Anthranilamide inhibitors of factor Xa

Article abstract of DOI:10.1016/j.bmcl.2007.06.051

SAR about the B-ring of a series of N²-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalkylether and B-ring p-amine probes of the S1′ and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in in vitro anticoagulation assays.

Full text of DOI:10.1016/j.bmcl.2007.06.051

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4832–4836
Anthranilamide inhibitors of factor Xa
David Mendel,* Angela L. Marquart, Sajan Joseph, Philip Waid, Ying K. Yee,
Anne Louise Tebbe, Andrew M. Ratz, David K. Herron, Theodore Goodson,
John J. Masters, Jeffry B. Franciskovich, Jennifer M. Tinsley, Michael R. Wiley,
Leonard C. Weir, Jeffrey A. Kyle, Valentine J. Klimkowski, Gerald F. Smith,
Richard D. Towner, Larry L. Froelich, John Buben and Trelia J. Craft
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 27 March 2007; revised 13 June 2007; accepted 14 June 2007
Available online 20 June 2007
Abstract—SAR about the B-ring of a series of N2-aroyl anthranilamide factor Xa (fXa) inhibitors is described. B-ring o-aminoalk-
ylether and B-ring p-amine probes of the S10 and S4 sites, respectively, afforded picomolar fXa inhibitors that performed well in
in vitro anticoagulation assays.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Thromboembolic diseases are well known as a major
R1
O
X
cause of morbidity and mortality throughout the world.
Standard therapeutics against these diseases such as
coumadin and heparin are limited by narrow therapeutic
windows and/or parenteral routes of administration,1,2
hence there is a major need to provide orally active
agents with a high safety margin suitable for both acute
and chronic treatment. Because of its central role in the
coagulation cascade, factor Xa (fXa) is a key target for
therapeutic intervention in diseases such as DVT and
PE.3 Numerous efforts to produce an orally active fXa
inhibitor are described in several reports and reviews.4,5
A
R1
c
O
N
O
X
C
H
a,b
Cl
NO2
NH
N
H
O
NH2
B
R3
1
2a-d
3a-f
Scheme 1. Reagents and conditions: (a) DCM, pyridine, and
p-methoxy aniline (2a), p-Cl aniline (2b), 2-amino-5-Cl-pyridine (2c),
or 4-t-Bu aniline (2d), rt; (b) Raney Ni, 4.1 bar H2, 1:1 THF/EtOAc, rt
16 h; (c) DCM, pyridine, and 4-OMe-, 4-i-Pr-, or 4-t-Bu-benzoyl
chloride, rt.
We previously described anthranilamide6 and 1,2-
dibenzamidobenzene7 inhibitors of fXa bearing amidine8
or neutral probes9 of the fXa S1 pocket. Others10,11 also
utilized the anthranilamide scaffold to produce potent
fXa inhibitors. Here we describe further SAR results
with anthranilamides. Scheme 1 depicts the general syn-
thetic route to N2-aroyl anthranilamides such as 3a,
which inhibits fXa with a Ki of about 1 lM.6 2-Nitro-
benzoyl chloride was coupled with a 4-substituted ani-
line or heterocyclic amine of choice in DCM–pyridine
and the resulting nitro amide reduced with Raney nickel
and hydrogen to provide amines 2a–d, typically 65% for
the two steps. Intermediates 2a–d were then coupled
with the indicated 4-substituted benzoyl chlorides in
DCM–pyridine to furnish N2-aroyl anthranilamides
3a–f (Table 1).
The A-ring R1 group of anthranilamides 3a–f likely
interacts with the fXa S1 pocket.6 Inhibitors equipped
with small R1 substituents such as methoxy and chloro
(3a–d) inhibit fXa with modest affinity, whereas bulky
groups are not well tolerated (Table 1). The R1 methoxy
to tert-butyl replacement (3a ! 3f) results in a thirtyfold
activity loss versus fXa. The 5-chloropyridin-2-yl deriv-
ative 3e is significantly more potent than the anilino
derivatives and serves as the prototype for further
SAR results. Based on modeling studies7 of 8b, we
Keywords: Protease inhibitor; Factor Xa; Anticoagulant.
*
Corresponding author. E-mail: Mendel_David@Lilly.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.051

