Investigation of factor Xa inhibitors containing non-amidine S1 elements

Article abstract of DOI:10.1016/j.bmcl.2005.07.030

Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their

Full text of DOI:10.1016/j.bmcl.2005.07.030

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4838–4841
Investigation of factor Xa inhibitors containing
non-amidine S1 elements
Jeffry B. Franciskovich,* John J. Masters, Jennifer M. Tinsley, Trelia J. Craft,
Larry L. Froelich, Donetta S. Gifford-Moore, Valentine J. Klimkowski,
Jeffrey K. Smallwood, Gerald F. Smith, Tommy Smith, Richard R. Towner,
Leonard C. Weir and Michael R. Wiley
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 27 April 2005; revised 8 July 2005; accepted 12 July 2005
Available online 2 September 2005
Abstract—Several non-amidino S1 derivatives of the 1,2-diaminobenzene-based scaffold (4) were synthesized and evaluated for their
ability to bind to the active site and inhibit the human protease factor Xa. A subset of these compounds were also evaluated for their
anticoagulant effects in human plasma as measured by prothrombin time (PT).
ꢀ 2005 Elsevier Ltd. All rights reserved.
Human factor Xa (fXa) is a trypsin-like serine protease
that plays a critical role in the blood coagulation cas-
cade.1 This enzyme is responsible for the generation of
thrombin that, in turn, produces fibrin and ultimately
leads to blood clot formation. Consequently, the discov-
ery of a small molecule inhibitor of fXa as an oral anti-
coagulant has been the focus of many pharmaceutical
research groups.2–5
O
NH2
NH
O
O
O
O
NH
NH
NH
NH
Our laboratories have previously disclosed a novel series
of 1,2-diaminobenzene-based fXa inhibitors (Fig. 1).6
that display lipophilic groups into both the S1 and S4
binding pockets of this protease. For example, a compu-
tational binding model of compound 1 in the active site
of factor Xa projects the 4-methoxyphenyl group into
the S1 site and the 4-t-butylphenyl group into the S4 site
(Fig. 2). Also, this model indicates that the 1,2-diamide
motif interacts with the S3 backbone of the enzyme.
This compound has reasonable binding affinity to fXa,
but displays a prothrombin time (2· PT) activity
>50 lM (Table 1). This difference may be attributed to
the highly lipophilic nature of 1 and enhanced non-spe-
cific interactions with plasma proteins, which detrimen-
tally impact the functional potency of this inhibitor. In
contrast, compound 2, which incorporates an amidine
1
2
O
O
O
O
O
O
NH
NH
NH
NH
O
N
N
N
N
4
3
Figure 1. Structures of factor Xa inhibitors 1–4.
into the S1 binding element of this scaffold, displays very
high binding affinity for fXa and potent anticoagulant
activity in vitro7 (Table 1). However, there is precedent
Keywords: Factor Xa; Thrombosis; Coagulation; 1,2-Diaminobenzene.
*
Corresponding author. Tel.: +1 317 27 79 499; fax: +1 317 276 14
17; e-mail: j.franciskovich@lilly.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.030

