 4.1 (m, 2H), 3.95 (m, 2H),
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzoic acid, ethyl ester, trifluoroacetic
acid salt (5a). NMR (DMSO-d6) d 11.25 (s, 1H), 10.3
(s, 1H), 9.1 (s, 2H), 9.0 (s, 2H), 8.1 (d, 1H), 7.7 (s, 1H),
7.6 (m, 1H), 7.5 (m,1H), 7.4 (m, 5H), 7.05 (d, 1H), 4.35
(q, 2H), 4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H), 1.35 (t,
.
3.0 (s, 3H). Anal. (C29H2F2N5O6 2.5 TFA) C, H, N.
.
3H). Anal. (C31H27F2N5O6 2.4 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethylbenzoic acid, trifluoroacetic
acid salt (5k). NMR (DMSO-d6) d 11.2 (s, 1H), 10.3 (s,
1H), 9.05 (s, 2H), 8.95 (s, 2H), 7.8 (s, 2H), 7.65 (s, 1H),
7.5 (m, 2H), 7.4 (m, 3H), 7.05 (d, 1H), 4.1 (m, 2H), 3.95
(m, 2H), 3.0 (s, 3H), 2.3 (s, 6H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phe-
noxy]pyridin-4-yl]benzoic acid, trifluoroacetic acid salt
(5b). NMR (DMSO-d6) d 11.25 (s, 1H), 10.3 (s, 1H),
9.04 (s, 2H), 8.84 (s, 2H), 8.04 (d, 1H), 7.66 (s, 1H), 7.58
(m, 1H), 7.5 (m, 2H), 7.4 (m, 2H), 7.32 (d, 2H), 7.03 (d,
1H), 4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H). Anal.
.
(C31H27F2N5O6 2.7 TFA) C, H, N.
.
.
(C29H23F2N5O6 2.5 TFA H2O) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethylbenzoic acid, ethyl ester,
trifluoroacetic acid salt (5l). NMR (DMSO-d6) d 11.2
(s, 1H), 10.3 (s, 1H), 9.04 (s, 2H), 8.94 (s, 2H), 7.82 (s,
2H), 7.65 (d, 1H), 7.58 (dd, 1H), 7.52 (dd, 1H), 7.4 (m,
3H), 7.02 (d, 1H), 4.35 (q, 2H), 4.1 (m, 2H), 3.95 (m,
2H), 3.0 (s, 3H), 2.3 (s, 6H), 1.35 (t, 3H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-hydroxybenzoic acid, trifluoroacetic
acid salt (5e). NMR (DMSO-d6) d 11.15 (s, 1H), 10.5
(s, 3H), 10.25 (s, 1H), 9.1 (s, 2H), 8.9 (s, 2H), 7.6 (m,
3H), 7.4 (m, 4H), 7.3 (m, 1H), 7.05 (d, 1H), 4.1 (m, 2H),
.
.
3.95 (m, 2H), 3.0 (s, 3H). Anal. (C29H23F2N5O7 2.5
.
TFA H2O) C, H, N.
(C33H31F2N5O6 2.7 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-methoxybenzeneacetic acid, ethyl
ester, trifluoroacetic acid salt (5m). NMR (DMSO-d6) d
11.25 (s, 1H), 10.3 (s, 1H), 9.1 (s, 2H), 8.95 (s, 2H), 7.65
(s, 1H), 7.6 (m, 2H), 7.4 (m, 3H), 7.2 (d, 1H), 7.15 (s,
1H), 7.0 (d, 1H), 6.9 (d, 1H), 4.15 (q, 2H), 4.1 (m, 2H),
3.95 (m, 2H), 3.85 (s, 3H), 3.7 (s, 2H), 3.0 (s, 3H), 1.25
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethoxybenzoic acid, methyl
ester, trifluoroacetic acid salt (5f). NMR (DMSO-d6) d
11.2 (s, 1H), 10.3 (s, 1H), 9.04 (s, 2H), 8.96 (s, 2H), 7.64
(m, 1H), 7.5 (m, 2H), 7.4 (m, 5H), 7.04 (d, 1H), 4.1 (m,
2H), 3.95 (m, 2H), 3.9 (s, 9H), 3.0 (s, 3H). Anal.
.
.
(C32H29F2N5O8 2.5 TFA) C, H, N.
