Thiophene-anthranilamides as highly potent and orally available factor Xa inhibitors

Article abstract of DOI:10.1021/jm070125f

There remains a high unmet medical need for a safe oral therapy for thrombotic disorders. The serine protease factor Xa (fXa), with its central role in the coagulation cascade, is among the more promising targets for anticoagulant therapy and has been the

Full text of DOI:10.1021/jm070125f

J. Med. Chem. 2007, 50, 2967-2980
2967
Thiophene-Anthranilamides as Highly Potent and Orally Available Factor Xa Inhibitors1
Bin Ye,* Damian O. Arnaiz, Yuo-Ling Chou, Brian D. Griedel, Rushad Karanjawala, Wheeseong Lee, Michael M. Morrissey,
Karna L. Sacchi, Steven T. Sakata, Kenneth J. Shaw, Shung C. Wu, Zuchun Zhao, Marc Adler, Sarah Cheeseman,
William P. Dole, Janice Ewing, Richard Fitch, Dao Lentz, Amy Liang, David Light, John Morser, Joseph Post,
Galina Rumennik, Babu Subramanyam, Mark E. Sullivan, Ron Vergona, Janette Walters, Yi-Xin Wang, Kathy A. White,
Marc Whitlow, and Monica J. Kochanny*
Berlex Biosciences, Post Office Box 4099, Richmond, California 94804-0099
ReceiVed January 31, 2007
There remains a high unmet medical need for a safe oral therapy for thrombotic disorders. The serine protease
factor Xa (fXa), with its central role in the coagulation cascade, is among the more promising targets for
anticoagulant therapy and has been the subject of intensive drug discovery efforts. Investigation of a hit
from high-throughput screening identified a series of thiophene-substituted anthranilamides as potent
nonamidine fXa inhibitors. Lead optimization by incorporation of hydrophilic groups led to the discovery
of compounds with picomolar inhibitory potency and micromolar in vitro anticoagulant activity. Based on
their high potency, selectivity, oral pharmacokinetics, and efficacy in a rat venous stasis model of thrombosis,
compounds ZK 814048 (10b), ZK 810388 (13a), and ZK 813039 (17m) were advanced into development.
Introduction
A wide variety of novel, potent, and selective small molecule
fXa inhibitors have been reported.9 Early small molecule fXa
inhibitors invariably contained a benzamidine or naphthylami-
dine group, initially thought to be necessary for binding in the
S1 pocket. The suboptimal oral absorption and/or pharmaco-
kinetic properties often associated with amidines directed efforts
to remove this functionality. Numerous reports of the identifica-
tion of potent, nonamidine, small molecule fXa inhibitors have
appeared in recent literature.10
Thrombotic disorders remain the leading cause of mortality
and morbidity in Western society.2 Anticoagulants are the
primary therapy for the treatment and prevention of thrombotic
disease, but have properties that limit their use. Heparin and
low molecular weight heparin must be dosed parenterally, and
heparin is associated with increased bleeding risk.3 Coumadin
(warfarin), although orally available, has a slow onset of action
and a narrow therapeutic window, resulting in the need for
careful monitoring.4 There remains an unmet clinical need for
orally active, safe, and efficacious anticoagulants for long-term
antithrombotic therapy.
One major area of focus in the effort to develop an oral
anticoagulant has been the development of inhibitors of factor
Xa (fXaa).5 fXa, a trypsin-like serine protease located at the
convergent point of the intrinsic and extrinsic pathways, plays
a pivotal role in the blood coagulation cascade. Together with
factor Va and calcium, fXa forms the prothrombinase complex,
which catalyzes the formation of thrombin via proteolysis of
prothrombin. Thrombin, the terminal enzyme in the cascade,
has several procoagulant functions including activation of
platelets, regulation of factors in the cascade, and the conversion
of fibrinogen to fibrin, which polymerizes to form the insoluble
matrix of a blood clot or thrombus. Because inhibition of fXa
prevents thrombin formation but does not affect pre-existing
thrombin, fXa inhibitors are predicted to cause less impairment
of hemostasis than direct thrombin inhibitors, leading to a wider
therapeutic window.6 Both proteinaceous and small molecule
inhibitors of fXa have been shown to be effective and safe in
preclinical animal thrombosis models.7 Data from animal models
has demonstrated a higher therapeutic ratio (antithrombotic
efficacy versus bleeding) for fXa inhibitors than for direct
thrombin inhibitors, leading to the expectation of a superior
clinical profile.8
High-throughput screening of our compound library identified
the anthranilamide compound 1 (Chart 1) as a novel, potent
(Ki,app ) 10 nM), nonamidine fXa inhibitor.11 Initial optimization
of this template resulted in the discovery of compound 2,12 with
improved fXa potency (Ki,app ) 1.0 nM) and micromolar
anticoagulant activity, as measured by the concentration of
compound required to cause a doubling of the prothrombin time
(PT) in vitro in human plasma (2×PT ) 12 µM). In this paper,
we present further optimization of template 2, leading to the
discovery of orally available fXa inhibitors with picomolar
potency.13
Chemistry. The synthesis of compounds 3a-c was similar
to that reported previously for compound 2.12 The synthesis of
3b and 3c is outlined in Scheme 1. For compound 3c,
commercially available 3-methoxy-2-nitrobenzoic acid 4 was
converted to the acid chloride and reacted with 5-chloro-2-
aminopyridine 5b to afford amide 6b in 97% yield. Following
reduction of the nitro group with sodium hydrosulfite, chlorina-
tion with N-chlorosuccinimide (NCS) occurred exclusively at
the 5-position of the anthranilamide ring as a result of the
directing effect of the aniline. The substitution pattern was
1
confirmed by H NMR. The thiophene-acid chloride 812 was
coupled with aniline 7b to afford 9b in 85% yield. Addition of
exactly one equivalent of base was critical in this coupling step
to avoid displacement of the halide. Reaction of 9b with
excess N-methylpiperazine afforded the desired product 3c.
Similarly, displacement of the chlorine with other amines or
nitrogen nucleophiles afforded compounds 10a-p, 17a-g, and
17j-p.
* To whom correspondence should be addressed. E-mail:
rickbinye@yahoo.com (B.Y.); mkochanny@sbcglobal.net (M.J.K.).
a Abbreviations: fXa, factor Xa; fIIa, factor IIa (thrombin); PT, pro-
thrombin time; 2×PT, concentration required to give a doubling of the
prothrombin time; tPA, tissue plasminogen activator; aPC, activated protein
C; EGF, epidermal growth factor; des-GLA-EGF1-factor Xaâ, factor Xa
construct with the EGF 2 domain and the serine protease domain.
Methylamino derivative 10b was further reacted to form
sulfonamide and urea analogs (Scheme 2). Treatment with the
10.1021/jm070125f CCC: $37.00 © 2007 American Chemical Society
Published on Web 05/31/2007

2968 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Ye et al.
Chart 1
branched chain hydroxy-substituted amines were investigated
(10j-10n), but none gave a significant change in fXa activity
compared to the aliphatic amines 10e and 10f. Aliphatic
diamines, however, gave an unexpected result. Compared to
the corresponding hydroxy-substituted amines 10k and 10j,
diamine compounds 10o and 10p showed a 10-fold improve-
ment in fXa potency, affording low picomolar fXa inhibitors.
A corresponding 6- to 7-fold improvement in PT potency was
also observed, giving the first submicromolar anticoagulants in
this series. This result is in contrast to the phenyl series, where
substitution of an amine for a hydroxy caused only about a
2-fold increase in fXa potency.12 The reason for this difference
between the two series is unclear. Compounds 10o and 10p
were 5- to 10-fold more potent against thrombin relative to the
monoamines (Ki,app ) 200-300 nM), but retained at least
10 000-fold selectivity for fXa.
appropriate sulfonyl chloride or isocyanate afforded compounds
12a-c directly, and 12f was prepared by hydrolysis of an
intermediate ester. Hydroxyethyl analog 12d was prepared by
the formation of an intermediate trichloromethyl carbamate and
displacement with 2-aminoethanol. Treatment of 10b with
2-bromoethyl isocyanate in THF at 0 °C afforded urea 11 in
quantitative yield. Displacement of the bromide using excess
pyrrolidine gave the substituted urea 12e in low yield. The major
product arose from intramolecular cyclization to afford oxazoline
13a. Modification of the reaction conditions failed to improve
the yield of urea 12e. When a tertiary amine was used instead
of a secondary amine, 13a was obtained as the sole product.
Other oxazoline (13b-e), oxazine (13h), keto-oxazoline (13i),
and thiazoline (13j) analogs were prepared from the corre-
sponding amines and isocyanates or thioisocyanates.
Structure-Activity Results and Pharmacological Activity.
Despite nanomolar fXa binding affinity, compound 2 is a weak
anticoagulant (2×PT ) 12 µM). Anticoagulant activity of fXa
inhibitors as well as thrombin inhibitors has been observed to
be a function not only of potency, but also of lipophilicity and
protein binding.14 Early optimization studies on this series
showed that the 4-chloroaniline could be replaced with 2-amino-
5-chloropyridine, and a polar substituent such as methoxy could
be added at C-3 of the central anthranilamide ring.11a Incorpora-
tion of these changes into inhibitor 2 afforded compounds 3a
and 3b, with similar or slightly improved fXa inhibitory and
anticoagulant activities compared to compound 2 (Table 1).
When both changes were made simultaneously, the effects were
additive, affording compound 3c with a further improvement
in fXa potency and low micromolar anticoagulant activity. All
compounds were selective for fXa versus thrombin and trypsin.
Experimental logD values did not differ substantially between
compounds 2, 3a, and 3c, suggesting that the improved PT
potency for 3c is primarily a function of increased potency
against fXa.
Given the improved activity of 3c, we returned to the
optimization of the thiophene ring C-4 substituent. Our previous
work indicated that this portion of the template binds in the S4
pocket of fXa and that amine substituents were preferred for
fXa potency.12 Initially, we investigated simple aliphatic and
alicyclic amines (Table 2). For aliphatic monoamines, adding
a small alkyl substituent increased fXa potency by up to 5-fold
compared to that of the unsubstituted compound (10b, 10c, vs
10a), while a branched alkyl substituent (10d) decreased potency
relative to the unbranched amines. Lower dialkylamine substit-
uents had similar fXa potency to the corresponding mono-
alkylamine compounds (10e, 10f). Alicyclic monoamines
gave similar (10h) or slightly reduced fXa potency com-
pared to piperazine and aliphatic amines (10g, 10i). These results
are consistent with previous observations for analogs of
compound 2.12
Simple sulfonamide (12a) and urea (12b, 12c) substituents
gave a 3- to 10-fold increase in fXa potency relative to the
closest amine analogs (10b, 10e), also affording picomolar
inhibitors (Table 3). However, in spite of their fXa potency,
these compounds were an order of magnitude less potent than
the diamino compounds 10o and 10p in the PT assay. A similar
disconnect between fXa potency and anticoagulant activity was
seen for our early neutral inhibitors in this series and was
attributed to poor solubility and high nonspecific protein
binding.11 To improve PT potency, we tried adding basic and
hydrophilic substituents to the urea (12d-f). These changes were
largely unsuccessful, affording only minor improvements in PT
potency in the best case. The pyrrolidinoethyl urea 12e had
5-fold better PT potency than the corresponding ethyl urea 12c,
consistent with the predicted lower lipophilicity of 12e. How-
ever, hydroxy (12d) or acid (12f) substitution had no effect on
PT.
As outlined in Scheme 2, the major product of the reaction
to form the substituted urea 12e was the cyclized oxazoline,
13a. Compound 13a was found to be surprisingly potent, having
single digit picomolar fXa activity and submicromolar antico-
agulant activity (Table 4). The potency of 13a led us to explore
modifications of the oxazoline substituent. Extension of the
N-methyl group to N-ethyl had little effect (13b), however,
replacement with trifluoroethyl caused a large drop in potency
(13c). Larger groups like t-butyl or 2-methoxyethyl also
significantly decreased potency (13d, 13e). Substitution on the
oxazoline ring decreased fXa and PT potency, although the
effect was smaller at C-4 than at C-5 (13f, 13g). Replacement
of the oxazoline with a number of closely related ring systems
also afforded potent fXa inhibitors (13h-l). However, only the
six-membered-ring analog 13h and the highly basic pyrroline
13l retained submicromolar PT potency. Compounds 13i and
13k are uncharged at neutral pH, and their PT potency is
comparable to that of the neutral sulfonamide and urea analogs
(12a, 12b). The reason for the reduced anticoagulant potency
of the thiazoline analog 13j, however, is unclear. Compounds
13a and 13i-k were more potent inhibitors of thrombin (Ki,app
) 50-100 nM) than other compounds from this template, but
remained highly selective for fXa.
The results for the urea and oxazoline analogs (Tables 3 and
4) suggest a preference for planar groups off the methyl-
thiophene. We explored this further by preparing a variety of
compounds in which the nitrogen substituent is sp2 hybridized
(Table 5). Consistent with our hypothesis, all compounds were
highly potent inhibitors of fXa, many with single digit picomolar
Ki,app values. In general, compounds in which the linking
nitrogen was part of a ring had better selectivity for fXa versus
We then examined the effect of aliphatic amine substituents
bearing an additional hydrophilic or basic group. Straight or