J. B. Franciskovich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4838–4841
4839
X
N
N
CO2Et
a
N
H
5; X = CO2Et
6; X = CH2OH
O
N+
b,c
NH2
NH
O
NCO
O
O
N
Figure 2. Proposed energy-minimized binding model of compound 1
complexed with the active site of factor Xa. The orientation is such
that Asp189 appears in the upper right-hand corner, Ser195 near the
top central, and Glu97 in the lower left-hand corner. Hydrogen bonds
are shown as dashed lines. All aliphatic hydrogens and waters have
been removed for clarity. The molecular modeling was performed, as
described previously in Herron et al.6 utilizing the X-ray structure
1hcg.pdb.12
N
7
R
O
O
NH
NH
d
O
N
Table 1. Human factor Xa binding affinity and prothrombin time
activity of inhibitors 1–3
N
8-36
Kass (in 106 L/mol)
PTb (lM)
a
Compounds
Scheme 1. Reagents and conditions: (a) EtOH, NEt3, 120 ꢁC, 2 days,
then LiAlH4, THF; (b) 6, CHCl3/THF; (c) H2, 10% Pd/C, EtOH;
(d) RCOCl, pyridine, CHCl3 or RCO2H, DCC, DMF.
1
2
3
4
7.0
242
2.0
>50
0.9
9.7
1.9
19
triethylamine in EtOH at 120 ꢁC. Reduction of ester 5
with LiAlH4 afforded the primary carbinol 6. Treatment
of 2-nitrophenylisocyanate with 6 in CHCl3/THF, fol-
lowed by reduction with H2 and Pd/C, afforded com-
pound 7. Acylation of aniline 7 was carried out by
treating with the corresponding acid chloride or by
carbodiimide-mediated coupling of the appropriate
carboxylic acid to obtain the final products.
a Kass represents the apparent association constant, as measured by the
methods of Smith et al.13
b PT is defined as the concentration of compound required to double
the time to clot formation in the prothrombin time assay.
that amidine containing molecules, in general, lack
desirable pharmacokinetic properties upon oral admin-
istration.9,10 Alternative structure–activity relationships
(SAR) on the 1,2-diaminobenzene scaffold have indicat-
ed, the 4-t-butylphenyl S4 binding element can be re-
placed with a cationic/basic S4 group (e.g., compound
3).8 Alternately, the 1-(4-pyridyl)piperidine moiety may
be positioned in the S4 site by the carbamate of com-
pound 4.11 This compound is of particular interest on
account of its improved binding affinity for fXa, but
more importantly, for its enhanced anticoagulant poten-
cy in vitro within this select group of molecules. The
good translation of binding affinity of the more hydro-
philic compounds 3 and 4 into functional anticoagulant
activity is consistent with reduced non-specific plasma
protein binding.
The SAR findings in the carbamate-linked series are giv-
en in Table 2. The unsubstituted phenyl compound 8
exhibits an association constant (Kass) of 1.6 · 106 L/
mol. Saturation of the aromatic ring affords a 40-fold
reduction in fXa binding affinity (9). Partial unsatura-
tion of the cyclohexyl construct (10 and 11) results in
a 5- and 22-fold gain in activity-relative to the complete-
ly saturated version 9. The mono-unsaturated enones,
10 and 12 display similar binding affinity as 8. In gener-
al, substitution at the 4 position enhances the binding
affinity to fXa compared to the unsubstituted phenyl
ring. For example, the 4-methyl, 4-methoxy, and 4-
OCHF2 display binding affinities of 6.3, 19, and
5.0 · 106 L/mol, respectively. Similarly, the 4-halogen-
substituted phenyl rings show increases in binding affin-
ity of 3- to 24-fold versus compound 8. Functionaliza-
tion of the 3 position is also tolerated (21–26), but the
enhancement in binding affinity is only modest when
compared to those the 4-position. The 2-methoxy and
2-chloro derivatives (27 and 28) display 0.02 and
0.30 · 106 L/mol binding affinities, respectively.
In this paper, we report the effects of varying the S1
binding portion of the carbamate scaffold with respect
to binding affinity and anticoagulant potency.
The syntheses of compounds 8–36 are shown in Scheme
1. Intermediate 6 was prepared by first heating ethyl
isonipecotate with 4-chloropyridine hydrochloride and