(t, 3H). Anal. (C33H31F2N5O6 2.4 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethoxybenzoic acid, ethyl ester,
trifluoroacetic acid salt (5g). NMR (DMSO-d6) d 11.25
(s, 1H), 10.3 (s, 1H), 9.05 (s, 2H), 9.0 (s, 2H), 7.6 (m,
3H), 7.4 (m, 5H), 7.05 (d, 1H), 4.35 (q, 2H), 4.1 (m, 2H),
3.95 (m, 2H), 3.9 (s, 6H), 3.0 (s, 3H), 1.35 (t, 3H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-methoxybenzeneacetic acid, trifluoro-
acetic acid salt (5n). NMR (DMSO-d6) d 11.25 (s, 1H),
10.3 (s, 1H), 9.05 (s, 4H), 7.65 (s, 1H), 7.6 (m, 2H), 7.4
(m, 3H), 7.2 (d, 1H), 7.15 (s, 1H), 7.05 (d, 1H), 6.9 (d,
1H), 4.1 (m, 2H), 3.95 (m, 2H), 3.8 (s, 3H), 3.6 (s,
.
.
(C33H31F2N5O8 2.8 TFA) C, H, N.
2H), 3.0 (s, 3H). Anal. (C31H27F2N5O7 2.4 TFA) C,
H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethoxybenzoic acid, trifluoroacetic
acid salt (5h). NMR (DMSO-d6) d 10.25 (s, 1H), 9.01
(s, 2H), 8.84 (s, 2H), 7.62 (d, 1H), 7.57 (d, 1H), 7.50 (t,
1H), 7.36 (m, 5H), 7.02 (d, 1H), 4.05 (m, 2H), 3.95 (m,
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethoxybenzene-3-propenoic acid,
ethyl ester, trifluoroacetic acid salt (5o). NMR (DMSO-
d6) d 11.1 (br, 1H), 10.25 (s, 1H), 9.0 (s, 2H), 8.9 (s, 2H),
7.6 (m, 3H), 7.5 (m, 1H), 7.35 (m, 3H), 7.25 (s, 2H), 7.0
(d, 1H), 6.75 (d, 1H), 4.2 (q, 2H), 4.05 (m, 2H), 3.9 (m,
.
2H), 3.9 (s, 6H), 3.0 (s, 3H). Anal. (C31H27F2N5O8 2.5
TFA) C, H, N.

H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
663
2H), 3.8 (s, 6H), 2.95 (s, 3H), 1.25 (t, 3H). Anal.
.
(C35H33F2N5O8 2.7 TFA) C, H, N.
1H), 7.14 (d, 1H), 4.1 (m, 2H), 3.95 (s, 3H), 3.95 (m, 2H),
.
3.0 (s, 3H). Anal. (C30H25F2N5O7 2.5 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3,5-dimethoxybenzene-3-propenoic acid,
trifluoroacetic acid salt (5p). NMR (DMSO-d6) d 11.2
(br, 1H), 10.25 (s, 1H), 9.05 (s, 2H), 8.95 (s, 2H), 7.6 (m,
3H), 7.5 (m, 1H), 7.35 (m, 3H), 7.2 (s, 2H), 7.0 (d, 1H),
6.65 (d, 1H), 4.05 (m, 2H), 3.9 (m, 2H), 3.8 (s, 6H), 2.95 (s,
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-4,5-dimethoxybenzoic acid, ethyl ester,
trifluoroacetic acid salt (5w). NMR (DMSO-d6) d 11.25
(s, 1H), 10.3 (s, 1H), 9.05 (s, 2H), 9.0 (s, 2H), 7.65 (s, 1H),
7.6 (d, 1H), 7.4–7.6 (m, 6H), 7.05 (d, 1H), 4.35 (q, 2H), 4.1
(m, 2H), 3.95 (s, 3H), 3.95 (m, 2H), 3.85 (s, 3H), 3.0 (s, 3H),
.
.
3H). Anal. (C33H29F2N5O8 2.5 TFA) C, H, N.
1.35 (t, 3H). Anal. (C33H31F2N5O8 2.4 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propenoic acid, trifluoroacetic
acid salt (5q). NMR (DMSO-d6/TFA) d 10.25 (s, 1H),
9.05 (s, 2H), 8.95 (s, 2H), 7.75 (d, 2H), 7.4–7.6 (m, 7H),
7.25 (d, 2H), 7.0 (d, 1H), 6.5 (d, 1H), 4.05 (m, 2H), 3.9
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-4,5-dimethoxybenzoic acid, trifluoro-
acetic acid salt (5x). NMR (DMSO-d6) d 11.25 (s, 1H),
10.3 (s, 1H), 9.1 (s, 2H), 9.05 (s, 2H), 7.65 (s, 1H), 7.6 (d,
1H), 7.4-7.6 (m, 6H), 7.05 (d, 1H), 4.1 (m, 2H), 3.95 (s,
3H), 3.95 (m, 2H), 3.85 (s, 3H), 3.0 (s, 3H). Anal.