D. Mendel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4832–4836
Table 1. Activity of N2-aroyl anthranilamides 3a–f versus fXa
4833
Phenol 4 was protected with methyl chloromethyl ether
in DCM at 0 ꢁC. Lithiation of the ether followed by car-
bonylation gave benzoic acid 5 in 65% yield. Treatment
of 5 with methanolic HCl provides methyl salicylate 6a
in 87% yield. Mitsunobu12 coupling of linear or cyclic
N-boc-protected aminoalcohols to 6a proceeded in good
yield, even for secondary alcohol N-boc-4-hydroxypi-
peridine 6d (73%). Esters 6b–e were then saponified,
converted to their acid chlorides, and coupled with 2c
in DCM–pyridine to give N2-aroyl anthranilamides
7b–e. Compound 7a was prepared by coupling 5 and
2c followed by acidic removal of the MOM ether pro-
tecting group. Compounds 7b–e were treated with acid,
then base, to afford free amines 8b–e.
R1
R3
fXa Kassoc
(in 106 L/mol)
a
Compound
X
3a
3b
3c
3d
3e
3f
CH
CH
CH
CH
N
OMe
OMe
OMe
Cl
OMe
i-Pr
1
7
t-Bu
t-Bu
t-Bu
OMe
5
0.5
42
0.03
Cl
t-Bu
CH
a Kassoc represents the apparent association constant between enzyme
and inhibitor and represents the average of at least two separate runs.
Ki may be approximated6 by 1/Kassoc
.
perceived that the fXa S10 site could possibly be reached
from the anthranilamide B-ring o-position (Fig. 1).
Accordingly, a series of B-ring o-aminoalkylether probes
was prepared according to Scheme 2.
Potency versus fXa in the anthranilamide series is not
greatly affected by addition of a B-ring o-phenol,
3e ! 7a, 42 versus 23 · 106 L/mol, respectively (Table
2). Whereas N-boc aminoethers 7b and 7d inhibit fXa
to a similar degree as proteo reference 3e, compounds
Table 2. Performance of compounds 7a–8e versus fXa, thrombin
(fIIa), and in in vitro anticoagulation assays
a
b
Compound fXa Kassoc
fIIa Kassoc
APTTc PTd
(in 106 L/mol) (in 106 L/mol) (lM)
(lM)
7a
7b
8b
7c
8c
7d
8d
7e
8e
23
54
0.02
0.2
>100
>70
0.85
>70
1.4
>100
>70
0.79
>70
1.4
1440
3.1
219
69
152
3.9
67
6
55
64
1112
2.9
76
114
0.14
1102
0.85
>70
2
0.88
>70
1.6
a,b
K
represents the apparent association constant between enzyme
assoc
and inhibitor.6
c,dAPTT and PT are defined as the concentration of compound
required to double clotting times in activated partial thromboplastin or
prothrombin clot formation assays, respectively.13
Figure 1. Molecular model of 8b docked into the fXa active site. In this
potential binding mode, the B-ring o-aminoalkylether chain is directed
toward the S10 region and hydrogen bonds to His57 and Ser214.
Cl
O
N
A
O
OH
O
OMe
R2
N
C
H
OH
e-g
O
a,b
c,d
NH
O
O
B
R2
4
5
6a-e
7a-e
Boc
h
Boc
N
R2=
O
N
OH
O
H
8b-e
6a
6b
6c
BocN
Boc
O
N
H
O
6d
6e
Scheme 2. Reagents and conditions: (a) DIEA, DCM, methyl chloromethyl ether, 0 ꢁC (54%); (b) t-BuLi, CO2, 0 ꢁC (65%); (c) MeOH, HCl, 0 ꢁC
(87%); (d) N-boc-aminoalcohol, PPh3, THF, DIAD, rt (73% for 6d); (e) LiOH, THF–water, 60 ꢁC (90%); (f) oxalyl chloride, DCM, pyridine, cat.
DMF; (g) 2c, DCM, pyridine, 0 ꢁC to rt (54%); (h) HCl dioxane, then K2CO3 in DCM–H2O (90%).

