Design, synthesis, and SAR of anthranilamide-based factor Xa inhibitors incorporating substituted biphenyl P4 motifs

Article abstract of DOI:10.1016/j.bmcl.2003.11.079

Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors. Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly potent in vitro, and show improved activity in human plasma-based thrombin generation assay.

Full text of DOI:10.1016/j.bmcl.2003.11.079

Bioorganic & Medicinal Chemistry Letters 14 (2004) 983–987
Design, synthesis, and SAR of anthranilamide-based factor Xa
inhibitors incorporating substituted biphenyl P4 motifs
Penglie Zhang,* Liang Bao, Jingmei F. Zuckett, Erick A. Goldman, Zhaozhong J. Jia,
Ann Arfsten, Susan Edwards, Uma Sinha, Athiwat Hutchaleelaha, Gary Park,
Joseph L. Lambing, Stanley J. Hollenbach, Robert M. Scarborough and Bing-Yan Zhu*
Millennium Pharmaceuticals, Inc., 256 E. Grand Avenue, South San Francisco, CA 94080, USA
Received 8 July 2003; accepted 26 November 2003
Abstract—Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors.
Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding
biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly
potent in vitro, and showimproved activity in human plasma-based thrombin generation assay.
# 2004 Published by Elsevier Ltd.
Thrombotic diseases such as deep vein thrombosis,
stroke and pulmonary embolism are a major cause of
mortality and morbidity in the developed world. The
approaches currently explored to regulate thrombosis
include reduction of thrombin generation and inhibition
of thrombin activity. Factor Xa is a serine protease that
links the extrinsic and intrinsic coagulation pathways of
the blood coagulation cascade. It is the enzyme that
converts prothrombin to thrombin. The central role of
factor Xa in the coagulation cascade has attracted tre-
mendous efforts to develop newanticoagulants as
heparin and warfarin replacements.1
linker.3 To further explore the structure–activity rela-
tionships (SARs) and to optimize in vitro and in vivo
properties, structural constraints were introduced to the
b-alanine template to give the anthranilamide-based
compound 4 as a potential factor Xa inhibitor by fol-
lowing the same underlying design of the diaryl ethers.
In this communication, we report the SAR studies
around compound 4, as well as our efforts to modify the
physicochemical properties of the resulting compounds
in order to improve their activity in antithrombotic
functional assay. It should be noted that other anthra-
nilamide-based factor Xa inhibitors have been reported
by several groups.4ꢀ6
We have previously reported a series of diaryl ether-
based factor Xa inhibitors derived from compound 1 by
rigidifying the flexible ethanolamine template (Fig. 1).2
The diaryl ethers, as represented by compound 2, are
potent against factor Xa in vitro and highly efficacious
in our rabbit deep vein thrombosis (DVT) model at sub-
micromolar plasma concentrations. Compound 2, like
many benzamidines, suffers from loworal bioavail-
ability (F<5%) in rat at a dose of 6 mg/kg. In a related
effort to discover factor Xa inhibitors with more favor-
able oral profiles, we have discovered a series of non-
benzamidines as exemplified by compound 3 which
contains a 5-bromo-2-aminopyridine P1 and a b-alanine
As shown in Table 1, compound 4 has an anti factor Xa
IC50 of 3.4 nM. The conformational restriction in com-
pound 4 likely contributes to its enhanced potency
* Corresponding author. Tel.: +1-650-246-7312 (P.Z.); tel.: +1-650-
244-6986 (B.-Y.Z.); e-mail: penglie.zhang@mpi.com; yan.zhu@mpi.
com
Figure 1. Design of anthranilamide 4 as a factor Xa inhibitor.
0960-894X/$ - see front matter # 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2003.11.079

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 983–987
compared to 3. Compound 4 is also very selective for
factor Xa over other serine proteases. The IC50 values
against thrombin, tissue plasminogen activator (tPA),
trypsin, activated protein C (APC) and plasmin are >10
mM, and its anti kallikrein IC50 is 2.1 mM. The P1
chloro analogue 5 is slightly less potent than compound
4. The enzyme selectivity profile of 5 is very similar to
that of 4.
Compounds 4 and 5 exhibit significantly improved
pharmacokinetic properties when compared to 2. For
example, 4 and 5 display oral bioavailability of 31%
and 35%, respectively, in Sprague–Dawley rats at 6 mg/kg.