.
(m, 2H), 2.95 (s, 3H). Anal. (C31H25F2N5O6 2.5
TFA.0.5 H2O) C, H, N.
.
(C31H27F2N5O8 2.5 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propenoic acid, methyl ester,
trifluoroacetic acid salt (5r). NMR (DMSO-d6/TFA) d
10.25 (s, 1H), 9.05 (s, 2H), 8.95 (s, 2H), 7.75 (d, 2H),
7.4–7.6 (m, 7H), 7.25 (d, 2H), 7.0 (d, 1H), 6.5 (d, 1H),
4.05 (m, 2H), 3.9 (m, 2H), 3.7 (s, 3H), 2.95 (s, 3H).
3-Aminocarbonyl-5-[2-[5-[Amino(imino)methyl]-2-hydroxy-
phenoxy]-3,5-difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imi-
dazol-2-yl)phenoxy]pyridin-4-yl]benzoic acid, ethyl ester,
trifluoroacetic acid salt (5y). NMR (DMSO-d6) d 11.25
(s, 1H), 10.3 (s, 1H), 9.1 (s, 2H), 8.95 (s, 2H), 8.35 (m,
2H), 8.0 (s, 1H), 7.9 (s, 1H), 7.4–7.8 (m, 7H), 7.05 (d, 1H),
4.35 (q, 2H), 4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H), 1.35
.
.
.
Anal. (C32H27F2N5O6 2.5 TFA) C, H, N.
(t, 3H). Anal. (C32H28F2N6O7 2.5 TFA H2O) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzoic acid, ethyl ester, trifluoroacetic
acid salt (5s). NMR (DMSO-d6) d 11.3 (br, 1H), 10.3
(s, 1H), 9.1 (s, 4H), 7.85 (s, 1H), 7.75 (d, 1H), 7.4–7.7
(m, 8H), 7.05 (d, 1H), 4.35 (q, 2H), 4.1 (m, 2H), 3.95 (m,
3-Aminocarbonyl-5-[2-[5-[Amino(imino)methyl]-2-hydroxy-
phenoxy]-3,5-difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imi-
dazol-2-yl)phenoxy]pyridin-4-yl]benzoic acid, trifluoroacetic
acid salt (5z). NMR (DMSO-d6) d 11.25 (s, 1H), 10.3
(s, 1H), 9.16 (s, 2H), 8.98 (s, 2H), 8.36 (m, 1H), 8.32 (m,
1H), 7.96 (s, 1H), 7.88 (s, 1H), 7.72 (m, 2H), 7.61 (dd,
1H), 7.4–7.6 (m, 4H), 7.05 (d, 1H), 4.1 (m, 2H), 3.95 (m,
.
2H), 3.0 (s, 3H), 1.35 (t, 3H). Anal. (C31H27F2N5O6 2.4
TFA) C, H, N.
.
2H), 3.0 (s, 3H). Anal. (C30H24F2N6O7 2.7 TFA) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzoic acid, trifluoroacetic acid salt
(5t). NMR (DMSO-d6) d 11.2 (s, 1H), 10.3 (s, 1H),
9.05 (s, 2H), 9.0 (s, 2H), 7.85 (d, 1H), 7.4–7.7 (m, 9H),
7.05 (d, 1H), 4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H).
5-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-1,3-dicarboxylic acid, trifluoro-
acetic acid salt (5aa). NMR (DMSO-d6) d 11.2 (s, 1H),
10.3 (s, 1H), 9.06 (s, 2H), 8.97 (s, 2H), 8.3 (m, 1H), 7.97
(m, 2H), 7.68 (d, 1H), 7.60 (dd, 1H), 7.4–7.6 (m, 4H),
7.05 (d, 1H), 4.1 (m, 2H), 3.95 (m, 2H), 3.0 (s, 3H).
.
Anal. (C29H23F2N5O6 2.7 TFA) C, H, N.
.
.