4834
D. Mendel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4832–4836
7c and 7e lose about 10-fold potency versus 3e. Factor
Xa inhibition improves when the N-boc groups of 7b–e
are removed to reveal amines 8b–e. For example, race-
mate 8c is about 5- to 10-fold more active than 3e and
7a, respectively. Greater improvement is observed for
amines 8b and 8d which inhibit fXa in the 1 nM range,
about 50 times more potent than 7a. Translation of en-
zyme inhibitory activity to in vitro anticoagulant activ-
ity is good for 8b and 8d, each of which double APTT
and PT clotting times when present at less than 1 lM
concentration.13 This result contrasts with the perfor-
mance of 7a–e that each requires more than 50 lM
concentration to double clotting times. Compound 8e
differs from 8b by the presence of two methyl groups
in the middle of the aminoalkylether sidechain. The
added methyl groups neutralize most of the fXa po-
tency gains realized with the aminopropyl ether chain
and produce potent thrombin inhibitor 8e, thus fXa-
fIIa selectivity may be tuned by varying B-ring ortho
substituents.
Compounds 8b–e show that adding aminoalkylethers to
the B-ring o-position produces very potent fXa and/or
fIIa inhibitors that perform well in anticoagulation as-
says using human plasma.13 Further SAR in this region
is illustrated by elaboration of 8d via reductive amina-
tion or acylation according to Scheme 3. Reductive ami-
nation of 8d with NaCNBH3 and aldehydes or acetone
in 95:5 MeOH/AcOH provides tertiary amines 12–20
(Table 3). Acylation of 8d was performed either with
acid chlorides and resin-bound base or with acids and
polymer-supported carbodiimide to provide 21–24.
Reaction of 8d with isocyanates or isothiocyanates in
chloroform provides urea and thiourea derivatives 25–
27.
a
Cl
Cl
O
N
O
N
N
N
H
b
c
H
NH O
NH
NH O
N X
Table 3 summarizes the SAR of substitution of the
amine of 8d. For alkyl derivatives 12–20, fXa inhibitory
activity changes slightly, increasing for isopropyl (13)
and methoxyethyl (15) derivatives, and decreasing for
thien-3-yl-methyl (18) and benzyl (19) derivatives. The
more polar alkyl derivatives 12–17 and 20 perform sim-
ilarly to unmodified 8d in in vitro anticoagulation as-
says, whereas 18 and 19 are much less potent in these
assays than would be expected based on their fXa
O
O
8d
12-27
Scheme 3. Reagents and condition: (a) XCHO or acetone (>2 equiv),
NaCNBH3 (3 equiv), 95:5 MeOH/AcOH; (b) XCOCl, piperidino-
methyl polystyrene, CHCl3 (amylenes stabilized), or XCOOH, resin-
bound carbodiimide, 4:1 CHCl3/t-BuOH, rt, or succinic anhydride,
CHCl3 (22); (c) XNCO or XNCS, CHCl3 (amylenes stabilized).
Table 3. Enzyme activity and anticoagulant performance of alkyl (12–20) and acyl (21–27) derivatives of amine 8d
fXa Kassoc (in 106 L/mol)
fIIa Kassoc (in 106 L/mol)
APTTc (lM)
PTd (lM)
a
b
Compound
X
8d
12
13
14
15
16
17
18
19
20
21
22
H
Me
1112
1630
3440
1940
3080
1370
2160
450
114
61
25
30
24
37
54
23
20
72
60
4
0.85
0.93
1.2
0.88
1.1
1.8
3.3
2.1
2.5
2.6
20
i-Pr
n-Pr
3.1
2.1
Methoxyethyl
Cyclopropylmethyl
Imidazo-2-yl-methyl
Thien-3-yl-methyl
Bn
2.4
1.8
14.8
28.1
0.57
n.t.
6.1
488
44.6
1.0
n.t.
5.3
2-Carboxybenzyl
–COCH2NMe2
–CO(CH2)2COOH
1160
176
714
O
23
24
25
513
448
307
21
5.7
8
7.6
10
N
N
N
O
N
134
27
N
N
O
N
21.5
30.2
H
F
26
27
–CSNH(CH2)3NMe2
170
733
145
34
2.9
2.9
S
13.4
21.8
N
N
H
represents the apparent association constant between enzyme and inhibitor.6
a,b
K
assoc
c,dAPTT and PT are defined as in Table 2.13

