Design, synthesis, and SAR of anthranilamide-based factor Xa inhibitors with improved functional activity

Article abstract of DOI:10.1016/j.bmcl.2003.11.080

Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure-activity relationship studies around 2 have led to a series of potent factor Xa inhibitors which are highly active in the human plasma-based thrombin generation assay with 2XTG values less than 1 μM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein thrombosis model, and also exhibits good oral bioavailability and a long half life in rats.

Full text of DOI:10.1016/j.bmcl.2003.11.080

Bioorganic & Medicinal Chemistry Letters 14 (2004) 989–993
Design, synthesis, and SAR of anthranilamide-based factor
Xa inhibitors with improved functional activity
Penglie Zhang,* Liang Bao, Jingmei F. Zuckett, Zhaozhong J. Jia, John Woolfrey,
Ann Arfsten, Susan Edwards, Uma Sinha, Athiwat Hutchaleelaha, Joseph L. Lambing,
Stanley J. Hollenbach, Robert M. Scarborough andBing-Yan Zhu *
Millennium Pharmaceuticals, Inc., 256 E. Grand Avenue, South San Francisco, CA 94080, USA
Received8 July 2003; revised26 November 2003
Abstract—Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate
hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure–activity relationship
studies around 2 have ledto a series of potent factor Xa inhibitors which are highly active in the human plasma-basedthrombin
generation assay with 2XTG values less than 1 mM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein
thrombosis model, andalso exhibits goodoral bioavailability anda long half life in rats.
# 2004 Publishedby Elsevier Ltd.
Factor Xa is an attractive target for the development of
new orally active anticoagulants to replace warfarin, a
vitamin K antagonist. The early factor Xa inhibitors—
such as BABCH,1 DABE,2 andDX-9065a 3—were
mostly bisamidines. The poor pharmacokinetics,
including low oral bioavailability, of these compounds
ledto the synthesis of a variety of mono-benzamidines
as factor Xa inhibitors in which the P1 amidino group is
retainedbecause it has been edemedcritical for the
strong binding of an inhibitor with factor Xa. However,
most mono-benzamidines still lack desirable level of
oral absorption to be developed as oral anticoagulants.4
with a para-aminomethyl group as in compound 2. The
amino group not only renders compound 2 a decreased
cLogD value (2.36), but is also expectedto bindto the
S4 pocket of factor Xa through a p-cation interaction.
We report here the synthesis of compound 2 andits
analogues, as well as the structure–activity relationship
(SAR) studies leading to factor Xa inhibitors which show
improvedactivity in the thrombin generation assay.
Anthranilamide-based factor Xa inhibitors incorporat-
ing aminomethyl substitutedchlorothiophene moieties
9
as P4 have recently been reportedby Berlex scientists.
Compound 2 was synthesizedandfoundto be less
potent (IC50=10 nM, Table 1) than 1 by about one
order of magnitude. SAR studies on substitution of the
benzylic amino group show that methylation increases
potency as demonstrated by compounds 7 and 8. Bulk-
ier substituents at the amino group reduce in vitro
activity as shown by 9 and 10. Compounds 11–16 were
prepared to probe additional interactions in the S4
Anthranilamide-based factor Xa inhibitors have been
5À7
reportedby several groups.
In the preceding paper,
we have described the discovery of compound 1 andits
analogues (Fig. 1). These compounds are highly potent
in in vitro binding assay (IC50 and Ki). However, they
display low anticoagulant activity in the human plasma-
basedthrombin generation assay as demonstratedby
their 2XTG values.8 The low anticoagulant activity is
partially attributedto the low hydrophilicity andhigh
plasma protein binding of this type of compound. In an
effort to design less lipophilic factor Xa inhibitors, we
sought to replace the distal phenyl ring in compound 1
* 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.
0960-894X/$ - see front matter # 2004 Publishedby Elsevier Ltd.
doi:10.1016/j.bmcl.2003.11.080