Anal. (C30H23F2N5O8 2.4 TFA H2O) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-4-methoxybenzoic acid, ethyl ester,
trifluoroacetic acid salt (5u). NMR (DMSO-d6) d 11.2
(s, 1H), 10.3 (s, 1H), 9.05 (s, 2H), 8.95 (s, 2H), 7.9 (dd,
1H), 7.78 (d, 1H), 7.66 (dd, 1H), 7.59 (dd, 1H), 7.5 (m,
1H), 7.4 (m, 3H), 7.04 (d, 1H), 4.35 (q, 2H), 4.1 (m, 2H),
3.95 (s, 3H), 3.95 (m, 2H), 3.0 (s, 3H), 1.35 (t, 3H).
5-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-1,3-dicarboxylic acid, ethyl
ester, trifluoroacetic acid salt (5bb). NMR (DMSO-d6) d
11.2 (s, 1H), 10.3 (s, 1H), 9.06 (s, 2H), 8.98 (s, 2H), 8.36
(m, 1H), 8.02 (m, 1H), 8.0 (m, 1H), 7.68 (d, 1H), 7.60
(dd, 1H), 7.4–7.6 (m, 4H), 7.06 (d, 1H), 4.4 (q, 2H), 4.1
(m, 2H), 3.95 (m, 2H), 3.0 (s, 3H), 1.35 (t, 3H). Anal.
.
Anal. (C32H29F2N5O7 2.4 TFA) C, H, N.
.
(C32H27F2N5O6 2.8 TFA) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-4-methoxybenzoic acid, trifluoroacetic
acid salt (5v). NMR (DMSO-d6) d 11.35 (s, 1H), 10.4
(s, 1H), 9.2 (s, 2H), 9.04 (s, 2H), 7.88 (dd, 1H), 7.72 (d,
2H), 7.66 (d, 1H), 7.56 (m, 1H), 7.44 (m, 3H), 7.36 (d,
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propanoic acid, ethyl ester,
trifluoroacetic acid salt (5cc). NMR (DMSO-d6/TFA) d
11.22 (s, 1H), 10.3 (s, 1H), 9.08 (s, 2H), 8.95 (s, 2H), 7.5

664
H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
(m, 7H), 7.1 (m, 4H), 4.0 (m, 6H), 3.05 (s, 3H), 2.92 (t,
.
2H). 2.68 (t, 2H), 1.15 (t, 3H). Anal. (C33H31F2N5O6 2.4
TFA) C, H, N.
3-[3,5-Difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-
yl)phenoxy]-4-(2-methoxyphenoxy)pyridin-2-yl]-4-hydro-
xycarboximidamide, trifluoroacetic acid salt (5kk).
NMR (DMSO-d6/TFA) d 10.35 (s, 1H), 9.0 (s, 2H), 8.8
(s, 2H), 7.65 (s, 1H), 7.55 (m, 2H), 7.4 (m, 3H), 7.2
(m, 2H), 7.0 (m, 2H), 4.1 (m, 4H), 3.95 (m, 2H), 3.8
5-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-4-(2-dimethylaminoethoxy)benzoic acid,
trifluoroacetic acid salt (5dd). NMR (DMSO-d6) d 9.70
(brs, 2), 8.95 (brs, 2), 7.92 (d, 1), 7.75–7.40 (m, 8), 7.15 (d,
1), 4.60 (m, 2), 4.20–3.90 (m, 4), 3.63 (m, 2), 3.05 (s, 3), 2.95
.
(s, 3H), 3.0 (s, 3H). Anal. (C29H25F2N5O5 2.5 TFA)
C, H, N.
3-[3,5-Difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2
-yl)phenoxy]-4-[2-methoxy-5-(trifluoromethylsulfonylami-
no)phenoxy]pyridin-2-yl]-4-hydroxycarboximidamide, tri-
fluoroacetic acid salt (5ll). NMR (DMSO-d6) d 10.26 (s,
1H), 9.0 (s, 2H), 8.8 (s, 2H), 7.1–7.7 (m, 9H), 7.0 (d,
1H), 4.0 (m, 4H), 3.8 (s, 3H), 2.94 (s, 3H). Anal.
.
.
(brs, 6). Anal. (C33H32F2N6O7 3.3 TFA 1.9 H2O) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propenamide, trifluoroacetic
acid salt (5ee). NMR (DMSO-d6/TFA) d 10.3 (s, 1H),
9.05 (s, 2H), 8.85 (s, 2H), 7.7 (s, 1H), 7.6 (d, 1H), 7.5 (m,
8H), 7.25 (m, 1H), 7.05 (d, 1H), 6.65 (d, 1H), 4.05 (m,
.
(C30H25F5N6O7S 3 TFA) C, H, N.