In the human plasma-based thrombin generation
assay,7 high concentrations of 4 and 5 were required to
Scheme 1. (a) SOCl2, reflux, 2 h; (b) methyl 2-NH2-5-NO2-benzoate (1
equiv), pyridine, CH2Cl2, rt, 12 h; (c) 5-Br-2-NH2-pyridine (4 equiv),
AlMe3 (20 equiv), CH2Cl2, rt, 12 h; (d) TFA, reflux, 2 h; (e)
.
SnCl2 2H2O (4 equiv), EtOAc, reflux, 2 h.
Table 1. In vitro data for compounds 4–15 as factor Xa inhibitors
Compd
X
IC50 (nM)
2XTG (mM)
4
5
Br
Cl
3.4
6.7
>5
>5
Scheme 2. (a) POCl3 (1 equiv), 5-Cl-2-NH2-pyridine (1 equiv), pyri-
dine, rt, 5 min; (b) N-methylpiperazine (1.5 equiv), Cs2CO3 (2 equiv),
ꢁ
.
DMF, 100 C, 1 h; (c) SnCl2 2H2O (4 equiv), EtOAc, reflux, 2 h; (d) 4-
(2-tert-butylaminosulfonyl)phenyl benzoyl chloride (1 equiv), pyridine,
CH2Cl2, rt, 12 h; (e) TFA, reflux, 2 h.
6
Br
Br
Cl
Br
Br
Br
Cl
Cl
Cl
Cl
6.2
4.6
>5
2.8
3.0
double the lag time of maximum thrombin generation
(2XTG>5 mM). The poor functional activities of 4 and
5 were partially attributed to their high lipophilicity that
led to extensive plasma protein binding.8 The cLogD
values of 4 and 5 at pH 7 are 3.97 and 3.78, respectively.
Both compounds are 99.8% protein bound in human
plasma.9 The detrimental effect of high plasma protein
binding of factor Xa inhibitors on antithrombotic
activity is consistent with recent literature dis-
closures.4,6c Similar observations have also been repor-
ted in the thrombin inhibitor area.10,11
7
8
6.7
a
9
370
1,548
128
48
—
a
10
11
12
13
14
—
We then focused on chemical modifications of com-
pounds 4 and 5 to enhance their hydrophilicity. The
initial attempt involved attaching an amino group to the
central phenyl ring as in compounds 6–8. Table 1 shows
these compounds retain the high in vitro binding
potency. The 4-amino substituted compounds (7 and 8)
are markedly more active in the thrombin generation
assay (2XTG=2.8 mM for 7 and 3.0 mM for 8) than the
3-substituted compound 6 (2XTG>5 mM). The cLogD
value of compound 7 (2.59) is indeed lower than that
of compound 6 (3.35). Further modifications of the
4-amino group in 7 led to substantially less potent
compounds 9–11.
a
—
>5
4.9
2.2
>5
23
18
In another approach aimed at increasing the water
solubility, a nitrogen atom was incorporated into the
central phenyl ring. The resulting compounds, 12–15,
are less potent than 5 (Table 1). The cLogD value of
15
26
a Not tested.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 983–987
985
Scheme 5. (a) 1H-Pyrazole-1-carboxamidine hydrochloride (1 equiv),
TEA (5 equiv), EtOH, reflux, 5 h.
Scheme 3. (a) 4-(CO2H)-PhB(OH)2 (1 equiv), Pd(PPh3)2Cl2 (0.05
equiv), 2 M aq K2CO3 (5 equiv), dioxane, reflux, 1 h; (b) SOCl2, reflux, 1
h; (c) 33 (1 equiv), THF, rt, 12 h; (d) NHMe2 (10 equiv), DMF, rt, 2 h.
Compounds 19a and 23a were orally dosed at 6 mg/kg
to Sprague–Dawley rats. The oral bioavailability and
half life are 100% and 8.3 h for compound 19a, and
32% and 1.5 h for compound 23a (Table 4). However,
both compounds displayed insignificant functional
activity (2xTG values >5 mM). Several more com-
pounds including 17a and 24a–28a were assayed in the
human plasma-based thrombin generation assay. All
showed weak anticoagulant activity even though they
have Ki values in the range of 12 pM to 2 nM (Table 4).