990
P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 989–993
43–45. The increase in ring size from five in 45 to six in
46 andseven in 47 leads to slight decreases in potency.
Methylation or dimethylation of the exocyclic nitrogen
Table 1. Compounds 7–24 as factor Xa inhibitors
Scheme 1. (a) POCl3 (1.1 equiv), 5-Cl-2-NH2-pyridine (1 equiv), pyri-
.
dine, rt, 5 min; (b) SnCl2 2H2O (4 equiv), EtOAc, reflux, 2 h; (c) 4-
chloromethyl-benzoyl chloride (1 equiv), THF, rt, 12 h; (d) NH(Boc)2
(5 equiv), NaH (5 equiv), DMF, rt, 3 h; (e) 4 N HCl in dioxane, rt, 3 h.
CompdR
IC
NH2
NHMe
NMe2
50 (nM)
2
7
8
10
7.0
4.0
9
12
10
11
12
13
14
15
16
17
35
82
53
156
176
6.8
6.9
1.1
Scheme 2. (a) NH2Me (5 equiv), DMF, rt,
1
h; (b)
NHMe(CH2)2NHMe (5 equiv), DMF, rt, 1 h; (c) 1H-pyrazole-1-car-
boxamidine hydrochloride (1 equiv), TEA (5 equiv), EtOH, reflux, 5 h;
(d) pyrrolidine (5 equiv), DMF, rt, 1 h; (e) MeI (neat), reflux, 5 h.
region. The higher potencies of 15 and 16 may result
from ionic or hydrogen bonding interactions between
the distal amino group and Glu97 at the region exten-
ded from factor Xa’s S4 pocket. These interactions may
also contribute to the enhancedpotency of compounds
17 and 18 due to the increased basicity at the P4 term-
inal. Compound 20 bearing a dimethylhydrazide group
is a sub-nanomolar factor Xa inhibitor. The high
potency of compounds 22–24 may arise from a p-cation
interaction between the positive charge andthe box-like
S4 pocket of Xa formedby Tyr99, Phe174 andTrp215.
18
19
20
21
22
23
24
2.0
40
Among the factor Xa Inhibitors containing cyclic amino
groups (Table 2), 25 and 26 are equipotent. A 4-amino
substituent at the piperidine moiety is tolerated as
shown by compound 28. Compounds 33–35 further
demonstrate that a basic moiety at the P4 terminus
helps retain factor Xa inhibitory activity.
0.9
40
Table 3 summarizes the in vitro binding potency of
compounds 36–49 which contain an amidino or guani-
dino group at the P4 position. The potency enhancing
effect of N1-methylation is again observed( 37 and 40 vs
36 and 39). Compound 38 with a phenyl at the P4
terminal is less potent than the methyl analogue 37.
Stepwise methylation of the guanidine nitrogen atoms
retains anti-factor Xa potency as demonstrated by
compounds 41 and 42. Cyclization with an ethylene unit
between two guanidine nitrogens results in compounds
0.8
2.3
5.8

P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 989–993
991
Scheme 3. (a) MeNCS (5 equiv), THF, EtOH, reflux, 12 h; (b) MeI (5
equiv), acetone, reflux, 2 h; (c) NH2Me (5 equiv), MeOH, reflux, 20
Scheme 4. (a) MeNCS (5 equiv), THF, EtOH, reflux, 12 h; (b) MeI (5
equiv), acetone, reflux, 2 h; (c) NH2(CH2)2OH (2 equiv), MeOH, rt, 2
h; (d) 2-pyrrolidinone (2 equiv), K2CO3 (5 equiv), DMF, 80 ꢀC; (e)
NH2(CH2)2NH2 (5 equiv), DMF, rt, 2 h; (f) Im2CS (1 equiv), Et3N (5
equiv), DMF, 70 ꢀC, 2 h; (g) HO(CH2)2NH2 (10 equiv), DMF, rt, 2 h;
(h) CNBr (3 equiv), DMF, rt, 12 h.
min; (d) NH2(CH2)2NHMe (2 equiv), MeOH, rt,
2 h; (e)
NH2(CH2)2NH2 (5 equiv), DMF, rt, 2 h; (f) CNBr (3 equiv), DMF, rt,
12 h; (g) Im2CS (1 equiv), Et3N (5 equiv), DMF, 70 ꢀC, 2 h; (h)
NHMe2 (5 equiv), MeOH, reflux, 20 min.
of compound 45 leads to compounds 48 and 49 which
retain high potency.
Table 2. Compounds 25–35 as factor Xa inhibitors
Continued modification of the guanidine moiety was
carriedout by replacing one or two nitrogens with a
carbon, oxygen or sulfur atom to obtain less basic ana-
logues (compounds 50–55 in Table 3). The most potent
compounds are 5010 and 55. Compound 50 is 7 times
more potent than its sulfur analogue, 51. The least
potent compounds in Table 3 are 52, 53, and 54. This
observation demonstrates again that more basic P4
motifs give more potent factor Xa inhibitors.
CompdR
IC
(nM)
16
50
25
26
27
28
29
30
31
32
33
34
35
15
The compounds discussed above show excellent selec-
tivity (IC50>11 mM) against thrombin, trypsin, tissue
plasminogen activator (tPA), activatedprotein C (APC)
andplasmin. Lower selectivity against kallikrein is
observedas exemplifiedby some of the most potent
compounds (Table 4).
136
15
76
Human plasma-basedthrombin generation assay was
performedon selectedpotent compounds. As shown in
Table 4, compound 8 doubled the lag time of maximum
thrombin generation at a fairly high plasma concen-
tration (2XTG=3.2 mM) despite its high in vitro
binding potency. Compound 20, which is more potent
171
230
140
17
in vitro andmore water soluble than
8, has a
2XTG=1.5 mM. Compound 22 bearing a quaternary
nitrogen; amidine and guanidine-based compounds 37,
40, 41, 44 and 45; as well as oxazolidine 55 are even more
active in this assay. Their 2XTG values are less than 1 mM.
The pharmacokinetics of compounds in Table 4 was
testedin Sprague–Dawley rats. Compound 8 is highly
absorbedfollowing oral administration (6 mg/kg). The
half life, volume of distribution and clearance of com-
pound 8 are respectively 7.5 h, 32.9 L/kg and20.6 mL/
min/kg (0.4 mg/kg dosed intravenously). The hydrazide
20; quaternary salt 22; amidine 37; guanidines 40, 41,
44, and 45 have poor oral bioavailability. Compound 55
has an F value of 44%. This compoundalso has a long
half life (8.5 h), reasonable volume of distribution (13.6
15
5