3-[3,5-Difluoro-4,6-bis[3-(4,5-dihydro-1-methyl-1H-imida-
zol-2-yl)phenoxy]pyridin-2-yl]-4-hydroxycarboximidamide,
trifluoroacetic acid salt (5mm). NMR (DMSO-d6) d
11.4 (br, 1H), 10.45 (s, 1H), 10.35 (s, 1H), 9.1 (br, 4H),
7.4–7.8 (m, 10H), 7.05 (d, 1H), 4.1 (m, 4H), 3.95 (m,
.
2H), 3.9 (m, 2H), 2.95 (s, 3H). Anal. (C31H26F2N6O6 2.5
TFA) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propenoic acid, methyl ester,
trifluoroacetic acid salt (5ff). NMR (DMSO-d6/TFA) d
10.25 (s, 1H), 9.05 (s, 2H), 8.85 (s, 2H), 7.4–7.7 (m,
10H), 7.3 (m, 1H), 7.05 (d, 1H), 6.7 (d, 1H), 4.05 (m,
2H), 3.9 (m, 2H), 3.7 (s, 3H), 3.0 (s, 3H). Anal.
.
2H), 3.05 (s, 3H), 3.0 (s, 3H). Anal. (C32H28F2N7O4 4.7
TFA) C, H, N.
3-[4-(2-Chlorophenoxy)-3,5-difluoro-6-[3-(4,5-dihydro-1-
methyl-1H-imidazol-2-yl)phenoxy]pyridin-2-yl]-4-hydroxy-
carboximidamide, trifluoroacetic acid salt (5nn). NMR
(DMSO-d6) d 11.3 (s, 1H), 10.45 (s, 1H), 9.2 (s, 2H), 9.0
(s, 2H), 7.7 (s, 1H), 7.6 (m, 2H), 7.5 (m, 1H), 7.4 (m, 5H),
7.25 (m, 1H), 7.1 (d, 1H), 4.05 (m, 2H), 3.9 (m, 2H), 3.0
.
(C32H27F2N5O6 2.2 TFA) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)-
phenoxy]pyridin-4-yl]benzene-3-propenoic acid, ethyl ester,
trifluoroacetic acid salt (5gg). NMR (DMSO-d6/TFA) d
10.3 (s, 1H), 9.05 (s, 2H), 8.85 (s, 2H), 7.4–7.7 (m, 10H),
7.3 (m, 1H), 7.05 (d, 1H), 6.7 (d, 1H), 4.2 (q, 2H), 4.05
(m, 2H), 3.9 (m, 2H), 3.0 (s, 3H), 1.25 (t, 3H). Anal.
.
.
(s, 3H). Anal. (C28H22ClF2N5O4 2.3 HCl H2O) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-chloro-N-phenylsulfonylbenzamide, tri-
fluoroacetic acid salt (5oo). NMR (DMSO-d6) d 10.26
(s, 1H), 9.01 (s, 2H), 8.84 (s, 2H), 8.2 (d, 1H), 8.0 (m,
2H), 7.9 (dd, 1H), 7.3–7.7 (m, 10H), 7.0 (d, 1H), 4.0 (m,
.
.
(C33H29F2N5O6 2 TFA H2O) C, H, N.
.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propenoic acid, trifluoroacetic
acid salt (5hh). NMR (DMSO-d6/TFA) d 10.25 (s, 1H),
9.05 (s, 2H), 8.85 (s, 2H), 7.4–7.7 (m, 10H), 7.3 (m, 1H),
7.05 (d, 1H), 6.6 (d, 1H), 4.05 (m, 2H), 3.9 (m, 2H), 3.0
4H), 2.94 (s, 3H). Anal. (C35H27ClF2N6O7S 2.5
.
TFA 1.5 H2O) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]-3-chloro-N-methylsulfonylbenzamide, tri-
fluoroacetic acid salt (5pp). NMR (DMSO-d6) d 10.26 (s,
1H), 9.0 (s, 2H), 8.8 (s, 2H), 8.26 (d, 1H), 7.96 (dd, 1H),
7.4–7.7 (m, 7H), 7.02 (d, 1H), 4.0 (m, 4H), 3.36 (s, 3H),
.
(s, 3H). Anal. (C31H25F2N5O6 2.5 TFA) C, H, N.
3-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-yl)phen-
oxy]pyridin-4-yl]benzene-3-propanoic acid, trifluoroacetic
acid salt (5ii). NMR (DMSO-d6/TFA) d 10.25 (s, 1H),
9.05 (s, 2H), 8.8 (s, 2H), 7.6 (s, 1H), 7.55 (d, 1H), 7.3–7.5
(m, 4H), 7.0–7.2 (m, 4H), 4.05 (m, 2H), 3.0 (s, 3H), 2.85
.