Scheme 4. (a) 4-(CO2H)-PhB(OH)2 (1 equiv), Pd(PPh3)2Cl2 (0.05 equiv),
2 M aq K2CO3 (5 equiv), dioxane, reflux, 1 h; (b) SOCl2, reflux, 1 h; (c) 33
(1 equiv), THF, rt, 12 h ; (d) LiNMe2 (5 equiv), THF, rt, 10 min.
Table 2. In vitro data for anthranilamides 17–28 as factor Xa
inhibitors
compound 14 (3.87) is higher than that of compounds
12, 13 and 15 (3.04, 3.05 and 3.23, respectively). What
leads to the higher antithrombotic activity of compound
14 (2XTG=2.2 mM) remains unclear.
The S4 binding biphenylsulfonamide moiety in com-
pounds 4 and 5 was originally developed by researchers
at DuPont. This moiety has been successfully employed
in a variety of factor Xa inhibitors.12 We chose to
replace the aminosulfonyl group with an aminomethyl
as in compound 16 in order to convert compounds 4
and 5 into more hydrophilic species. As bromo sub-
stituted compounds tend to have higher cLogD values
than the corresponding chloro analogues, we focused on
compounds with a 5-chloro-pyridine as the P1 moiety.
Compound 16 is, however, 10 times less potent than 5.
Compd
R
IC50 (nM)
20 (a)
30 (b)
17
18
19
NH2
NHMe
NMe2
1.3
4.1
0.9
1.9
2.3
3.5
20
21
22
23
24
25
26
27
28
2.8
2.8
4.1
1.0
0.4
4.1
3.5
5.6
3.1
5.3
16
74
7.0
1.8
39
In a study of factor Xa inhibitors with structures closely
related to 16, a -Cl at the 4-position of the central
phenyl ring was shown to significantly improve the anti
factor Xa potency.13 The observation is consistent with
recent reports on other anthranilamide-based com-
pounds.4,5 Incorporation of such -Cl into compound 16
results in 17a, which regains the lost potency (IC50=1.3
nM, Table 2) as anticipated.
23
SAR endeavors around 17a were then carried out. As
shown in Table 2, compounds 18a–28a showhigh anti
factor Xa potency with the most potent ones being 19a,
23a and 24a, which bear a dimethylamino, a 4-hydroxy-
piperidinyl and a 4-carboxypiperidinyl, respectively.
Moving the P4 substituents to the 30-position results in
less potent compounds 18b–28b.
32
22

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 983–987
Table 3. In vitro data for anthranilamides 29–32 as factor Xa inhi-
bitors
Compounds 29b, 31a, 32a, and 32b were tested for oral
bioavailability in rats at 6 mg/kg. They were all poorly
absorbed with F values <1% (Table 4). This result
demonstrates that the improvement in anticoagulant
activity would probably be at the expense of oral
biovailablity.
Selected compounds described here were synthesized
according to Schemes 1 to 5. Other compounds were
obtained using slightly modified procedures.
Compd
R
IC50 (nM)
20 (a)
30 (b)
40 (c)
In summary, we have designed and synthesized potent
and orally bioavailable anthranilamide-based com-
pounds 4 and 5 as factor Xa inhibitors. Their lack of
activity in the human plasma-based thrombin generation
assay led to the synthesis of more hydrophilic, and hence
less protein bound factor Xa inhibitors. The strategies
employed to enhance hydrophilicity included attaching
an amino group to the central phenyl ring (6–11), sub-
stituting the central phenyl ring with a pyridyl (12–15),
and replacing the sulfonamide moiety in the P4 region
with substituted aminomethyl (18–28), amidino (29–30)
or guanidino (31–32) groups. These compounds retain
in vitro binding potency and some compounds (7, 8, 14,
30a, and 32b) showimproved anticoagulant activity
with 2XTG values between 1–5 mM. In the accompany-
ing report, we describe our efforts at further reducing
the lipophility of this type of compound in order to
obtain factor Xa inhibitors which would show
antithrombotic activity at therapeutically viable plasma
levels.14
a
29
30
—
1.6
2.0
66
0.2
0.7
1.2
60
a
31
4.4
9.5
—
a
32
—
a Not synthesized.