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2969
Scheme 1a
a Reagents and conditions: (a) (COCl)2, DMF (cat.), CH2Cl2, 0 °C to rt; (b) pyridine, DMAP (cat), 0 °C to rt; (c) Na2S2O4, THF/dioxane/H2O, rt; (d)
NCS, benzene, 50 °C; (e) pyridine, CH2Cl2, 0 °C to rt; (f) N-methylpiperazine, DMF, rt.
Scheme 2a
a Reagents and conditions: (a) MeSO2Cl, pyridine, 0 °C to rt (for 12a); (b) isocyanate, dioxane or MeOH, rt (for 11, 12b,c,e); (Cl3CO)2C(O), CH2Cl2,
then 2-aminoethanol, rt (for 12d); OdCdNCH2CH2CO2Et, dioxane, rt; then LiOH.H20, water (for 12f); (c) pyrrolidine, DMF, rt or NEt3, THF, 0 °C to rt
(to give 13a exclusively).
thrombin than compounds with an exocyclic linking nitrogen
(e.g., 17a vs 13a, 17b vs 13j, and 17g vs 17h). While the
compounds containing nonaromatic amine substituents were
highly potent fXa inhibitors and anticoagulants, most of the
substituents are highly basic (cpKa 10-14). This was expected
to lead to poor pharmacokinetic properties, given the experience
with amidine and guanidine containing fXa inhibitors. In the
search for substituents with moderate basicity, we prepared
imidazole derivatives 17j and 17k. These compounds were also
found to be highly potent fXa inhibitors, but were weaker
anticoagulants (2×PT ) 1.4 and 2.0 µM) than the corresponding
imidazolines 17f and 17g. Adding an amino group at C-2 to
increase hydrophilicity maintained fXa activity and improved
anticoagulant activity (17l, 2×PT ) 0.77 µM). Alkylation of
the exocyclic amine (17m-o) also afforded highly potent fXa
inhibitors. The anticoagulant potency dropped off slightly as
substituent size increased. The substituted imidazoles were
among the most potent fXa inhibitors and anticoagulants we
identified. Furthermore, selectivity against thrombin was sig-
nificantly improved for these compounds relative to the oxazo-
line series.
Pharmacokinetic profiles were determined for selected inhibi-
tors (Table 6). Compounds were dosed in conscious beagle dogs
intravenously at 1 mg/kg and by oral gavage at 10 mg/kg, and
plasma samples were taken at intervals after dosing. Inhibitor
concentrations were determined ex vivo using an established
chromogenic assay measuring fXa inhibition15,16 or by HPLC,
with peak area measured by UV absorption and compared to a
standard solution of known inhibitor concentration. The simple
methylamino compound 10b was among the best compounds

2970 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Table 1. Addition of Hydrophilic Substituents
Ye et al.
Table 3. Effect of Sulfonamide and Urea Substitution on Enzyme
Inhibition and Anticoagulant Activity
Ki,appa,b (nM)
Ki,appa,b (nM)
cmpd
X
Y
fXa
fIIa
2×PTc (µM)
logD (7.4)
2×PTc
(µM)
2
C
N
C
N
H
H
OMe
OMe
0.76
0.36
0.43
0.16
840
680
1300
1500
12
2.96
3.11
NDd
2.75
cmpd
R1
fXa
fIIa
3a
3b
3c
6.9
4.2
1.6
10b
10e
12a
12b
12c
12d
12e
12f
-H
0.21
0.22
3700
1700
340
340
450
360
140
630
1.6
1.4
2.2
2.6
5.8
4.1
1.1
7.0
-CH2CH3
-SO2CH3
-C(O)NH2
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is
determined at a substrate concentration equal to the Km. Ki,app values are
averaged from multiple determinations (n g 2), and the standard deviations
are <30% of the mean. b All compounds had Ki,app values for bovine trypsin
of >5000 nM. c Concentration that gives a 2-fold extension of human
prothrombin time in vitro. d ND ) not determined.
0.045
0.026
0.020
0.050
0.036
0.022
-C(O)NHCH2CH3
-C(O)NHCH2CH2OH
-C(O)NHCH2CH2-(1-pyrrolidine)
-C(O)NHCH2CH2CO2H
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is
determined at a substrate concentration equal to the Km. Ki,app values are
averaged from multiple determinations (n g 2), and the standard deviations
are <30% of the mean. b All compounds had Ki,app values for bovine trypsin
of >5000 nM. c Concentration that gives a 2-fold extension of human
prothrombin time in vitro.
Table 2. Effect of Amine-Substituted Thiophenes on Enzyme Inhibition
and Anticoagulant Activity
measurement. After a 10 mg/kg oral dose, compound 13a
prolonged PT 3.2-, 5.0-, and 4.0-fold at 1, 2, and 6 h.
Compounds 10b and 17m prolonged PT to a lesser extent
following oral dosing (data not shown), but were also weaker
anticoagulants in vitro in dog plasma (Table 7).
Selectivity data against a panel of human proteases are shown
in Table 8. Compounds 10b, 13a, and 17m all had >20 000-
fold selectivity relative to trypsin, tPA, factor XIa, urokinase,
aPC, plasmin, chymotrypsin, cathepsin G, and neutrophil
elastase. Compound 17m was also highly selective against
thrombin, while thrombin selectivity for 10b and 13a was
somewhat lower (>17 000- and >12 000-fold). The lowest
selectivity observed was against plasma kallikrein, however, all
three compounds retained >4000-fold selectivity versus this
enzyme.
Compounds 10b, 13a, and 17m were tested in the rat vena
cava stasis model of thrombosis. The results are shown in Figure
1. All three compounds exhibited a dose-dependent inhibition
of experimental vena cava thrombosis in anesthetized rats. For
compounds 13a and 17m, inhibition was statistically significant
at 1 mg/kg, and no clot was detected in any of the animals at
3 mg/kg. For compound 10b, inhibition was statistically
significant only at the highest dose of 10 mg/kg. Estimated ED50
values in this model were approximately 4.4 mg/kg, i.v., for
10b, 0.4 mg/kg, i.v., for 13a, and 0.36 mg/kg, i.v., for 17m. In
general, these data are consistent with in vitro data for these
compounds, which show that compound 10b is a substantially
weaker inhibitor of rat fXa than 13a and 17m and also requires
higher concentrations to prolong PT in rat plasma (Table 7).
Based on their potency, selectivity, efficacy, and pharmacoki-
netic profiles, compounds 10b (ZK 814048), 13a (ZK 810388),
and 17m (ZK 813039) were all identified as candidates meeting
the selection criteria for development.17
Ki,appa,b (nM)
2×PTc
(µM)
cmpd
Y
fXa
fIIa
3c
4-methyl-1-piperazine
-NH2
0.16
1.0
0.21
0.40
1.4
0.22
0.32
1.4
0.52
2.2
1500
>5000
3700
2600
5400
1700
1500
1400
1400
770
1.6
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
4.2
1.6
2.2
NDd
1.4
2.4
4.9
1.6
3.2
1.6
1.5
1.5
5.3
3.2
0.24
0.24
-NHCH3
-NHCH2CH3
-NHCH(CH3)2
-N(CH3)CH2CH3
-N(CH2CH3)2
1-morpholine
1-pyrrolidine
1-(4-hydroxy)piperidine
-N(CH3)CH2CH2OH
-N(CH3)-(CH2)3-OH
-N(CH3)CH2CH(CH3)OH
-N(CH3)CH2C(CH3)2OH
-N(CH3)CH2CH(OH)CH2OH
-N(CH3)-(CH2)3-N(CH3)2
-N(CH3)CH2CH2-(1-pyrrolidine)
0.24
0.18
0.40
0.71
0.26
0.024
0.019
1400
1100
2100
1100
1200
300
190
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is
determined at a substrate concentration equal to the Km. Ki,app values are
averaged from multiple determinations (n g 2), and the standard deviations
are <30% of the mean. b All compounds had Ki,app values for bovine trypsin
of >5000 nM. c Concentration that gives a 2-fold extension of human
prothrombin time in vitro. d ND ) not determined.
in this series, affording high plasma levels and maintaining levels
close to peak out to at least 12 h. Adding a hydroxy substituent
(10j) led to slightly lower oral exposure, while the potent
diamino compound 10o gave no measurable plasma levels after
oral dosing. The highly potent oxazoline compound 13a and
imidazole 17m also gave quite promising results, with plasma
concentration profiles similar to the aliphatic monoamines (10b,
10j). Anticoagulant activity was determined ex vivo by PT
Crystallography. The crystal structure of compound 17m
bound to fXa is shown in Figure 2A (pdb code 2P3T). The
inhibitor binds in an L-shaped conformation with the chloro-
pyridine buried in the S1 pocket and the imidazole substituent

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2971
Table 5. SAR of sp2 Hybridized Nitrogen Substituents
Table 4. SAR of Oxazoline Analogs
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is
determined at a substrate concentration equal to the Km. Ki,app values are
averaged from multiple determinations (n g 2), and the standard deviations
are <30% of the mean. b All compounds had Ki,app values for bovine trypsin
of >5000 nM. c Concentration that gives a 2-fold extension of human
prothrombin time in vitro. d ND ) not determined.
extending into the S4 binding site. The chlorine atom makes
contact with Tyr228 in the S1 pocket. This chloro binding mode
has been reported for other fXa inhibitors,18 as well as for
inhibitors of thrombin,14b trypsin,19 and urokinase.20 Compound
17m forms only one well-defined hydrogen bond to fXa between
the amide NH attached to the chloropyridine ring and the
carbonyl of Glycine 218. There are no direct charge-charge
interactions.
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is
determined at a substrate concentration equal to the Km. Ki,app values are
averaged from multiple determinations (n g 2), and the standard deviations
are <30% of the mean. b All compounds had Ki,app values for bovine trypsin
of >5000 nM. c Concentration that gives a 2-fold extension of human
prothrombin time in vitro.
The crystal structures of compounds in this series provide
insight into the observed SAR (pdb entries 1MQ5,21 1MQ6,21
and 2P3T). Figure 2B shows the methyl group of 13a (1MQ6)
is buried deep in the S4 pocket, where it is in close contact
with the indole ring of Trp215 (3.5 Å). The methyl group also
makes contact with a water molecule (HOH722 in 1MQ6, 3.4
Å). This water donates two hydrogen bonds to the backbone
carbonyls of Thr98 (2.9 Å) and Ile175 (2.7 Å). Similar waters
have been seen in other fXa structures.21,22 Replacing methyl
with ethyl (13b) may force this water to shift away from the
S4 pocket, but it still should maintain the hydrogen bonds to
Thr98 and Ile175 (see HOH16 in 1EZQ22a), consistent with the
similar potency of 13a and 13b. More bulky groups such as
t-butyl (13d) or CH2CH2OCH3 (13e) would entirely displace
this water molecule. For these compounds, however, the
space between Phe174 and Trp215 may have insufficient volume
to accommodate the larger residues, resulting in the observed
10-fold loss of potency. Electronegative groups such CH2CF3
(13c) would have unfavorable interactions with the carbonyls
of Thr98 and Ile175. In 17m, part of the imidazole ring occupies
the same space as the methyl group of 13a. Most of the
compounds shown in Table 5 could maintain the same contacts
with Trp215 and HOH722. However, the oxygen of 17d would
have unfavorable contacts with HOH722, accounting for the
6-fold loss in potency for this inhibitor. Planar substituents
likely have a better fit in the space between Tyr99 and
Phe174, accounting for the high potency of the compounds in
Table 5.
It has been hypothesized that basic substituents in the S4
pocket interact with electronegative groups of residues 96-98