2.95 (s, 3H). Anal. (C30H25ClF2N6O7S 2.5 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]phenoxy]-2-[5-[Amino(imi-
no)methyl]-2-hydroxyphenoxy]-3,5-difluoropyridin-4-yl]-
3-methoxybenzoic acid, trifluoroacetic acid salt (8a).
NMR (DMSO-d6) d 11.2 (s, 1H), 9.3 (s, 2H), 9.2 (s,
2H), 9.05 (s, 2H), 8.95 (s, 2H), 7.4–7.7 (m, 8H), 7.4 (d,
1H), 7.05 (d, 1H), 3.95 (s, 3H). Anal.
.
(t, 2H), 2.55 (m, 2H). Anal. (C31H27F2N6O6 2.5 TFA)
C, H, N.
.
3-[3,5-Difluoro-6-[3-(4,5-dihydro-1-methyl-1H-imidazol-2-
yl)phenoxy]-4-[2-methoxy-5-(1H-tetrazol-5-yl)phenoxy]-
pyridin-2-yl]-4-hydroxycarboximidamide, trifluoroacetic
acid salt (5jj). NMR (DMSO-d6/TFA) d 9.08 (s, 2H),
8.95 (s, 2H), 7.95 (s, 2H), 7.5 (m, 7H), 7.1 (d, 1H), 4.08
(C27H21F2N5O7 2.6 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-(3-dimethylamino)phenoxy]pyridin-4-yl]oxy]-2-
methoxybenzoic acid, trifluoroacetic acid salt (8b).
NMR (DMSO-d6) d 11.05 (s, 1H), 9.05 (s, 2H), 8.8 (s,
2H), 7.7 (m, 2H), 7.6 (m, 2H), 7.35 (d, 1H), 7.05 (m,
.
(m, 7H), 3.05 (s, 3H). Anal. (C30H25F2N9O5 2.5 TFA)
C, H, N.

H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
665
2H), 6.45 (d, 1H), 6.3 (m, 2H), 3.95 (s, 3H), 2.85 (s, 6H).
.
Anal. (C28H24F2N4O7 2.5 TFA) C, H, N.
1H), 9.03 (s, 2H), 8.8 (s, 2H), 6.8–7.7 (m, 10H), 3.86 (s,
.
6H). Anal. (C28H24F2N6O8 2.0 TFA H2O) C, H, N.
.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[(3-dimethylaminocarbonyl)phenoxy]pyridin-4-yl]-
oxy]-2-methoxybenzoic acid, trifluoroacetic acid salt
(8c). NMR (DMSO-d6) d 11.05 (br, 1H), 9.05 (s, 2H),
8.75 (s, 2H), 7.6 (m, 4H), 7.35 (m, 2H), 7.1 (m, 4H), 3.95
(s, 3H), 3.0 (s, 3H), 2.8 (s, 3H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[[3-pyrolidin-1-yl(imino)methyl]amino]phenoxy]-
pyridin-4-yl]oxy]-2,6-dimethoxybenzoic acid, 2.7 tri-
fluoroacetic acid (8i). NMR (DMSO-d6) d 9.16 (s, 1H),
9.04 (s, 2H), 8.86 (s, 2H), 7.5–7.7 (m, 4H), 7.36 (s, 2H),
7.28 (t, 1H), 7.07 (d, 1H), 6.8–7.0 (m, 3H), 3.9 (s, 6H),
.
.
(C29H24F2N4O8 1.1 TFA) C, H, N.
3.5 (br, 4H), 1.9 (br, 4H). Anal. (C32H30F2N6O8 2.7
TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[(3-dimethylaminomethyl)phenoxy]pyridin-4-yl]-
oxy]-2-methoxybenzoic acid, trifluoroacetic acid salt
(8d). NMR (DMSO-d6) d 9.05 (s, 2H), 9.0 (s, 2H), 7.7
(m, 2H), 7.6 (m, 2H), 7.35 (m, 2H), 7.1 (m, 3H), 7.05 (d,
1H), 4.2 (s, 2H), 3.95 (s, 3H), 2.7 (s, 6H). Anal.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-[(4,5-dihydro-1H-imidazol-2-yl)amino]phen-
oxy]pyridin-4-yl]oxy]-3,5-dimethoxybenzoic acid, 2.5
trifluoroacetic acid salt (8j). NMR (DMSO-d6) d 10.48
(s, 1H), 9.0 (s, 2H), 8.8 (s, 2H), 8. 4 (s, 2H), 7.62 (d, 1H),
7.55 (dd, 1H), 7.32 (s, 2H), 7.22 (t, 1H), 7.02 (d, 1H),
6.8–6.9 (m, 3H), 3.84 (s, 6H), 3.62 (s, 4H). Anal.