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P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 989–993
Table 3. Compounds 36–55 as factor Xa inhibitors
Table 3 (continued)
CompdR
IC
50 (nM)
35
54
55
CompdR
IC
(nM)
3.6
50
0.2
36
Table 4. Data for selectedcompounds as factor Xa inhibitors
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
0.9
CompdXa
Ki
(nM)
kallikrein,
IC50 (mM)
2XTG
(mM)
cLogD
@pH 7
F
(%)
11
8
1.3
0.3
1.0
0.6
0.1
0.2
0.3
0.5
1.5
0.50
0.64
0.13
0.50
0.38
0.79
0.30
0.21
0.56
3.2
1.5
0.3
0.5
0.38
0.79
0.30
0.58
0.56
3.61
3.91
—
0.71
0.17
0.50
0.04
100
4
20
22
37
40
41
44
45
55
a
<1
<1
<1
1.4
1.0
0.7
0.8
2.0
0.3
1.5
3.8
6.3
3.5
1.3
1.5
b
—
<1
<1
44
À0.07
1.63
a By definition, a cationic compound does not have a cLogD value.
b Not tested.
L/kg) andclearance (18.3 mL/min/kg) at an intravenous
dose of 0.4 mg/kg. Inhibitor 55 shows 25% and40%
inhibition of thrombus formation at a plasma concen-
tration of 0.25 mM and0.83 mM, respectively, in our
rabbit deep vein thrombosis model.11 The cLogD of
compound 55 (1.63) is in the middle of the range shown
in Table 4. The favorable in vivo profile of compound
55 strongly implies that a balance between antico-
agulant activity andoral absorption can be achievedby
fine tuning the physical chemical properties of the
anthranilamide-based factor Xa inhibitors and their in
vitro binding potency.
The preparation of compounds in Tables 1 to 3 is
exemplifiedby the synthesis of compounds shown in
Schemes 1–4. Other compounds were obtained according
to slightly modified procedures.
In summary, in order to modulate the lipophilicity of
the previous series of anthranilamide-based factor Xa
inhibitors with biphenyl P4 groups, we have designed
andsynthesizedcompounds which contain substituted
aminomethylbenzoyl moieties as the S4 binding motifs.
These compounds are potent against factor Xa in vitro,
selective over other serine proteases andmore active in
the human plasma-basedthrombin generation assay.
Compound 55 not only shows strong antithrombotic
activity in our rabbit deep vein thrombosis model, but
also has goodoral bioavailability andlong half life in
rat. The amidine or guanidine containing compounds
are poorly absorbed, but exhibit potent activity in the
functional thrombin generation assay. Further modifi-
cations of this type of compoundresultedin P1 neutral,
P4 benzamidine-based factor Xa inhibitors which display
pharmacokinetic profiles suitable for oral anticoagulants.
The details will be reported in due course.
10
552
30

P. Zhang et al. / Bioorg. Med. Chem. Lett. 14 (2004) 989–993
993
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