2972 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Table 6. Pharmacokinetic Profile in Dogs
Ye et al.
Corp., Indianapolis, IN. All peptide-p-nitroanilide substrates were
purchased from Kabi Pharmacia Hepar, Inc., Franklin, OH. TrisHCl,
NaCl, and CaCl2 were from J. T. Baker Inc., Jackson, TN, and
polyethylene glycol 6000 was from BDH Laboratory Supplies,
Poole, England.
N-(5-Chloro-2-pyridinyl)-3-methoxy-2-nitrobenzamide (6b).
To a suspension of 3-methoxy-2-nitrobenzoic acid 4 (197 g, 1.0
mol) in CH2Cl2 (1 L) at 0 °C were added several drops of DMF,
followed by oxalyl chloride (114 mL, 1.3 mol). The cooling bath
was removed, and the reaction was stirred at room temperature.
After 16 h, the mixture was concentrated to give the crude acid
chloride. The solid was pulverized and dried under vacuum.
3-Methoxy-2-nitrobenzoyl chloride (216 g, quant.) was obtained
as a light yellow solid, mp 83-85 °C: 1H NMR (CDCl3) δ 7.8 (d,
1H), 7.6 (t, 1H), 7.4 (d, 1H), 3.9 (s, 3H).
To a stirred solution of 2-amino-5-chloropyridine, 5b (129 g,
1.0 mol), in dry pyridine (1 L) at 0 °C was added a small amount
of 4-dimethylaminopyridine, followed by 3-methoxy-2-nitrobenzoyl
chloride (215 g, 1.0 mol). The reaction was allowed to warm to
room temperature and stirred overnight. The mixture was poured
into water (6 L) and filtered. The filter cake was collected, slurried
in water (3 L), and filtered. The solid was dried to give 6b (301 g,
97%) as an off-white powder, mp 215-217 °C: 1H NMR (CDCl3)
δ 8.8 (br s, 1H), 8.3 (d, 1H), 8.1 (s, 1H), 7.7 (d, 1H), 7.5 (t, 1H),
7.3 (m, 2H), 3.9 (s, 3H); 13C NMR (DMSO-d6) δ 164.4, 151.4,
151.0, 147.2, 138.8, 132.7, 130.4, 126.9, 121.2, 117.3, 116.3, 57.7.
Anal. (C13H10ClN3O4) C, H, N.
N-(4-Chlorophenyl)-3-methoxy-2-nitrobenzamide (6a). Com-
pound 6a was prepared from 4-chloroaniline and 4 by a similar
procedure as for compound 6b (4.4 g, 48%): 1H NMR (DMSO-
d6/TFA) δ 10.8 (s, 1H), 7.7 (m, 3H), 7.5 (d, 1H), 7.4 (t, 2H), 3.9
(s, 3H), 3.4 (br s, 1H).
2-Amino-5-chloro-N-(5-chloro-2-pyridinyl)-3-methoxybenza-
mide (7b). To a stirred solution of sodium hydrosulfite (300 g, 1.7
mol) in water (4 L) was added 6b (140 g, 0.45 mol). THF (2 L)
and 1,4-dioxane (2 L) were added, and the reaction became
homogeneous. The reaction was stirred overnight, then potassium
carbonate was added until basic, at which point the organic and
aqueous phases separated. The organic phase was concentrated to
give an off-white solid. The solid was slurried in water, filtered,
and dried under vacuum at 40 °C to give the aniline, 2-amino-N-
(5-chloro-2-pyridinyl)-3-methoxybenzamide (111 g, 88%): 1H
NMR (CDCl3) δ 8.8 (br s, 1H), 8.3 (d, 1H), 8.1 (s, 1H), 7.7 (dd,
1H), 7.1 (dd, 1H), 6.8 (d, 1H), 6.6 (t, 1H), 5.9 (br s, 1H), 3.9 (s,
3H).
To a stirred solution of the aniline (39.1 g, 140 mmol) in benzene
(2 L) was added NCS (20 g, 148 mmol). The reaction was heated
at 50-55 °C for 24 h, then cooled to room temperature and
concentrated. The crude solid was dissolved in EtOAc (1 L), washed
with water (3 × 100 mL), dried over Na2SO4, and concentrated.
The crude product was recrystallized from benzene to afford 7b
(40 g, 90%) as off-white needles, mp 146-148 °C: 1H NMR
(CDCl3) δ 8.6 (br s, 1H), 8.3 (m, 2H), 7.7 (dd, 1H), 7.1 (d, 1H),
6.8 (d, 1H), 5.9 (br s, 1H), 3.9 (s, 3H); 13C NMR (CDCl3) δ 166.4,
149.8, 148.3, 146.6, 139.3, 137.9, 126.7, 120.1, 118.0, 114.7, 113.9,
112.9, 56.0. Anal. (C13H11Cl2N3O2) C, H, N.
a
a
b
Cla
Vss
t1/2
Cmax
cmpd
(mL/min/kg)
(L/kg)
(h)
(µg/mL)
%Fc
10bd
10jd
1.6
27
1.4
4.4
ND
1.6
1.0
10.5
2.1
ND
3.2
5.1
1.2
75
>100
ND
56
10od
13ad
17me
NDf
5.8
4.4
BLQg
1.9
3.4
3.7
98
a Cl, Vss, and t1/2 values were determined based on a 1 mg/kg iv dose
b
(10% DMSO/saline, dosing volume 1 mL/kg). Cmax was based on a 10
mg/kg po dose (40% w/w hydroxypropyl-â-cyclodextrin/water, dosing
volume 1 mL/kg). c %F was calculated by normalizing the AUC after 10
mg/kg po and dividing by the AUC after 1 mg/kg iv. AUC values were
calculated based on extrapolation to infinity. d Inhibitor concentrations were
determined by chromogenic assay. e Inhibitor concentrations were deter-
mined by HPLC-UV. f ND ) not determined. g BLQ indicates that levels
were below the limit of quantitation for the method employed.
(Figure 2B). The positive charge on the imidazole ring of 17m
interacts with the backbone carbonyls of residues 96-98. For
compounds 10o and 10p, the tertiary amine at the end of the
chain may directly interact with the carboxylic acid of Glu97.
It is harder to explain the relative potency of other modifications
in the S4 binding region (e.g., 12a-f and 13f-l, Tables 3 and
4). Potent inhibitors with neutral amide groups at this position
such as 12b and 12c may form hydrogen bonds to the backbone
carbonyls that line the S4 pocket. However, there is no easy
explanation for the 45 pM potency of compound 12a. This
inhibitor may adopt a unique conformation that places the
sulfonamide group outside of the S4 pocket. The X-ray
structures of fXa complexes provide important insight into the
activity of this thiophene template. However, the electrostatic
interactions in the S4 pocket remain difficult to predict.
Conclusions
Replacement of the chlorobenzene ring of 2 with chloropy-
ridine and addition of a methoxy substituent to the anthranil-
amide ring afforded compound 3c with improved potency and
anticoagulant activity. Modification of the thiophene C-4
substituent led to the discovery of a series of highly potent and
orally available fXa inhibitors. Pharmacokinetic studies in dogs
identified the picomolar inhibitors 13a and 17m as having low
clearance and moderate to high oral bioavailability. Compound
10b, while a weaker inhibitor, gave high plasma levels after
oral dosing and had a prolonged half-life. All three compounds
had good selectivity against a panel of human serine proteases
and were efficacious in a venous thrombosis model in rats.
Compound 13a was initially selected for development, but was
terminated for reasons that included insufficient chemical
stability of the oxazoline moiety. Following the termination of
13a, compounds 10b and 17m were advanced into development.
Experimental Section
2-Amino-5-chloro-N-(4-chlorophenyl)-3-methoxybenzamide
(7a). Compound 7a was prepared from 6a by a similar procedure
as for compound 7b (1.4 g, 61%): 1H NMR (CDCl3) δ 7.7 (br s,
1H), 7.4 (dd, 4H), 7.1 (d, 1H), 6.8 (d, 1H), 5.8 (br s, 2H), 3.9 (s,
3H).
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-chloromethyl-2-thiophenecarboxam-
ide (9b). To a stirred solution of 3-chloro-4-chloromethyl-2-
thiophenecarbonyl chloride (8;12 60 g, 260 mmol) in dry CH2Cl2
(450 mL) at 0 °C was added 7b (63 g, 200 mmol) in portions,
followed after 5 min by pyridine (33 mL, 330 mmol). The reaction
changed from a bright yellow suspension to a homogeneous brown
solution within a few minutes after the pyridine addition, and a
precipitate began to form within 1 h. After 17 h at room temperature,
the reaction was concentrated and dried under vacuum. The crude
All reactions were run under an atmosphere of dry nitrogen. All
starting materials not described below were purchased from
commercial sources or synthesized following the noted literature
procedure. All reagents and solvents were used as received from
commercial sources without additional purification. Elemental
analysis and logD determinations were performed by Robertson
Microlit Laboratories; Madison, NJ, and elemental analysis results
were within (0.4% of the calculated values. NMR spectra were
obtained with a Varian XL-300 spectrometer and were consistent
with the assigned structures. HPLC was performed with a Rainin
SD-1 Dynamax system and a C-18 reverse phase Dynamax 60 A
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, and bovine trypsin was from Boehringer Mannheim