.
(C29H26F2N4O7 2.4 TFA) C, H, N.
.
4-[2-[3-[Amino(imino)methyl]amino]phenoxy]-6-[5-cyano-
2-(phenylmethoxy)phenoxy]-3,5-difluoropyridin-4-yl]oxy]-
3-methoxybenzoic acid, ethyl ester, trifluoroacetic acid
salt (7, R=2-OCH3-4-COOCH2CH3). To 4-[2-(3-amino-
phenoxy)-6-[5-cyano-2-(phenylmethoxy)phenoxy]-3,5-
difluoropyridin-4-yl]oxy]-3-methoxybenzoic acid, ethyl
ester (0.50 g, 0.80 mmol) dissolved in EtOH (15 mL) was
added cyanamide (0.6 g, 14 mmol) and 6 Maq HCl
(0.7 mL). After refluxing for 16 h, cyanamide (1.2 g, 28
mmol) and 6 Maq HCl (0.1.4 mL) was added. After
stirring for 56 h, the reaction mixture was concentrated.
The residue was purified by HPLC using a Dynamax
column and a gradient of CH3CN in H2O with 0.1%
TFA. The resulting fractions were combined and the
solvent was removed by lyophylization to give 0.42 g of
the title compound.
(C30H26F2N6O8 2.5 TFA) C, H, N.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-[(4,5-dihydro-1-methyl-1H-imidazol-2-yl)ami-
no]phenoxy]pyridin-4-yl]oxy]-3,5-dimethoxybenzoic acid,
2.5 trifluoroacetic acid salt (8k). NMR (DMSO-d6) d
9.98 (s, 1H), 9.0 (s, 2H), 8.8 (s, 2H), 8.24 (s, 1H), 7.65 (d,
1H), 7.58 (dd, 1H), 7.35 (s, 2H), 7.26 (t, 1H), 7.03 (d,
1H), 6.9–7.0 (m, 3H), 3.85 (s, 6H), 3.6 (m, 4H), 3.0 (s,
.
3H). Anal. (C31H28F2N6O8 2.5 TFA) C, H, N. Enzyme
assay procedures.11 The activities of human fXa, human
fIIa, and bovine trypsin were determined as the initial
rate of the cleavage of peptide p-nitroanilide by the
enzyme. The assay was performed at room temperature
in flat-bottom microtiter plates in a final volume of
200 mL. The reaction mixture consisted of 50 mM
TrisHCl (pH 7.5), 150 mMNaCl, 2.5 mMCaCl 2, and
0.1% polyethylene glycol 6000, with enzyme and sub-
strate at the following concentrations: (1) FXa assay:
1 nMfXa and 164 mMS2222, (2) FIIa assay: 16 nMfIIa
and 300 mMS2302, and (3) trypsin assay: 16 nMbovine
trypsin and 127 mMS2266. All substrate concentrations
used are equal to their Km values under the present
assay conditions. Controls without the test inhibitors or
with a reference compound were also run in each assay
plate. Enzyme was incubated with test compounds for
10 min, the reaction was then started by the addition of
the substrate. Reaction rates were determined by mea-
suring the rate of the absorbance change at 405 nm in a
ThermoMax microplate reader (Molecular Devices
Corp., Sunnyvale, CA, USA).
4-[2-[3-[Amino(imino)methyl]amino]phenoxy]-6-[5-[ami-
no(imino)methyl]-2-hydroxyphenoxy]-3,5-difluoropyridin-
4-yl]oxy]-3-methoxybenzoic acid, 2.0 trifluoroacetic acid
salt, 1.0 hydrate, (8e). NMR (DMSO-d6) d 11.2 (s, 1H),
9.9 (s, 1H), 9.07 (s, 2H), 8.9 (s, 2H), 7.7 (m, 2H), 7.6 (m,
5H), 7.36 (d, 1H), 7.28 (t, 1H), 7.1 (d, 1H), 6.95 (m,
.
2H), 3.95 (s, 3H). Anal. (C27H22F2N6O7 2.0 TFA) C,
H, N, F.