D. Mendel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4832–4836
4835
Potassium N-boc-n-aminopropoxide was condensed
with ethyl 2,4-difluorobenzoate in THF at ꢀ65 ꢁC, then
warmed to room temperature to give a 4:1 mixture of o/p
adducts. The o-substituted product was isolated in 65%
yield after flash chromatography, then subjected to a
second round of nucleophilic substitution in neat mor-
pholine to provide ester 28. Ester 28 was saponified,
converted to its acid chloride, then condensed with 2c
and deprotected to give amine 33.
Cl
O
OEt
F
O
OEt
O
O
N
O
a-b
c-f
N
H
NH
NH2
O
F
N
O
NHBoc
O
N
28
33
Scheme 4. Reagents and conditions: (a) HO(CH2)3NHBoc, t-BuOK,
THF, ꢀ65 ꢁC to rt (4:1 o/p isomers); (b) morpholine, >80 ꢁC (56%); (c)
LiOH, dioxane-H2O, 60 ꢁC (91%); (d) oxalyl chloride, DCM, pyridine,
cat. DMF; (e) 2c, DCM, pyridine, 0 ꢁC to rt (55%); (f) HCl-dioxane,
then K2CO3 in DCM–H2O.
The SAR for B-ring p-amines is shown in Table 4.
Replacing tert-butyl with pyrrolidine (8b ! 30) in-
creases fXa affinity and in vitro anticoagulant potency,
but a further increase in ring size to piperidine 32 and
homopiperidine 37 diminishes both activities. Morphol-
ine derivative 33 is slightly less potent versus fXa than its
tert-butyl comparator 8b, but 33 is a slightly more po-
tent anticoagulant, consistent with reduced protein or
phospholipid binding. Remarkably, N-methylpiperazine
35 loses significant fXa affinity, but ring expansion to
N-methylhomopiperazine 36 increases potency one hun-
dredfold versus 35. Compound 36 is a picomolar fXa
inhibitor with nanomolar in vitro anticoagulation
potency.
inhibition. This observation may reflect increased bind-
ing to proteins and/or phospholipids by these more lipo-
philic derivatives. Of the acyl analogs 21–27, none are as
potent versus fXa as 8d and their anticoagulation per-
formance lags both 8d and the simpler alkyl derivatives.
Replacing the p-tert-butyl group of 8b with more polar
amino groups affords further SAR results related to
the fXa S4 binding site. In general, amines were installed
at the B-ring p-position according to the route illus-
trated for morpholine in Scheme 4.
In summary, relative to the prototypical N2-aroyl
anthranilamides such as 3a, we describe SAR for the
Table 4. R3 group SAR: enzyme activity and anticoagulant performance of compounds 30–37 versus comparator 8b
Cl
O
N
O
N
H
NH
NH2
O
R3
8b, 30-37
R3
fXa Kassoc (in 106 L/mol)
fIIa Kassoc (in 106 L/mol)
APTTc (lM)
PTd (lM)
a
b
Compound
8b
t-Bu
1440
152
0.85
0.79
30
31
32
33
34
35
3010
874
323
516
709
82
40
7.3
17
5.8
17
2
0.41
0.41
0.45
0.93
0.34
0.61
n.t.
N
NH2
0.4
N
N
1.0
0.26
0.69
n.t.
N
N
N
O
S
N
N
36
37
8970
130
24
25
0.1
3.1
0.14
2.7
N
N
represents the apparent association constant between enzyme and inhibitor.13
a,b
K
assoc
c,dAPTT and PT are defined as in Table 2.13

4836
D. Mendel et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4832–4836
8. Wiley, M. R.; Weir, L. C.; Briggs, S.; Bryan, N. A.;
Buben, J.; Campbell, C.; Chirgadze, N. Y.; Conrad, R.
C.; Craft, T. J.; Ficorilli, J. V.; Franciskovich, J. B.;
Froehlich, L. L.; Gifford-Moore, D. S.; Goodson, T.,
Jr.; Herron, D. K.; Klimkowski, V. J.; Kurz, K. D.;
Kyle, J. A.; Masters, J. J.; Ratz, A. M.; Milot, G.;
Shuman, R. T.; Smith, T.; Smith, G. F.; Tebbe, A. L.;
Tinsley, J. M.; Towner, R. D.; Wilson, A.; Yee, Y. K.
J. Med. Chem. 2000, 43, 883.
B-ring which provides more potent fXa inhibitors.
Elaboration of the B-ring o-position with a series of
aminoalkylethers produces several ꢁ1 nM fXa inhibitors
that perform well in in vitro anticoagulation assays.
Additional modification of the B-ring p-position with
various amines affords pM fXa inhibitor 36 with in vitro
anticoagulant potency in the 100–140 nM range.
9. Masters, J. J.; Franciskovich, J. B.; Tinsley, J. M.;
Campbell, C.; Campbell, J. B.; Craft, T. J.; Froelich, L.
L.; Gifford-Moore, D. S.; Hay, L. A.; Herron, D. K.;
Klimkowski, V. J.; Kurz, K. D.; Metz, J. T.; Ratz, A. M.;
Shuman, R. T.; Smith, G. F.; Smith, T.; Towner, R. D.;
Wiley, M. R.; Wilson, A.; Yee, Y. K. J. Med. Chem. 2000,
43, 2087.
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