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2973
2×PTb (µM)
Table 7. Species Specificity of fXa Inhibition and Anticoagulant Activity
fXa Ki,appa (nM)
cmpd
human
dog
rat
human
dog
rat
10b
13a
17m
0.21 ( 0.07
0.007 ( 0.001
0.005 ( 0.001
1.7 ( 0.0
0.15 ( 0.05
0.31 ( 0.02
2.5 ( 0.2
0.29 ( 0.17
0.06 ( 0.01
1.64 ( 0.11
0.36 ( 0.02
1.25 ( 0.07
4.35 ( 0.05
0.35c
7.3 ( 0.28
0.93c
4.2c
2.56c
a Ki,app is the apparent Ki value, defined as IC50/2 when the IC50 value is determined at a substrate concentration equal to the Km. Ki,app values are averaged
from multiple determinations (n g 2). b Concentration that gives a 2-fold extension of the PT in vitro. Values are averaged from multiple determinations
unless otherwise indicated. c n ) 1.
Table 8. Human Protease Selectivity Profiles
enzyme Kia (nM)
10b
13a
17m
fXa
thrombin
trypsin
0.21b
3700
0.007b
0.005b
990
>5000
>5000
45
>5000
>5000
>5000
>5000
>5000
>5000
>5000
90
>10 000
>10 000
tPA
>5000
>5000
kallikrein
factor XIa
urokinase
aPC
1000
37
>10 000
>5000
>10 000
>10 000
>10 000
>10 000
>10 000
>10 000
>5000
>10 000
>10 000
>10 000
>10 000
>10 000
plasmin
chymotrypsin
cathepsin G
neutrophil elastase
a Ki values are averaged from multiple determinations (n g 2), and the
b
standard deviations are <30% of the mean. Ki,app value. Ki,app is the
apparent Ki value, defined as IC50/2 when the IC50 value is determined at
a substrate concentration equal to the Km.
Figure 2. (A) The conformation of 17m bound to fXa (PDB entry
2P3T). Hydrogen bonds are shown as green lines. The carbon atoms
of 17m are colored green and the chlorines are displayed in purple.
Asp189 at the base of the S1 pocket is dark green and Gly218 is light
blue. The hydrophobic residues that line the S4 pocket, Tyr99, Phe174,
and Trp215, are shown with magenta carbons. Two residues from the
active site triad, His57 and Ser195, are gold. (B) A detailed view of
the S4 pocket from the fXa/13a complex (PDB entry 1MQ6). Glu97
is shown in dark green. R1 and R2 refer to the substitution scheme
from Table 4.
Figure 1. The effects of compounds on vena cava thrombosis after
i.v. dosing in anesthetized rats. *p < 0.05 compared to the vehicle
control group.
flash chromatography. Alternatively, the product 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 lyophilization to afford the product. In
the case of HPLC purification, the product was obtained as the
trifluoroacetic acid salt.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]phenyl]-4-[(4-methyl-1-piperazinyl)methyl]-2-thiophenecar-
boxamide Trifluoroacetic Acid Salt (3a). The aniline precursor,
2-amino-5-chloro-N-(5-chloro-2-pyridinyl)benzamide, was prepared
from 5-chloro-2-nitrobenzoic acid and 2-amino-5-chloropyridine
following the coupling and reduction procedures reported above
for compounds 6b and 7b. Reaction with 8 according to the
procedure for compound 9b afforded the chloromethylthiophene
intermediate, 3-chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]-
carbonyl]phenyl]-4-chloromethyl-2-thiophenecarboxamide, (2.9 g,
90%): 1H NMR (DMSO-d6/TFA) δ 11.4 (s, 1H), 11.0 (s, 1H),
7.6-8.4 (m, 7H), 4.8 (s, 2H).
solid was ground, triturated with water (4 × 100 mL) and dried
under vacuum to give 9b (113 g, 85%) as a light brown powder,
mp 196-198 °C: 1H NMR (CDCl3) δ 9.3 (br s, 1H), 9.1 (br s,
1H), 8.3 (d, 1H), 8.0 (d, 1H), 7.7 (d, 1H), 7.6 (s, 1H), 7.2 (d, 1H),
7.0 (d, 1H), 4.6 (s, 2H), 3.9 (s, 3H); 13C NMR (CDCl3) δ 164.9,
158.5, 153.4, 149.9, 146.3, 137.9, 136.8, 132.9, 132.5, 129.7, 126.8,
123.9, 121.7, 119.8, 115.1, 113.9, 56.6, 38.6. Anal. (C19H13-
Cl4N3O3S) C, H, N.
3-Chloro-N-[4-chloro-2-[[(4-chlorophenyl)amino]carbonyl]-
6-methoxyphenyl]-4-chloromethyl-2-thiophenecarboxamide (9a).
Compound 9a was prepared from 7a by a similar procedure as for
compound 9b (0.27 g, 67%): 1H NMR (DMSO-d6/TFA) δ 10.4
(s, 1H), 9.5 (s, 1H), 8.1 (s, 1H), 7.7 (d, 2H), 7.3-7.4 (m, 4H), 4.8
(s, 2H), 3.9 (s, 3H).
General Method for Preparation of Amine-Substituted Com-
pounds from Chloromethylthiophenes. To a 0.1 M solution of a
chloromethylthiophene intermediate (e.g., 9a or 9b) in dry DMF
at 0 °C was added an amine (5 equiv), the mixture was stirred for
0.5 h at 0 °C, and then the mixture was warmed to room
temperature. After 4-18 h, the reaction mixture was poured into
water, the resulting solid was collected by filtration or extracted
into a solvent such as EtOAc, and the organic solution was dried
over Na2SO4 and concentrated. The crude product was purified by
This chloromethylthiophene intermediate was reacted with 1-me-
thylpiperazine according to the general method to afford compound
3a, purified by HPLC, giving a white solid (0.38 g, 64%): 1H NMR
(DMSO-d6/TFA) δ 11.4 (br s, 1H), 11 (s, 1H), 8.4 (d, 1H), 8.3 (s,
1H), 8.2 (d, 1H), 8.1 (d, 1H), 8.0 (m, 2H), 7.8 (dd, 1H), 4.4 (s,
2H), 3.1-3.8 (m, 8H), 2.9 (s, 3H). Anal. (C23H22Cl3N5O2S‚3.0C2-
HF3O2) C, H, N.

2974 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Ye et al.
3-Chloro-N-[4-chloro-2-[[(4-chlorophenyl)amino]carbonyl]-
6-methoxyphenyl]-4-[(4-methyl-1-piperazinyl)methyl]-2-thiophen-
ecarboxamide (3b). Compound 3b was prepared from 9a and
1-methylpiperazine and purified by flash chromatography, giving
a white solid (0.77 g, 64%): 1H NMR (DMSO-d6/TFA) δ 10.4 (s,
1H), 9.5 (s, 1H), 8.1 (s, 1H), 7.6 (d, 2H), 7.3 (m, 4H), 4.3 (s, 2H),
3.8 (s, 3H), 3.1-3.8 (m, 8H), 2.9 (s, 3H). Anal. (C24H23-
Cl3N4O3S‚0.4CH2Cl2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(4-methyl-1-piperazinyl)methyl]-2-
thiophenecarboxamide Trifluoroacetic Acid Salt (3c). Compound
3c was prepared from 9b and 1-methylpiperazine and purified by
HPLC, giving a white solid (0.1 g, 35%): 1H NMR (DMSO-d6/
TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.3 (d, 1H), 8.2 (s, 1H), 8.1 (d,
1H), 7.8 (dd, 1H), 7.3 (d, 2H), 4.4 (s, 2H), 3.8 (s, 3H), 3.1-3.8
(m, 8H), 2.9 (s, 3H). Anal. (C24H24Cl3N5O3S‚3.4C2HF3O2‚0.5 H2O)
C, H, N.
4-Aminomethyl-3-chloro-N-[4-chloro-2-[[(5-chloro-2-pyridi-
nyl)amino]carbonyl]-6-methoxyphenyl]-2-thiophenecarboxam-
ide (10a). Compound 10a was prepared from 9b and ammonia and
purified by flash chromatography, giving a white solid (0.51 g,
53%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.4
(s, 1H), 8.1 (d, 1H), 7.9 (s, 1H), 7.8 (dd, 1H), 7.3 (m, 2H), 4.0 (m,
2H), 3.8 (s, 3H). Anal. (C19H15Cl3N4O3S) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-(4-morpholinylmethyl)-2-thiophene-
carboxamide (10g). Compound 10g was prepared from 9b and
morpholine and purified by flash chromatography, giving a white
solid (0.68 g, 62%): 1H NMR (CDCl3) δ 9.1 (s, 1H), 8.7 (s, 1H),
8.2 (d, 1H), 8.1 (s, 1H), 7.6 (dd, 1H), 7.4 (s, 1H), 7.2 (d, 1H), 7.0
(s, 1H), 3.9 (s, 3H), 3.7 (br s, 4H), 3.5 (s, 2H), 2.5 (br s, 4H).
Anal. (C23H21Cl3N4O3S‚0.4C4H8O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-(1-pyrrolidinylmethyl)-2-thiophenecar-
boxamide (10h). Compound 10h was prepared from 9b and
pyrrolidine and purified by flash chromatography, giving a white
solid (0.90 g, 84%): 1H NMR (CDCl3) δ 9.1 (s, 1H), 8.7 (s, 1H),
8.3 (d, 1H), 8.1 (s, 1H), 7.6 (d, 1H), 7.5 (s, 1H), 7.2 (s, 1H), 7.0 (s,
1H), 3.9 (s, 3H), 3.8 (s, 2H), 2.6 (m, 4H), 1.8 (m, 4H). Anal.
(C23H21Cl3N4O3S‚0.1CH2Cl2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(4-hydroxy-1-piperidinyl)methyl]-2-
thiophenecarboxamide (10i). Compound 10i was prepared from
9b and 4-hydroxypiperidine and purified by flash chromatography,
giving a white solid (1.0 g, 59%): 1H NMR (DMSO-d6/TFA) δ
10.9 (s, 1H), 9.5 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.2 (d, 1H), 8.1
(s, 1H), 7.8 (d, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.3 (m, 2H), 3.9 (s,
3H), 3.8 (br s, 1H), 3.4 (m, 1H), 3.2 (m, 2H), 3.0 (m, 1H), 1.8 (m,
4H). Anal. (C24H23Cl3N4O4S‚0.22 CH2Cl2‚0.5H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(2-hydroxyethyl)methylamino]methyl]-
2-thiophenecarboxamide (10j). Compound 10j was prepared from
9b and 2-(methylamino)ethanol and purified by flash chromatog-
raphy, giving a white solid (0.48 g, 57%): 1H NMR (DMSO-d6/
TFA) δ 10.9 (s, 1H), 9.9 (s, 1H), 9.4 (s, 1H), 8.4 (s, 1H), 8.1 (d,
1H), 7.9 (d, 1H), 7.8 (s, 1H), 7.4 (s, 1H), 7.2 (s, 1H), 4.4 (t, 1H),
3.9 (s, 3H), 3.5 (m, 4H), 3.3 (d, 2H), 2.4 (m, 3H). Anal. (C22H21-
Cl3N4O3S‚0.3C4H8O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(3-hydroxypropyl)methylamino]m-
ethyl]-2-thiophenecarboxamide Trifluoroacetic Acid Salt (10k).
Compound 10k was prepared from 9b and 3-methylamino-1-
propanol and purified by HPLC, giving a white solid (0.96 g,
29%): 1H NMR (DMSO-d6/TFA) δ 10.9 (br s, 1H), 9.6 (br s, 1H),
7.2-8.5 (m, 6H), 4.2-4.5 (m, 4H), 3.9 (s, 3H), 3.0-3.4 (m, 2H),
2.7 (s, 3H), 2.2 (m, 2H). Anal. (C23H23Cl3N4O4S‚2.45C2HF3O2) C,
H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(2-hydroxypropyl)methylamino]m-
ethyl]-2-thiophenecarboxamide Trifluoroacetic Acid Salt (10l).
Compound 10l was prepared from 9b and 1-methylamino-2-
propanol and purified by HPLC, giving a white solid (2.2 g, 68%):
1H NMR (DMSO-d6/TFA) δ 11.1 (s, 1H), 9.6 (br s, 1H), 9.5 (s,
1H), 8.3 (s, 1H), 8.2 (m, 1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.4 (d, 1H),
4.1-4.4 (m, 2H), 3.8 (s, 3H), 3.14 (m, 1H), 2.74 (m, 2H), 2.4 (s,
3H), 1.0 (d, 3H). Anal. (C23H23Cl3N4O4S‚2.1C2HF3O2‚1.0H2O) C,
H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(2-hydroxy-2-methylpropyl)methy-
lamino]methyl]-2-thiophenecarboxamide Trifluoroacetic Acid
Salt (10m). Compound 10m was prepared from 9b and 2-methyl-
1-(methylamino)-2-propanol and purified by HPLC, giving a white
solid (2.3 g, 62%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.6
(s, 1H), 7.2-8.5 (m, 6H), 4.2-4.6 (m, 2H), 3.9 (s, 3H), 3.0-3.3
(m, 2H), 2.9 (s, 3H), 1.3 (s, 3H), 1.2 (s, 3H). Anal. (C24H25-
Cl3N4O4S‚2.7C2HF3O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(2,3-dihydroxypropyl)methylamino]-
methyl]-2-thiophenecarboxamide (10n). Compound 10n was
prepared from 9b and 3-(methylamino)-1,2-propanediol and purified
by flash chromatography, giving a white solid (0.60 g, 35%): 1H
NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.5 (s, 1H), 8.4 (s, 1H), 8.2
(m, 1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.3 (s, 1H), 7.2 (s, 1H), 4.0-4.4
(m, 3H), 3.9 (s, 3H), 3.0-3.5 (m, 3H), 2.5 (s, 3H). Anal. (C23H23-
Cl3N4O5S‚0.5H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(methylamino)methyl]-2-thiophene-
carboxamide (10b). Compound 10b was prepared from 9b and
methylamine and purified by flash chromatography, giving a white
solid (22 g, 74%), mp 166-168 °C: 1H NMR (DMSO-d6/TFA) δ
11.0 (s, 1H, C(O)NH), 9.33 (s, 1H, C(O)NH), 9.1 (br s, 1H, NH),
8.23 (d, 1H, Ar-H), 8.03 (d, 1H, Ar-H), 8.01 (s, 1H, Ar-H), 7.79
(dd, 1H, Ar-H), 7.24 (d, 1H, Ar-H), 7.22 (d, 1H, Ar-H), 4.06 (s,
2H, CH2-thiophene), 3.81 (s, 3H, OCH3), 2.55 (s, 3H, NCH3); 13
C
NMR (DMSO-d6/TFA) δ 166.4, 159.6, 155.8, 151.2, 146.2, 140.1,
135.2, 133.6, 133.1, 132.9, 132.5, 127.2, 125.9, 123.3, 121.4, 116.8,
115.4, 57.6, 45.6, 33.3. Anal. (C20H17Cl3N4O3S) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(ethylamino)methyl]-2-thiophenecar-
boxamide Trifluoroacetic Acid Salt (10c). Compound 10c was
prepared from 9b and ethylamine, giving a yellow solid (22 g, 57%).
A sample was further purified by HPLC, giving a white solid: 1H
NMR (DMSO-d6) δ 10.90 (s, 1H), 9.40 (s, 1H), 8.80 (br s, 2H),
8.75 (d, 1H), 8.20 (d, 1H), 8.10 (s, 1H), 7.80 (dd, 1H), 7.48 (d,
1H), 7.60 (d, 1H), 4.15 (s, 2H), 3.85 (s, 3H), 3.05 (br s, 2H), 1.20
(t, 3H). Anal. (C21H19Cl3N4O3S‚1.25C2HF3O2‚0.5H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(1-methylethyl)amino]methyl]-2-
thiophenecarboxamide (10d). Compound 10d was prepared from
9b and isopropylamine and purified by flash chromatography,
giving a white foam (0.56 g, 53%): 1H NMR (CDCl3) δ 9.1 (s,
1H), 8.9 (s, 1H), 8.3 (d, 1H), 8.1 (d, 1H), 7.6 (dd, 1H), 7.4 (s, 1H),
7.3 (s, 1H), 7.05 (s, 1H), 3.9 (s, 3H), 3.8 (s, 2H), 2.9 (m, 1H), 1.0
(d, 6H). Anal. (C22H21Cl3N4O3S) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(ethylmethylamino)methyl]-2-thiophen-
ecarboxamide (10e). Compound 10e was prepared from 9b and
N-ethylmethylamine and purified by flash chromatography, giving
a white solid (0.24 g, 47%): 1H NMR (DMSO-d6) δ 10.90 (s, 1H),
9.38 (s, 1H), 8.30 (d, 1H), 8.20 (d, 1H), 7.90 (dd, 1H), 7.70 (s,
1H), 7.40 (d, 1H), 7.25 (d, 1H), 3.90 (s, 3H), 3.30 (s, 2H), 2.40 (q,
2H), 2.10 (s, 3H), 1.00 (t, 3H). Anal. (C22H21Cl3N4O3S‚0.05CH2-
Cl2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(diethylamino)methyl]-2-thiophene-
carboxamide (10f). Compound 10f was prepared from 9b and
diethylamine and purified by flash chromatography, giving a white
solid (0.42 g, 54%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.5 (s, 1H), 9.4 (s, 1H), 8.4 (s, 1H), 8.2 (s, 1H), 8.1 (d, 1H), 7.9 (d,
1H), 7.3 (s, 1H), 7.2 (s, 1H), 4.2 (s, 2H), 3.8 (s, 3H), 3.1 (br s,
4H), 1.2 (m, 6H). Anal. (C24H23Cl3N4O3S‚1.2H2O) C, H, N.