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[3-(1-methyl-1H-imidazol-2-yl)phenoxy]pyridin-
4-yl]-3-methoxybenzoic acid, trifluoroacetic acid salt
(8f). NMR (DMSO-d6) d 8.90 (brs, 2), 8.70 (brs, 2),
7.77–7.31 (m, 10), 7.21 (d, 1), 6.85 (d, 1), 3.85 (s, 3), 3.78
.
.
(s, 3). Anal. (C30H23F2N5O7 2.0 TFA 1.7 H2O) C, H, N.
Data analysis methods
4-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3,5-di-
fluoro-6-[(3-hydroxycarbonyl)phenoxy]pyridin-4-yl]oxy]-
2-methoxybenzoic acid, trifluoroacetic acid salt (8g).
NMR (DMSO-d6) d 11.05 (s, 1H), 9.05 (s, 2H), 8.95 (s,
2H), 7.6 (m, 6H), 7.35 (m, 3H), 7.05 (d, 1H), 3.95 (s,
IC50 values for inhibitors were obtained by log-logit
analysis with a computer spreadsheet. For inhibitors
with Ki less than 3 nM, IC50 values were determined by
fitting data to a modification of the Morrison equation
to correct for the proportion of inhibitor bound to the
enzyme relative to the free inhibitor.7 Since the inhibition
mechanism is competitive, Ki values were then obtained
by dividing the IC50 values by a factor of 1+[S]/Km. Ki
values are the mean of multiple determinations (nꢂ2).
Standard deviations are <30% of the mean.
.
3H). Anal. (C27H19F2N3O9 1.3 TFA) C, H, N.
4-[2-[3-[Amino(imino)methyl]amino]phenoxy]-6-[5-[ami-
no(imino)methyl]-2-hydroxyphenoxy]-3,5-difluoropyridin-
4-yl]oxy]-3,5-dimethoxybenzoic acid, 2.0 trifluoroacetic
acid salt, 1.0 hydrate, (8h). NMR (DMSO-d6) d 9.8 (s,

666
H. P. Ng et al. / Bioorg. Med. Chem. 10 (2002) 657–666
Wu, S.; Xu, W.; Morrissey, M. M. J. Med. Chem. 1998, 41,
3557.
References and Notes
1. Some of this data has been previously reported: Ng, H.;
Buckman, B. O.; Davey, D. D.; Eagen, K.; Guilford W. J.;
Kochanny, M.; Mohan, R.; Phillips, G. B.; Shaw, K. J.; Wu,
S.; Xu, W.; Liang, A.; Trinh, L.; Ho, E.; Smith, D.; Sub-
ramanyam, B.; Vergona, R.; Walters, J.; White, K. A.; Sulli-
van, M. E.; Morrissey, M. M. Presented at the 215th
American Chemical Society National Meeting, March 29–
April 2, 1998, Dallas, TX; MEDI-124.
6. Phillips, G. B.; Davey, D. D.; Eagen, K. A.; Koovakkat, S.;
Liang, A.; Ng, H. P.; Pinkerton, M.; Trinh, L.; Whitlow, M.;
Beatty, A. M.; Morrissey, M. M. J. Med. Chem. 1999, 42, 1749.
7. (a) Jordan, S. P.; Waxman, L.; Smith, D. E.; Vlasuk, G. P.
Biochemistry 1990, 29, 11095. (b) Morrison, J. F. Biochim.
Biophys. Acta 1969, 185, 269.
8. Xu, W., Davey, D. D., Eagen, K., Guilford, W. J., Ng, H.,
Phillips, G. B., Pinkerton, M., Wu, S. C., Liang, A., Trinh, L.,
Hinchman, J., Post, J., Sullivan, M. E., Morrissey, M. M.
Book of Abstracts, 215th ACS National Meeting, Dallas, TX,
March 29–April 2, 1998; MEDI-123.
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3. Broze, G. J., Jr. Sem. Hematol. 1992, 29, 159.
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K. A.; Guilford, W. J.; Hinchman, J.; Ho, E.; Koovakkat, S.;
Liang, A.; Light, D. R.; Mohan, R.; Ng, H. P.; Post, J. M.;
Shaw, K. J.; Smith, D.; Subramanyam, B.; Sullivan, M. E.;
Trinh, L.; Vergona, R.; Walters, J.; White, K.; Whitlow, M.;
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10. Although this method has a shortcoming in that active
metabolites could alter results, the speed at which we were able
to generate data to influence the design process outweighed
this shortcoming.
11. Lottenberg, R.; Christensen, U.; Jackson, C. M.; Cole-
man, P. L. Methods Enzymol. 1981, 80, 341.