4840
J. B. Franciskovich et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4838–4841
Table 2. Human factor Xa binding affinity and prothrombin time
activity of compounds 8–36
Table 3. Binding affinity of compound 17 for various enzymes
Kass (in 106 L/mol) for 17
a
Enzyme
R
O
fXa
fIIa
35
0.02
NH
NH
t-PA
<0.01
<0.01
<0.01
<0.01
Plasmin
Urokinase
Trypsin (bovine)
O
O
N
a Kass represents the apparent association constant as measured by the
methods of Smith et al.13
N
a
Compounds R
Kass
PT
constant of 33 · 106 L/mol. These data suggest that the
specific placement of the substituent in the S1 pocket
is critical. There exists a general correlation between
fXa binding affinity and enhanced plasma anticoagulant
potency (PT) within this set of inhibitors (R2 = 0.54). In
addition, the most active compounds show a high degree
of selectivity versus similar proteases. For example,
compound 17 showed minimal binding to serine prote-
ases involved in hemostasis (fIIa (thrombin), t-PA, plas-
min, and urokinase), and to bovine trypsin (Table 3).
(in 106 L/mol) (lM)
8
Phenyl
Cyclohexyl
1.6
—
—
—
—
—
8
9
0.04
0.90
0.20
0.70
6.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1-Cyclohexenyl
2-Cyclohexenyl
1-Cyclopentenyl
Phenyl(4-Me)
Phenyl(4-OMe)
Phenyl(4-OCHF2)
Phenyl(4-F)
19
1.9
5.0
4.6
6
—
Phenyl(4-Cl)
Phenyl(4-Br)
Phenyl(4-I)
35
1.9
38
14
1.8
6
In summary, structural modification of the S1 binding
element in a carbamate-based series affords human fac-
tor Xa inhibitors that do not contain an amidino func-
tionality, but display both high binding affinity and
anticoagulant potency in human plasma. The most
potent derivatives contain some type of substitution at
the 4 position of the phenyl S1 binding element. These
derivatives also display a high degree of specificity
against similar enzymes. In general, within this series
of inhibitors, there is a correlation between binding
affinity for factor Xa and anticoagulant potency as mea-
sured by prothrombin time (PT).
Phenyl(4-CN)
Phenyl(3-Me)
Phenyl(3-OMe)
Phenyl(3-F)
0.60
—
5.9
3.3
2.5
4.1
1.0
5.1
0.02
0.30
—
10
17
12
—
8
Phenyl(3-Cl)
Phenyl(3-OH)
Phenyl(3-NH2)
Phenyl(2-OMe)
Phenyl(2-Cl)
—
—
Phenyl(3-F, 4-OMe)
Phenyl(3-F, 4-Me)
Phenyl(3-Cl, 4-Me)
Phenyl(2,3-diCl)
46
1.1
5.8
2.3
7
22
5
19
2,3-[Dihydro]benzofuran-5-yl 0.60
—
Acknowledgments
6-Indoyl
5-Cl-furan-2-yl
5-Cl-thiophen-2-yl
54
0.70
33
1.6
—
The authors thank Dr. John J. Masters, Dr. Michael
R. Wiley, Dr. Scott Sheehan, and Dr. Thomas E.
Jackson for their helpful discussions and review of this
manuscript.
2.6
PT is defined as the concentration of compound required to double the
time to clot formation in the prothrombin time assay.
a Kass represents the apparent association constant as measured by the
methods of Smith et al.13
References and notes
These data suggest that substitution in the ortho posi-
tion negatively impacts binding to fXa. The 3,4-disubsti-
tuted compounds display different results when
compared to their 4-substituted counterparts. The 3-flu-
oro-4-methoxy analog (29) has a more than 2-fold high-
er binding affinity than the 4-methoxy compound (14),
whereas the 3-fluoro-4-methyl analog (30) has a binding
affinity similar to the mono-substituted 4-methyl com-
pound (13). Bicyclic groups display mixed results. The
6-indoyl derivative (34) shows the highest binding affin-
ity for fXa of 54 · 106 L/mol and the 2,3-[dihydro]ben-
zofuran-5-yl compound (33) has one of the lowest
affinities of 0.6 · 106 L/mol. Heterocyclic replacements
for the phenyl group display very different effects on
fXa binding. For example, the 5-chloro-furan-2-yl com-
pound (35) has a binding affinity of 0.7 · 106 L/mol and
the 5-chloro-thiophen-2-yl compound (36) has a binding
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