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2975
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[[3-(dimethylamino)propyl]methy-
lamino]methyl]-2-thiophenecarboxamide (10o). Compound 10o
was prepared from 9b and N,N,N′-trimethyl-1,3-propanediamine
and purified by flash chromatography, giving a white solid (0.76
g, 33%): 1H NMR (DMSO-d6) δ 8.3 (d, 1H), 8.1 (d, 1H), 7.9 (dd,
1H), 7.7 (s, 1H), 7.4 (d, 1H), 7.2 (d, 1H), 3.9 (s, 3H), 3.4 (s, 2H),
2.4 (t, 2H), 2.2 (t, 2H), 2.1 (s, 3H), 2.0 (s, 6H), 1.5 (m, 2H). Anal.
(C25H28Cl3N5O3S) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[methyl[2-(1-pyrrolidinyl)ethyl]ami-
no]methyl]-2-thiophenecarboxamide (10p). Compound 10p was
prepared from 9b and 1-(2-methylaminoethyl)pyrrolidine (prepared
by reductive amination of N-(2-aminoethyl)pyrrolidine with 37%
formaldehyde using NaBH3CN) and purified by flash chromatog-
raphy, giving a white solid (0.48 g, 37%): 1H NMR (DMSO-d6/
TFA) δ 11.2 (s, 1H), 10.8 (s, 1H), 10.1 (br s, 1H), 8.4 (d, 1H), 8.3
(s, 1H), 7.9 (s, 1H), 7.7 (d, 2H), 7.6 (d, 1H), 7.4 (d, 2H), 4.4 (s,
2H), 3.6 (m, 2H), 3.4 (br s, 6H), 2.8 (s, 3H), 2.0 (br s, 4H). Anal.
(C26H28Cl3N5O3S‚1.0H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-iminotetrahydrooxazol-3-
yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide Trifluoroacetic Acid Salt (17a). Compound
17a was prepared from 9b and 2-aminooxazoline and purified by
HPLC, giving a white solid (0.80 g, 30%): 1H NMR (DMSO-d6/
TFA) δ 10.9 (s, 1H), 9.6 (br s, 1H), 9.4 (s, 1H), 9.2 (br s, 1H),
7.2-8.3 (m, 6H), 4.7 (t, 2H), 4.6 (s, 2H), 3.8 (s, 3H), 3.7 (t, 2H).
Anal. (C22H18Cl3N5O4S‚1.6C2HF3O2‚0.5H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-iminotetrahydrothiazol-
3-yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide Trifluoroacetic Acid Salt (17b). Compound
17b was prepared from 9b and 2-aminothiooxazoline and purified
by HPLC, giving a white solid (0.16 g, 4%): 1H NMR (DMSO-
d6/TFA) δ 8.3 (s, 1H), 8.1 (d, 1H), 7.8 (s, 1H), 7.8 (dd, 1H), 7.3
(dd, 2H), 4.7 (s, 2H) 3.9 (t, 2H), 3.8 (s, 3H) 3.5 (t, 2H). Anal.
(C22H18Cl3N5O3S‚2.3C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-(methylthio)imidazolin-1-
yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide Trifluoroacetic Acid Salt (17e). Compound
17e was prepared from 9b and 2-thiomethyl-4,5-dihydroimidazole
and purified by HPLC, giving a white solid (0.15 g, 37%): 1H
NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 10.1 (s, 1H), 9.4 (s, 1H),
8.3 (s, 1H), 8.1 (d, 1H), 7.9 (s, 1H), 7.8 (d, 1H), 7.3 (s, 1H), 7.2 (s,
1H), 4.6 (s, 2H), 3.8 (s, 3H), 3.8 (s, 4H), 2.6 (s, 3H). Anal. (C23H20-
Cl3N5O3S2‚3.5C2HF3O2‚2.0H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-methylimidazolin-1-yl)m-
ethyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chlo-
robenzamide Trifluoroacetic Acid Salt (17f). Compound 17f was
prepared from 9b (2.00 g, 4 mmol), 2-methyl-4,5-dihydroimidazole
(1.50 g, 17.8 mmol), and K2CO3 (2.50 g, 18.1 mmol) and purified
by HPLC, giving a white solid (1.0 g, 33%): 1H NMR (DMSO-
d6/TFA) δ 10.9 (s, 1H), 10.2 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.1
(d, 1H), 8.0 (s, 1H), 7.8 (d, 1H), 7.3 (s, 1H), 7.2 (s, 1H), 4.6 (s,
2H), 3.8 (s, 3H), 3.7 (s, 4H), 2.3 (s, 3H). Anal. (C23H20-
Cl3N5O3S‚1.7C2HF3O2‚0.5 H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-aminoimidazol-1-yl)methyl)-
3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chloroben-
zamide (17l). Compound 17l was prepared from 9b and 2-ami-
noimidazole and purified by flash chromatography, giving a white
solid (6.1 g, 87%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 10.4
(s, 1H), 8.3 (d, 1H), 8.1 (d, 1H), 7.9 (dd, 1H), 7.8 (br s, 1H), 7.6
(s, 1H), 4 (d, 1H), 7.3 (d, 1H), 6.9 (dt, 1H), 5.0 (s, 2H), 4.8 (s,
3H). Anal. (C22H17Cl3N6O3S‚0.4CH2Cl2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-(ethylamino)imidazol-1-
yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide Trifluoroacetic Acid Salt (17n). Compound
17n was prepared from 9b and 2-(ethylamino)imidazole and purified
by HPLC, giving a white solid (0.31 g, 21%): 1H NMR (DMSO-
d6/TFA) δ 10.9 (s, 1H), 9.4 (d, 1H), 8.3 (d, 1H), 8.1 (d, 2H), 7.8
(d, 1H), 7.6 (s, 1H), 7.35 (s, 1H), 7.3 (s, 1H), 7.0 (s, 1H), 6.9 (s,
1H), 5.1 (s, 2H), 3.9 (s, 3H), 3.2 (m, 2H), 1.2 (t, 3H). Anal. (C24H21-
Cl3N6O3S‚1.6C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-(isopropylamino)imidazol-
1-yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide (17o). Compound 17o was prepared from 9b
and 2-(isopropylamino)imidazole and purified by flash chroma-
tography, giving a white solid (1.16 g, 49%): 1H NMR (DMSO-
d6/TFA) δ 10.9 (s, 1H), 9.4 (br d, 1H), 8.38 (s, 1H), 8.1 (d, 2H),
7.8 (d, 1H), 7.42 (s, 1H), 7.35 (s, 2H), 7.0 (s, 1H), 6.5 (s, 1H),
6.45 (s, 1H), 5.43 (d, 1H), 4.94 (s, 2H), 3.9 (s, 3H), 3.2 (m, 2H),
1.2 (d, 6H). Anal. (C25H23Cl3N6O3S‚0.1CH2Cl2‚0.5H2O) C, H, N.
3-Chloro-N-[4-chloro-2-{[(5-chloro-2-pyridinyl)amino]carbo-
nyl}-6-methoxyphenyl]-4-{2-(methylamino)-1H-imidazol-1-yl]-
methyl}-2-thiophenecarboxamide (17m) and 3-Chloro-N-[4-
chloro-2-{[(5-chloro-2-pyridinyl)amino]carbonyl}-6-
methoxyphenyl]-4-[(1H-imidazol-2-ylmethylamino)methyl]-2-
thiophenecarboxamide (17p). To a suspension of 9b (14.7 g, 29
mmol, 1 equiv) in DMSO (50 mL) was added a solution of
2-(methylamino)imidazole hydrochloride (17.5 g, 131 mmol, 4.5
equiv) in DMSO (30 mL), followed by K2CO3 (26.1 g, 190 mmol,
6.5 equiv, powdered form, -325 mesh). The reaction mixture was
stirred at room temperature for 16 h and then poured into water
(500 mL). The pink solid was collected by filtration and washed
with water (2 L). The solid was dissolved in CH2Cl2 (500 mL),
containing a small amount of MeOH, and washed with water. The
aqueous layer was extracted with CH2Cl2 (2 × 500 mL), and the
extracts were combined with the original organic layer. The
combined organic layers were dried over Na2SO4 and concentrated.
The crude product was dissolved in 20:1 (v/v) EtOAc-MeOH and
purified by flash chromatography, eluting with 20:1 EtOAc-MeOH,
then 9:1 EtOAc-MeOH, to afford 13 g of a light red oil. The oil
was dissolved in CH2Cl2 and added dropwise into stirred hexane.
Filtration and drying afforded 17m as a white solid (9 g, 55%):
1H NMR (DMSO-d6/TFA) δ 12.46 (br s, 1H, NH), 10.85 (s, 1H,
C(O)NH), 9.36 (s, 1H, C(O)NH), 8.28 (d, 1H, Ar-H), 8.08 (d, 1H,
Ar-H), 7.83 (dd, 1H, Ar-H), 7.57 (s, 1H, Ar-H), 7.31 (d, 1H, Ar-
H), 7.24 (d, 1H, Ar-H), 7.04 (d, 1H, Ar-H), 6.94 (d, 1H, Ar-H),
4.99 (s, 2H, -CH2-thiophene), 3.83 (s, 3H, OCH3), 2.85 (s, 3H,
NCH3); 13C NMR (DMSO-d6/TFA) δ 165.2, 158.6, 155.2, 150.8,
147.3, 146.6, 138.3, 134.9, 134.4, 133.0, 132.1, 128.4, 126.0, 123.7,
122.5, 120.5, 117.4, 115.7, 114.7, 113.3, 57.0, 43.3, 29.6. Anal.
(C23H19Cl3N6O3S‚0.1C6H14‚1.2H2O) C, H, N.
Also isolated by chromatography was 17p (white solid, 0.20 g,
1%): 1H NMR (DMSO-d6) δ 10.9 (br s, 2H), 9.3 (br s, 1H), 8.4
(d, 1H), 8.1 (d, 1H), 7.9 (dd, 1H), 7.6 (s, 1H), 7.4 (s, 1H), 7.2 (s,
1H), 6.6 (s, 2H), 4.4 (s, 2H), 3.9 (s, 3H), 2.9 (s,3H). Anal. (C23H19-
Cl3N6O3S‚0.05C6H14‚0.8H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-imino-tetrahydroimidazol-
1-yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide Trifluoroacetic Acid Salt (17c). Reaction of
9b with 1,2-diaminoethane afforded N-(5-chloropyridin-2-yl)-2-[((4-
((N′-(2-aminoethyl)amino)methyl)-3-chlorothiophen-2-yl)carbonyl)-
amino]-3-methoxy-5-chlorobenzamide (0.97 g, 93%), which was
used without purification: 1H NMR (DMSO-d6/TFA) δ 10.9 (s,
1H), 9.4 (d, 1H), 9.2 (br s, 1H), 8.4 (d, 1H), 8.2 (d, 1H), 8.1 (s,
1H), 7.8-8.0 (m, 2H), 7.4 (d, 1H), 7.3 (d, 1H), 4.3 (s, 2H), 3.9 (s,
3H), 3.2-3.4 (m, 4H).
To a solution of this amine (0.57 g, 1.1 mmol) in MeOH (20
mL) were added NaOAc (0.18 g, 2.2 mmol) and cyanogen bromide
(0.26 mL, 5 M solution in CH3CN, 1.3 mmol). After stirring for 3
h at room temperature, the reaction mixture was concentrated and
saturated NaHCO3 (aq) was added. The reaction mixture was
extracted with CH2Cl2, and the combined extracts were dried over
Na2SO4. Purification by HPLC afforded 17c (0.37 g, 24%) as a
white solid: 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H),
8.3 (s, 1H), 8.1 (d, 2H), 8.0 (br s, 1H), 7.8 (dd, 1H), 7.75 (s, 1H),
7.3 (d, 2H), 4.5 (s, 2H), 3.8 (s, 3H), 3.5 (s, 4H). Anal. (C22H18-
Cl3N6O3S‚1.8C2HF3O2‚1.7H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-imino-4-oxoimidazolin-1-
yl)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-
5-chlorobenzamide (17d). Prepared by the reaction of 9b with