Table 4. Data for selected anthranilamides as factor Xa inhibitors
Compd
Ki (nM)
2XTG (mM)
cLogD @pH=7
F
a
17a
19a
23a
24a
25a
26a
27a
28a
29b
30a
30b
31a
31b
32a
32b
0.2
0.1
0.2
0.012
1.0
0.9
1.8
0.8
0.7
0.1
1.0
0.1
1.8
0.3
3.9
>5
>5
>5
>5
>5
>5
>5
>5
>5
1.4
5
3.57
5.01
4.50
4.08
3.77
4.83
3.11
3.80
2.52
2.03
2.39
2.40
2.40
1.85
1.85
—
100%
32%
a
—
—
a
a
—
—
a
a
References and notes
—
<1%
a
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—
—
<1%
a
>5
>5
>5
2.2
a
—
<1%
<1%
a Not tested.
The substituted aminomethyl group was replaced with
an amidine or the likes in order to further decrease
lipophlicity. Table 3 summarizes the in vitro data for
compounds 29–32 with amidino and guanidino sub-
stituted P4 biphenyls. The 20-isomers are again more
potent than the 30-isomers. Further loss of potency is
observed with substituents at the 40-position.
As shown in Table 4, the amidines and guanidines 29–
32 have reduced theoretical lipophilicity (cLogD) than
the aminomethyl analogues. Certain compounds
including 30a and 32b showed improved activity in the
thrombin generation assay. The remaining ones (e.g.,
32a) do not display expected 2XTG values. The results
indicate that the anticoagulant activity of the factor Xa
inhibitors discussed above depends not only on their in
vitro potency and hydrophilicity but also on other
pharmacodynamic parameters including their Kon and
Koff values.
5. Shrader, W. D.; Young, W. B.; Sprengler, P. A.;
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mixed and incubated at 37 ꢁC for 30 min. At the conclusion
of the incubation three aliquots (450 mL each) are added
to a Microcon1 YM-10 Centrifugal Filter Device fitted to
a 96-well plate. A standard is prepared in protein free
human plasma and transferred to the Microcon1 YM-10
Centrifugal Filter Device fitted to the same plate. The
plate is centrifuged (3690 rpm) for 25 min at 32 ꢁC in a
Beckman CS-6R centrifuge. 15 mL each of the filtrate is
transferred to a round bottom 96 well plate and 15 mL of
ACN including KN1022 (1 mg/mL) as a internal standard
is added followed by 60 mL of di-water. The plate is
placed on a Multi-Tube Vortexer and vortexed for 30 s.
Concentrations in the filtrate are determined by LC/MS/
MS and using standard curves prepared in ultra filtered
plasma. The plasma protein binding value is expressed as
a percentage.
7. Thrombin generation in human plasma assay: Potential
factor Xa inhibitors are dissolved in DMSO, and serial
dilutions are made in wells of 96 well plates. Reptilase-
treated human plasma is added to the inhibitors, followed
by substrate (H-b-Ala-Gly-Arg-p-nitroanilide) and CaCl2.
The thrombin generation reaction is started by addition of
640 pmol/L of recombinant tissue factor. The absorbance
is monitored at 405 nm at 37 ꢁC for 22 min. The results
are expressed as ‘2XTG’, the concentration of an inhibitor
required to double the time of maximum thrombin
generation. See also: Hemker, H. C.; Wielders, S.; Kessels,
H.; Beguin, S. Thromb. Haemost. 1993, 70, 617.
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S. J.; Malinowski, J.; Arfsten, A. E. Arterios. Thromb.
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9. Factor Xa inhibitor is dissolved in DMSO (1 mg/mL) as a
stock solution. The stock solution is diluted in 1M
HEPES (pH 7.4) to yield a 1 mM working solution. The
working solution is added to a human plasma sample
(EDTA is used as an anticoagulant) in a ration of 1:100 to
yield a final concentration of 10 mM. The mixture is gently
13. Zhang, P. et al., unpublished results.
14. Zhang, P.; Bao, L.; Zuckett, J. F.; Jia, Z. J.; Woolfrey, J.;
Arfsten, A.; Edwards, S.; Sinha, U.; Hutchaleelaha, A.;
Lambing, J. L.; Hollenbach, S. J.; Scarborough, R. M.;
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paper in this issue. doi: 10.1016/j.bmcl.2003.11.080.