2976 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Ye et al.
2-aminoacetoamide, followed by cyanogen bromide, in a similar
manner as for 17c, and purified by HPLC, giving a white solid
(0.37 g, 15%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.3 (s,
1H), 8.3 (d, 1H), 8.1 (m, 2H), 7.7 (d, 1H), 7.6 (d, 1H), 7.5 (s, 1H),
7.3 (d, 1H), 7.2 (d, 1H), 4.4 (s, 2H), 4.3 (s, 2H), 3.8 (s, 3H). Anal.
(C22H17Cl3N6O4S‚0.25C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((imidazolin-1-yl)methyl)-3-
chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chloroben-
zamide Trifluoroacetic Acid Salt (17g). To a stirred solution of
9b (1.5 g, 3.0 mmol) in DMF (15 mL) was added 1,2-diaminoethane
(0.9 g, 15 mmol) at room temperature. After 2 h, the reaction was
poured into water and extracted with EtOAc, dried (Na2SO4) and
concentrated to afford the crude amine intermediate. To the amine
was added triethyl orthoformate (1.33 g, 9 mmol) in acetic acid
(20 mL). After stirring at room temperature for 1 h, the reaction
was poured into water and extracted with EtOAc. The organic layer
was dried (Na2SO4), concentrated, and purified by HPLC to afford
17g as a white solid (0.87 g, 39%): 1H NMR (DMSO-d6/TFA) δ
10.9 (s, 1H), 10.3 (s, 1H), 9.4 (s, 1H), 8.6 (d, 2H), 8.3 (d, 1H), 8.1
(d, 1H), 7.8 (dd, 1H), 7.3 (d, 1H), 7.2 (d, 1H), 4.6 (s, 2H), 3.7-3.9
(m, 7H). Anal. (C22H18Cl3N5O3S‚1.6C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((imidazol-1-yl)methyl)-3-
chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chloroben-
zamide Trifluoroacetic Acid Salt (17j). A solution of 9b (0.87 g,
1.73 mmol) and imidazole (0.35 g, 5.18 mmol) in DMF (6 mL)
was heated at 45 °C for 15 h. Additional imidazole (0.25 g, 3.67
mmol) was added, and heating continued for 5 days. Acidification
with trifluoroacetic acid (0.5 mL) and purification by HPLC
afforded 17j as a white solid (0.77 g, 50%): 1H NMR (DMSO-
d6/TFA) δ 10.85 (s, 1H), 9.40 (s, 1H), 9.20 (s, 1H), 8.25 (d, 1H),
8.05 (d, 1H), 7.95 (s, 1H), 7.80 (dd, 1H), 7.70 (s, 1H), 7.60 (s,
1H), 7.30 (d, 1H), 7.20 (d, 1H), 5.40 (s, 2H), 3.80 (s, 3H). Anal.
(C22H16Cl3N5O3S‚2.0C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((2-methylimidazol-1-yl)methyl)-
3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chloroben-
zamide Trifluoroacetic Acid Salt (17k). A mixture of 9b (0.8 g,
1.6 mmol) and 2-methylimidazole (0.39 g, 5.4 mmol) in DMF (7
mL) was heated at 45 °C for 2 days. Acidification with trifluoro-
acetic acid (0.5 mL) and purification by HPLC afforded 17k as a
white solid (0.70 g, 59%): 1H NMR (DMSO-d6/TFA) δ 10.85 (s,
1H), 9.40 (s, 1H), 8.25 (d, 1H), 8.05 (d, 1H), 7.80 (m, 2H), 7.50
(s, 2H), 7.30 (d, 1H), 7.20 (d, 1H), 5.30 (s, 2H), 3.80 (s, 3H), 2.60
(s, 3H). Anal. (C23H18Cl3N5O3S‚1.7C2HF3O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[methyl(methylsulfonyl)amino]methyl]-
2-thiophenecarboxamide (12a). To a solution of 10b (1.3 g, 2.5
mmol) in pyridine (20 mL) at 0 °C was added methanesulfonyl
chloride (0.20 mL, 2.8 mmol). The solution was allowed to warm
to room temperature with stirring. After 16 h, the mixture was
concentrated, and the resulting oil was purified by flash chroma-
tography to afford 12a (1.1 g, 75%) as a white solid: 1H NMR
(DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.1 (d,
1H), 7.9 (d, 1H), 7.8 (s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.2 (s, 2H),
3.9 (s, 3H), 3.0 (s, 3H), 2.7 (s, 3H). Anal. (C21H19Cl3N4O5S2‚
0.42C4H8O2) C, H, N.
4-[[(Aminocarbonyl)methylamino]methyl]-3-chloro-N-[4-chloro-
2-[[(5-chloro-2-pyridinyl)amino]carbonyl]-6-methoxyphenyl]-2-
thiophenecarboxamide Trifluoroacetic Acid Salt (12b). A so-
lution of potassium cyanate (0.70 g, 8.6 mmol) in MeOH (4 mL)
was added dropwise to a solution of 10b (0.10 g, 0.20 mmol) in
acetic acid (1.5 mL), and the mixture was stirred at room
temperature for 20 h. Concentration and purification by HPLC
afforded 12b as a white solid (0.090 g, 58%): 1H NMR (DMSO-
d6/TFA) δ 10.9 (br s, 1H), 9.3 (s, 1H), 8.2 (s, 1H), 8.1 (s, 1H), 7.8
(d, 1H), 7.5 (s, 1H), 7.2 (s, 2H), 4.4 (s, 2H), 3.8 (s, 3H), 2.9 (s,
3H). Anal. (C21H18Cl3N4O4S‚2.0C2HF3O2‚1.0H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[[(ethylamino)carbonyl]methylamino]-
methyl]-2-thiophenecarboxamide (12c). To a solution of 10b (1.0
g, 2.0 mmol) in 1,4-dioxane (20 mL) was added ethyl isocyanate
(0.18 mL, 2.2 mmol), and the reaction was stirred at room
temperature. After 16 h, the mixture was concentrated. The residual
solid was purified by flash chromatography to afford 12c (0.85 g,
74%) as a white solid: 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.4 (s, 1H), 8.4 (s, 1H), 8.1 (d, 1H), 7.9 (d, 1H), 7.5 (s, 1H), 7.4 (s,
1H), 7.3 (s, 1H), 4.4 (s, 2H), 3.9 (s, 3H), 3.1 (q, 2H), 2.8 (s, 3H),
1.0 (t, 3H). Anal. (C23H22Cl3N5O4S‚0.5H2O‚0.19CH2Cl2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[[[(2-hydroxyethyl)amino]carbonyl]-
methylamino]methyl]-2-thiophenecarboxamide Trifluoroacetic
Acid Salt (12d). To a solution of bis(trichloromethyl) carbonate
(0.15 g, 0.51 mmol) in CH2Cl2 (5 mL) at 0 °C was added 10b
(0.10 g, 0.20 mmol), and the mixture was stirred for 0.5 h.
Ethanolamine (0.40 mL, 6.6 mmol) was then added, and the mixture
was stirred at room temperature for 4 h. Concentration and
purification by HPLC afforded 12d as a white solid (0.080 g,
22%): 1H NMR (DMSO-d6/TFA) δ 10.9 (br s, 1H), 9.3 (s, 1H),
8.3 (d, 1H), 8.1 (d, 1H), 7.8 (dd, 1H), 7.7 (br s, 1H), 7.5 (s, 1H),
7.3 (s, 1H), 7.2 (s, 1H), 4.4 (s, 2H), 3.8 (s, 3H), 3.4 (t, 2H), 3.1 (t,
2H), 2.8 (s, 3H). Anal. (C23H22Cl3N5O5S‚1.2C2HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((N′-methyl-N′′-(2-carboxy-
ethyl)ureido)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-
methoxy-5-chlorobenzamide Trifluoroacetic Acid Salt (12f). To
a solution of N-(5-chloropyridin-2-yl)-2-[((4-((methylamino)m-
ethyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chlo-
robenzamide 10b (0.42 g, 0.87 mmol) in 1,4-dioxane (5 mL) was
added ethyl 3-isocyanatopropionate (0.15 mL, 1.0 mmol), and the
mixture was stirred at room temperature. After 0.5 h, water (2 mL)
was added. Excess LiOH‚H2O was added, and the mixture was
stirred for 2 h. Concentration and purification by HPLC afforded
12f as a white solid (0.39 g, 62%): 1H NMR (DMSO-d6/TFA) δ
10.9 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.1 (d, 1H), 7.9 (d, 1H), 7.5
(s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.3 (s, 2H), 3.9 (s, 3H), 3.3 (t,
2H), 2.8 (s, 3H), 2.4 (t, 2H). Anal. (C24H22Cl3N5O6S‚0.9C2HF3O2‚
0.5H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[methyl[[[2-(1-pyrrolidinyl)ethyl]ami-
no]carbonyl]amino]methyl]-2-thiophenecarboxamide (12e). To
a stirred solution of 10b (0.7 g, 1.4 mmol) in THF (10 mL) at 0 °C
was added 2-bromoethylisocyanate (0.23 g, 1.54 mmol), and the
mixture was stirred for 0.5 h. Concentration afforded bromoethyl-
urea 11 (0.91 g, quant.), which was used without purification.
To a stirred solution of 11 (0.91 g, 1.4 mmol) in DMF (4 mL)
at room temperature was added pyrrolidine (0.50 g, 7 mmol). After
1 h, the reaction was diluted with EtOAc (60 mL) and washed with
brine (2 × 15 mL). The organic phase was dried over Na2SO4 and
concentrated. Purification by flash chromatography afforded 13a
as a white solid (0.48 g, 60%) and impure 12e. Compound 12e
was further purified by HPLC to afford a white solid (0.20 g, 22%).
1
12e: H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.5 (br s, 1H),
9.4 (s, 1H), 8.4 (s, 1H), 8.1 (d, 1H), 7.9 (d, 1H), 7.5 (s, 1H), 7.4 (s,
1H), 7.3 (s, 1H), 6.8 (br s, 1H), 4.4 (s, 2H), 3.9 (s, 3H), 3.6 (m,
2H), 3.4 (m, 2H), 3.2 (m, 2H), 3.0 (m, 2H), 2.8 (s, 3H), 2.0 (m,
2H), 1.8 (m, 2H). Anal. (C27H29Cl3N6O4S‚3.3C2HF3O2) C, H, N.
Optimized Conditions for the Synthesis of 3-Chloro-N-[4-
chloro-2-[[(5-chloro-2-pyridinyl)amino]carbonyl]-6-methoxyphe-
nyl]-4-[[(4,5-dihydro-2-oxazolyl)methylamino]methyl]-2-thiophen-
ecarboxamide (13a). To a stirred solution of 10b (0.40 g, 0.8
mmol) in THF (5 mL) was added 2-bromoethylisocyanate (0.144
g, 0.96 mmol) at 0 °C. After 2 h, Et3N (0.16 g, 1.6 mmol) was
added. The reaction was allowed to warm to room temperature,
stirred overnight, and concentrated. Purification by flash chroma-
tography afforded 13a as a white solid (0.40 g, 89%): 1H NMR
(DMSO-d6/TFA) δ 10.9 (s, 1H, C(O)NH), 10.3 (br s, 1H, NH),
9.4 (s, 1H, C(O)NH), 8.3 (d, 1H, Ar-H), 8.0 (dd, 2H, Ar-H), 7.9
(dd, 1H, Ar-H), 7.4 (d, 1H, Ar-H), 7.3 (s, 1H, Ar-H), 4.8 (t, 2H,
oxazoline CH2O), 4.5 (s, 2H, CH2-thiophene), 3.9 (t, 2H, oxazoline
CH2N), 3.8 (s, 3H, OCH3), 3.0 (s, 3H, NCH3); 13C NMR (DMSO-
d6/TFA) δ 164.8, 161.1, 158.4, 154.7, 150.5, 146.3, 137.8, 136.9,
134.4, 131.9, 131.4, 127.34, 125.5, 123.6, 122.3, 120.0, 115.3,
114.3, 68.4, 56.8, 52.4, 47.6, 35.4. Anal. (C23H20Cl3N5O4S‚0.2CH2-
Cl2) C, H, N.

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2977
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-2-oxazolyl)ethylamino]-
methyl]-2-thiophenecarboxamide Trifluoroacetic Acid Salt (13b).
Compound 13b was prepared from 10c (1.53 g, 2.97 mmol) in a
similar manner as for 13a and purified by HPLC, giving a white
solid (0.56 g, 23%): 1H NMR (CDCl3) δ 9.0 (s, 1H), 8.9 (s, 1H),
8.2 (d, 1H), 8.1 (d, 1H), 7.6 (dd, 1H), 7.4 (s, 1H), 7.3 (d, 1H), 7.0
(d, 1H), 4.4 (s, 2H), 4.3 (t, 2H), 3.9 (s, 3H), 3.8 (t, 2H), 3.3 (q,
2H), 1.1 (t, 3H). Anal. (C24H22Cl3N5O4S‚2.15C2HF3O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-2-oxazolyl)(2,2,2-trifluo-
roethyl)amino]methyl]-2-thiophenecarboxamide (13c). To a
stirred solution of 9b (2.1 g, 4.1 mmol) in DMF (10 mL) was added
2,2,2-trifluoroethanamine (2.0 g, 20 mmol) at room temperature.
The reaction was heated at 75 °C overnight, then cooled to room
temperature, and poured into ice water (50 mL). The mixture was
extracted with CH2Cl2 (3 × 40 mL), and the organic phase was
dried over Na2SO4. Concentration and purification by flash chro-
matography afforded the trifluoroethylamino intermediate (2.1 g,
90%). H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.3
(d, 1H), 8.1 (d, 2H), 7.8 (dd, 1H), 7.2 (d, 2H), 4.3 (s, 2H), 4.1 (m,
2H), 3.9 (m, 1H), 3.8 (s, 3H).
The intermediate (2.1 g, 3.68 mmol) was reacted in a similar
manner as for 13a. Purification by flash chromatography afforded
13c (1.1 g, 43%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4
(s, 1H), 8.3 (d, 1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.75 (d, 1H), 7.2 (d,
2H), 4.9 (t, 2H), 4.7 (s, 2H), 4.4 (br s, 2H), 3.9 (t, 2H), 3.8 (s, 3H).
Anal. (C24H19Cl3F3N5O4S‚0.1C4H8O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-4-oxo-2-oxazolyl)methy-
lamino]methyl]-2-thiophenecarboxamide (13i). Compound 13i
was prepared from 10b (0.5 g, 1.0 mmol) and bromoacetylisocy-
anate (0.59 g, 3.6 mmol; prepared by reaction of 2-bromoacetamide
with oxalyl chloride) in a similar manner as for 13a and purified
by flash chromatography, giving a white solid (0.12 g, 21%): 1H
NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.1
(dd, 1H), 7.9 (dd, 1H), 7.7 (s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.8 (m,
2H), 4.6 (m, 2H), 3.8 (s, 3H), 3.0 (s, 3H). Anal. (C23H18-
Cl3N5O5S‚0.4CH2Cl2‚0.3C4H8O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-2-thiazolyl)methylami-
no]methyl]-2-thiophenecarboxamide (13j). Compound 13j was
prepared from 10b (1 g, 2 mmol) and 2-bromoethylisothiocyanate
(1 g, 6 mmol) in a similar manner as for 13a and purified by flash
chromatography, giving a white solid (0.97 g, 87%): 1H NMR
(DMSO-d6/TFA) δ 11.0 (s, 1H), 10.1 (br d, 1H), 9.5 (s, 1H), 7.3-
8.5 (m, 6H), 4.7 (m, 2H), 4 (m, 2H), 3.9 (s, 3H), 3.6 (m, 2H), 3.2
(s, 3H). Anal. (C23H20Cl3N5O3S2‚0.05C4H8O2) C, H, N.
1
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-5-methyl-2-oxazolyl)m-
ethylamino]methyl]-2-thiophenecarboxamide Trifluoroacetic Acid
Salt (13f). To a stirred solution of 1-amino-2-propanol (2.0 g, 27
mmol) in THF (20 mL) was added thiocarbonyldiimidazole (5.3
g, 27 mmol) at room temperature. After 3 h, the reaction mixture
was concentrated and purified by flash chromatography to afford
5-methyl-2-oxazolidinethione (2.9 g, 93%).
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-2-oxazolyl)(1,1-dimeth-
ylethyl)amino]methyl]-2-thiophenecarboxamide Trifluoromethyl
Acetic Acid Salt (13d). To a stirred solution of 9b (2.83 g, 5.5
mmol) in DMF (10 mL) was added tert-butylamine (2.0 g, 28
mmol) at room temperature. After 12 h, the reaction mixture was
poured into ice water (70 mL). The mixture was extracted with
CH2Cl2 (3 × 50 mL), and the organic phase was dried over Na2-
SO4. Concentration and purification by flash chromatography
afforded the tert-butylamino intermediate (2.5 g, 83%).
The intermediate (2.43 g, 4.5 mmol) was reacted in a similar
manner as for 13a. Purification by HPLC afforded 13d as a white
solid (1.49 g, 39%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.9 (s, 1H), 9.4 (s, 1H), 8.3 (s, 1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.7 (s,
1H), 7.4 (d, 2H), 7.3 (s, 1H), 4.9 (t, 2H), 4.6 (s, 2H), 3.9 (s, 3H),
3.8 (t, 2H), 1.4 (s, 9H). Anal. (C26H26Cl3N5O4S‚2.0C2HF3O2) C,
H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-2-oxazolyl)(2-methoxy-
ethyl)amino]methyl]-2-thiophenecarboxamide Trifluoromethyl
Acetic Acid Salt (13e). To a stirred solution of 9b (2.0 g, 4.0 mmol)
in DMF (10 mL) was added 2-methoxyethanamine (1.46 g, 20
mmol) at 0 °C. The ice bath was removed, and the reaction was
stirred at room temperature overnight. The solvent was removed,
and the resulting residue was purified by flash chromatography to
afford the methoxyethylamino intermediate (1.2 g, 55%).
The intermediate (1.2 g, 2.2 mmol) was reacted in a similar
manner as for 13a. Purification by HPLC afforded 13e (0.58 g,
28%): 1H NMR (DMSO-d6/TFA) δ 11.0 (s, 1H), 10.3 (d, 1H), 9.4
(s, 1H), 7.2-8.4 (m, 6H), 4.8 (m, 2H), 4.6 (s, 2H), 3.9 (s, 6H), 3.5
(s, 2H), 3.4 (s, 2H), 3.2 (d, 2H). Anal. (C25H24Cl3N5O5S‚2.9C2-
HF3O2) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(5,6-dihydro-4H-1,3-oxazin-2-yl)m-
ethylamino]methyl]-2-thiophenecarboxamide Trifluoroacetic Acid
Salt (13h). Compound 13h was prepared from 10b (1.5 g, 3.0
mmol) and 3-bromopropyl isocyanate (0.59 g, 3.6 mmol) in a
similar manner as for 13a and purified by HPLC, giving a white
solid (0.56 g, 40%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.4 (s, 1H), 9.3 (br s, 1H), 8.3 (s, 1H), 8.1 (d, 1H), 7.9 (dd, 1H),
7.4 (s, 1H), 7.2 (s, 1H), 4.5 (m, 2H), 4.4 (s, 2H), 3.8 (s, 3H), 3.3
(m, 2H), 3.0 (s, 3H), 2.1 (m, 2H), 1.3 (m, 2H). Anal. (C24H22-
Cl3N5O4S‚1.8C2HF3O2‚0.5H2O) C, H, N.
The 5-methyl-2-oxazolidinethione (2.7 g, 23 mmol) was dis-
solved in POCl3 (40 mL), and PCl5 (4.8 g, 23 mmol) was added at
room temperature. The reaction was heated at 100 °C for 3 h, then
cooled to room temperature, concentrated, and dried under vacuum.
The resulting product, 2-chloro-4,5-dihydro-5-methyloxazole (2.7
g, 100%), was used without purification.
To a stirred solution of 2-chloro-4,5-dihydro-5-methyloxazole
(0.36 g, 3 mmol) in DMF (5 mL) was added 10b (1.5 g, 3 mmol),
followed by Et3N (0.57 g, 5.6 mmol) at room temperature. After
16 h, the reaction mixture was concentrated and purified by HPLC
to afford 13f (0.66 g, 31%) as a white solid: 1H NMR (DMSO-
d6/TFA) δ 10.9 (s, 1H), 9.4 (s, 1H), 8.4 (d, 1H), 8.1 (d, 1H), 7.9
(dd, 1H), 7.5 (s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.4 (s, 2H), 4.2 (m,
1H), 3.8 (s, 3H), 3.2 (m, 2H), 2.8 (s, 3H), 1.4 (d, 3H). Anal. (C24H22-
Cl3N5O4S‚0.9C2HF3O2‚0.8H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[[(4,5-dihydro-4-methyl-2-oxazolyl)-
ethylamino]methyl]-2-thiophenecarboxamide (13g). Reaction of
10c with 2-chloro-4,5-dihydro-4-methyloxazole (prepared by a
similar method as for 13f) afforded 13g, which was purified by
flash chromatography, giving a white solid (0.12 g, 47%): 1H NMR
(CDCl3) δ 9.1 (s, 1H), 8.7 (s, 1H), 8.3 (d, 1H), 8.2 (s, 1H), 7.7 (d,
1H), 7.5 (s, 1H), 7.3 (s, 1H), 4.4 (s, 2H), 4.3 (m, 1H), 4.1 (m, 1H),
3.9 (s, 3H), 3.8 (t, 1H), 3.3 (m, 2H), 1.3 (d, 3H), 1.2 (t, 3H). Anal.
(C25H24Cl3N5O4S‚0.3H2O) C, H, N.
3-Chloro-N-[4-chloro-2-[[(5-chloro-2-pyridinyl)amino]carbo-
nyl]-6-methoxyphenyl]-4-[(methyl-2-oxazolylamino)methyl]-2-
thiophenecarboxamide Trifluoroacetic Acid Salt (13k). To a
suspension of 12d (0.10 g, 0.18 mmol) in EtOH (10 mL) was added
chloroacetaldehyde diethylacetal (0.28 g, 1.84 mmol). The reaction
mixture was refluxed for 4 days. After cooling to room temperature,
water and saturated NaHCO3 (aq.) were added. The solid was
collected by filtration and purified by HPLC to afford 13k (0.03 g,
24%) as a white solid: 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.4 (s, 1H), 8.4 (s, 1H), 8.1 (dd, 1H), 7.9 (dd, 1H), 7.8 (s, 1H), 7.6
(s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 7.0 (s, 1H), 4.4 (s, 2H), 3.9 (s,
3H), 3.6 (m, 2H), 3.0 (s, 3H). Anal. (C23H18Cl3N5O4S‚1.0C2HF3O2‚
1.0H2O) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((N′-methyl-N′-(3,4-dihydro-
2H-pyrrol-5-yl)amino)methyl)-3-chlorothiophen-2-yl)carbonyl)-
amino]-3-methoxy-5-chlorobenzamide Trifluoroacetic Acid Salt
(13l). To a solution of 10b (3.0 g, 6.0 mmol) in DMF (30 mL)
were added N,N-diisopropylethylamine (1.94 g, 15 mmol) and

2978 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Ye et al.
2-methylthioimidazoline hydroiodide (1.9 g, 7.8 mmol). The mixture
was heated at 90 °C for 20 h. The cooled mixture was poured into
water, extracted with EtOAc, dried (MgSO4) and concentrated.
Purification by HPLC afforded 13l (1.3 g, 27%) as a white solid:
1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H), 9.7 (d, 1H), 9.4 (d, 1H),
8.3 (m, 1H), 8.2 (d, 1H), 8.1 (d, 1H), 7.8 (d, 1H), 7.7 (d, 1H), 7.3
(d, 1H), 7.2 (d, 1H), 4.6 (d, 2H), 3.9 (s, 3H), 3.6 (m, 2H), 2.9-3.2
(m, 5H), 2.2 (m, 2H). Anal. (C24H22Cl3N5O3S‚1.65C2HF3O2) C,
H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-((N′-(1-iminoethyl)-N′-methy-
lamino)methyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-meth-
oxy-5-chlorobenzamide (17h). To a stirred solution of 10b (0.70
g, 1.4 mmol) in MeOH (30 mL) was added NEt3 (3 mL, 22 mmol)
and ethyl acetimidate hydrochloride (large excess). The reaction
was stirred at room temperature for 16 h and then concentrated.
Purification of the residual oil by HPLC afforded 17h as a white
solid (0.80 g, 75%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.4 (s, 1H), 9.3 (br s, 1H), 8.6 (br s, 1H), 8.4 (s, 1H), 8.1 (d, 1H),
7.9 (d, 1H), 7.8 (s, 1H), 7.4 (s, 1H), 7.3 (s, 1H), 4.6 (s, 2H), 3.9 (s,
3H), 3.1 (s, 3H), 2.3 (s, 3H). Anal. (C22H20Cl3N5O3S‚0.5H2O‚1.9C2-
HF3O2) C, H, N.
N-(5-Chloropyridin-2-yl)-2-[((4-(((amidino)(methyl)amino)m-
ethyl)-3-chlorothiophen-2-yl)carbonyl)amino]-3-methoxy-5-chlo-
robenzamide (17i). To a stirred solution of 10b (0.10 g, 0.20 mmol)
in DMF (3 mL) were added NEt3 (0.28 mL, 2.0 mmol) and 1H-
pyrazole-1-carboxamidine hydrochloride (0.30 g, 2.0 mmol). The
mixture was stirred at room temperature for 15 h and then heated
at 45 °C for 3 h. The cooled mixture was acidified with
trifluoroacetic acid and purified by HPLC to afford 17i as a white
solid (0.080 g, 56%): 1H NMR (DMSO-d6/TFA) δ 10.9 (s, 1H),
9.4 (s, 1H), 8.3 (d, 1H), 8.1 (d, 1H), 7.8 (dd, 1H), 7.6 (s, 1H), 7.4
(br s, 4H), 7.3 (s, 1H), 7.2 (s, 1H), 4.5 (s, 2H), 3.8 (s, 3H), 2.9 (s,
3H). Anal. (C21H19Cl3N6O3S‚1.5C2HF3O2) C, H, N.
for all coagulation assays was measured by taking the average of
two measurements using an Electra 1400C automated coagulometer
(Medical Laboratory Automation, Inc., Pleasantville, NY).
Pharmacokinetic Studies in Dogs. Fasted (18 h) male beagle
dogs were instrumented with indwelling jugular catheters on the
day of the experiment for the purpose of withdrawing blood
samples. Dogs were allowed water ad lib during the experiment.
Drugs were administered orally by gavage via a feeding tube
temporarily inserted into the stomach or intravenously by a bolus
(ca. 30 s) injection via the cephalic vein. For oral dosing, the
compound was dissolved in a 40% w/w solution of hydroxypropyl-
â-cyclodextrin in water acidified to pH 5 with 1 N HCl. Final
compound concentration was 10 mg/mL, and dosing volume was
1 mL/kg. For intravenous dosing, the compound was dissolved in
a 10% v/v solution of DMSO in unbuffered saline, acidified to pH
5 with 0.1 N HCl. Final compound concentration was 1 mg/mL,
and the dosing volume was 1 mL/kg.
Blood samples were drawn at predetermined time intervals out
to 12 h. For each time point, 2.7 mL of blood was collected from
the jugular vein catheter and placed in a test tube containing 0.3
mL of a 5% sodium citrate solution in phosphate buffered saline
(without calcium or magnesium). The blood samples were kept on
ice until centrifuged (1000 × g for 10 min at 4 °C) to obtain plasma.
The plasma samples were kept frozen (-20 °C) until assayed for
determination of drug levels.
Anti-fXa activity of the test compound in plasma was determined
ex vivo using a chromogenic assay.15 The assay was performed at
room temperature in flat-bottom microtiter plates in a final volume
of 200 µL. The assay buffer consisted of 50 mM Tris-HCl, 150
mM NaCl, and 0.1% polyethylene glycol 6000, pH 7.5. Solutions
of human fXa (6 nM in assay buffer) and substrate S-2222 (948
µM in assay buffer) were prepared. A standard curve was prepared
with five dilutions of test compound in pooled dog plasma. Plasma
samples were thawed and diluted 1:100 with assay buffer. To each
well of a 96-well microtiter plate were added 20 µL of diluted
plasma sample, 30 µL of buffer, and 50 µL of substrate solution.
Two wells of each plate received pooled dog plasma instead of
diluted plasma sample (negative control), and four wells received
a diluted solution of a standard fXa inhibitor of known activity
(positive control). The plate was shaken for 30 s, then 100 µL of
fXa solution added. Reaction rates were determined by measuring
the rate of the absorbance change at 405 nm at 10 s intervals for 2
min. Plasma samples and standards were tested in duplicate. The
rate of reaction in mOD/min was calculated based on the kinetic
readings. The rate was converted to compound concentration (µM)
based on the standard curve using log-logit curve fit analysis.
Thrombosis in Rats: Vena Cava Stasis Model. Male Wistar
rats were anesthetized with 90 mg/kg, i.p., Nembutal. Five minutes
prior to the ligature of the vena cava, the test compound was
administered by intravenous bolus injection. Three minutes prior
to application of the ligature, thromboplastin (Thromborel, Behring
Werke, Marburg, Germany) was infused over 5 min at a dose of
100 µL/kg/min. The vena cava was ligated immediately below the
bifurcation of the left vena renalis. The thrombi were excised 60
min following ligation, and thrombus wet weight was determined.
Venous blood samples for the determination of coagulation
parameters were taken immediately before and five and 60 min
following drug administration. Statistical analysis was performed
by a one-way analysis of variance and subsequent comparison of
the treatment groups with controls employing Dunn’s method, as
the data are not normally distributed. P < 0.05 was taken as a
significant difference.
Enzyme Assay Procedures.23 The activities of human fXa,
human thrombin and bovine trypsin were determined kinetically
as the initial rate of cleavage of a peptide p-nitroanilide by the
enzyme. The assay was performed at room temperature in flat-
bottom microtiter plates in a final volume of 200 µL. The reaction
mixture consisted of 50 mM Tris-HCl, 150 mM NaCl, 2.5 mM
CaCl2, and 0.1% polyethylene glycol 6000, pH 7.5, with enzyme
and substrate at the following concentrations: (1) fXa assay, 0.04-1
nM fXa and 164 µM S-2222; (2) thrombin assay, 16 nM thrombin
and 300 µM S-2302; and (3) trypsin assay, 16 nM bovine trypsin
and 127 µM S-2266. Standard techniques with at least four substrate
dilutions were used to determine the Km for a given enzyme and
substrate. The substrate concentration listed is equal to the Km.
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 measuring the
rate of the absorbance change at 405 nm in a ThermoMax
microplate reader (Molecular Devices Corp., Sunnyvale, CA). Assay
conditions used to determine the selectivity for the remaining
proteases shown in Table 8 have been previously published.11b
Data Analysis Methods. IC50 values for inhibitors were deter-
mined from the dose response curve by fitting the data to the Hill
equation with an automated analysis method using a computer
spreadsheet. Ki,app values were calculated as the IC50/2, with the
IC50 value determined at a substrate concentration equal to Km (Ki,app
) IC50/(1 + [S]/Km) ) IC50/2). For inhibitors with Ki,app values
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.24
Ki,app values are the mean of multiple determinations (n g 2).
Standard deviations are <30% of the mean.
X-ray Crystallography. Crystals of des-GLA-EGF1-factor Xaâ
were grown using protocols described previously.21 This reference
describes a proprietary inhibitor used to grow the initial crystals.
This proprietary inhibitor is compound 17m.
Prothrombin Time (PT) Assay. For in vitro coagulation studies,
pooled, lyophilized human plasma (SARP, Helena Laboratories,
Beaumont, TX) was used following reconstitution. Thromboplastin
HS (Sigma, St. Louis, MO) was used for PT assays. For construction
of concentration-response curves, plasma was serially diluted at
half-log increments ranging from 0.01 to 100 µg/mL. Clot time
Supporting Information Available: Combustion analysis data
for newly synthesized target compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.

Thiophene-Anthranilamides as Factor Xa Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 2979
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to influence the design process outweighed this shortcoming.
JM070125F