Factor VIIa inhibitors: Target hopping in the serine protease family using X-ray structure determination

Article abstract of DOI:10.1016/j.bmcl.2008.07.044

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing l-Gln or l-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

Full text of DOI:10.1016/j.bmcl.2008.07.044

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4533–4537
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Factor VIIa inhibitors: Target hopping in the serine protease family
using X-ray structure determination
*
Takuya Shiraishi , Shojiro Kadono, Masayuki Haramura, Hirofumi Kodama, Yoshiyuki Ono, Hitoshi Iikura,
Tohru Esaki, Takaki Koga, Kunihiro Hattori, Yoshiaki Watanabe, Akihisa Sakamoto, Kazutaka Yoshihashi,
Takehisa Kitazawa, Keiko Esaki, Masateru Ohta, Haruhiko Sato, Toshiro Kozono
Fuji Gotemba Research Labs, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2008
Revised 9 July 2008
Accepted 11 July 2008
Available online 16 July 2008
Selective factor VIIa–tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for
developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine pro-
tease-directed compounds from our internal collection. Using parallel synthesis supported by structure-
based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4–11) containing
L-Gln
or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in
the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out
with a focus on the P4 moiety. Among the optimized compounds, 12b–f showed improved selectivity.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
FactorVIIa
Thromboembolic disorder
Serine protease
Thrombin
X-ray
Peptidemimetic inhibitor
Factor VIIa (FVIIa) is a serine protease and one of the key en-
zymes of blood coagulation. FVIIa in complex with tissue factor
(TF) initiates the blood coagulation cascade. This complex activates
factors IX to IXa and X to Xa, which activates factors X to Xa and
prothrombin to thrombin, respectively.1 Thrombin cleaves fibrino-
gen to fibrin, which forms blood clots with activated platelets.
Inappropriate thrombus formation in blood vessels causes cardio-
vascular disease (myocardial infarction, stroke, pulmonary embo-
lism, and so on), which is the most common cause of mortality
in industrialized countries.2 Therefore, the prevention of blood
coagulation has become a major target for treatment of thrombo-
embolic disorders, and much research has focused on the genera-
tion of thrombin or factor Xa selective inhibitors.3–6 Recent
studies on the inhibition of the FVIIa/TF complex have shown that
selective FVIIa/TF complex inhibition may provide effective antico-
agulation and low risk of bleeding compared to other antithrom-
botic mechanisms.7–12 Thus, selective FVIIa/TF complex inhibition
is regarded as a promising target for developing new anticoagulant
drugs.13–21
P2
P2
O
H
N
H
N
Cl
P3
N
N
P3
O
O
O
O
N
H
NH2
P1
HN
H2N
P1
H2N
NH
NH
PPACK
1
FVIIa IC50 2400 nM
Thrombin IC50 88 nM
Figure 1. Structure of compound 1 and PPACK.
and thrombin,22–26 we attempted to improve inhibition activity
for FVIIa/TF and selectivity against thrombin. Although both FVIIa
and thrombin belong to the serine protease family and their 3D
structures are similar, several regions are structurally different
especially the S2 site. Thrombin has an insertion loop at the S2 site,
protected by hydrophobic residues Tyr60A and Trp60D. The small,
hydrophobic residue proline in P2 of PPACK fits the S2 site of
thrombin25,26 and thus would also fit compound 1. First, using so-
lid-phase synthesis, we conducted a search for modifications of P2
which would improve inhibition activity for FVIIa/TF and selectiv-
ity against thrombin (Scheme 1).
Compound 1 was discovered from focused screening of serine
protease-directed compounds from our internal collection (Fig.
1). Compound 1 showed more potent inhibition activity for throm-
bin than for FVIIa/TF. Using 3D-structural information of FVIIa/TF
* Corresponding author. Tel.: +81 055087 67227.
E-mail address: shiraishitky@chugai-pharm.co.jp (T. Shiraishi).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.044

4534
T. Shiraishi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4533–4537
O
OH
R
O
HN
NO2
OH
O
H2N
CO2Me
O
O
N
O
O
BSA
N-Methylmorpholine
PyBOP
DMAP
Wang resin
NH
H
N
H
N
O
O
O
O
CO2H
CO2Me
H2N
NHBoc
NaOHaq
R
O
N
R
R
O
O
N
PyBOP
DMAP
O
H
N
O
O
H
N
O
O
N
H
TFA
N
H
N
R
HN
NH
NH
BocHN
H2N
Scheme 1. Synthetic scheme of the derivatization of P2 using solid-phase synthesis.
Table 1
O
NH2
In vitro inhibitory activities against FVIIa/TF, thrombin, and FXa28
S
H
N
H
N
R2
H
N
NH
N
NH
O
O
O
R3
O
O
N
H
N
H
O
O
N
H
S O
H2N
NH
H2N
NH
3
R4
2
H2N
NH
FVIIa IC50 5000 nM
Thrombin IC50 83000 nM
FVIIa IC50 6000 nM
Thrombin IC50 10000
Compound
R2
R3
R4
Ki (nM)
Thrombin
FVIIa
FVIIa
Thrombin
Fxa
2700
330
NT*
NT*
1500
160
25,000
1800
Figure 2. Structures of primary lead compounds containing L-Gln or L-Met.
4
5
6
7
8
9
10
11
L
L
L
L
L
L
L
L
-Gln
D
D
D
D
D
D
D
D
-Ile
-Ile
-Thr
-Thr
-Ile
-Ile
-Thr
-Thr
Propyl
Benzyl
Propyl
Benzyl
Propyl
benzyl
Propyl
Benzyl
69
25
198
114
35
15
130
42
500
150
13,342
3362
220
55
3400
760
7.2
6.0
67.4
29.5
6.3
3.7
26.2
18.1
-Gln
-Gln
-Gln
-Met
-Met
-Met
-Met
L-Gln and L-Met were considered favorable for the S2 site of
FVIIa/TF but compounds 2 and 3 lacked the selectivity of the other
serine proteases in the coagulation cascade and anti-FVIIa activity
(Fig. 2). Therefore, we carried out further optimization focusing on
P3 and P4. Here, we report the preparation and structure–activity
relationships (SAR) of these compounds.27
*
First, we searched for modifications of P3 supported by struc-
ture-based drug design. We found that in reference to thrombin
NT, not tested.
inhibitors,29,30 P2 contained
L-Gln or L-Met, and P4 was fixed with
The X-ray structure of compound 5 suggested that the gluta-
mine side chain in P2 of compound 5 formed potent hydrogen
bonds with Asp60, Tyr94, and Thr98 in the hydrophilic pocket of
the S2 site of FVIIa/sTF (Fig. 3). However, the glutamine side chain
in P2 could not form such hydrogen bonds in the S2 site of throm-
bin because instead of the corresponding hydrophilic pocket,
thrombin has hydrophobic residues Tyr60A and Trp60D in the S2
site that well fit the small, hydrophobic proline moiety in P2 of
PPACK. Therefore, the introduction of a relatively large glutamine
moiety in P2 seemed to cause a steric repulsion to the residues
and a reduction of inhibition activity for thrombin.
either a propylsulfonyl or a benzylsulfonyl group.
The S3 site of thrombin, which consists of Leu99, Ile174, and
Trp215, is larger than that of FVIIa/TF because the side chain of
Leu99 in thrombin extends to the S2 site whereas the side chain
of Thr99 in FVIIa occludes the S3 site of FVIIa/TF.25,26,31 Hydropho-
bic interactions of the D-Phe side chain in P3 of PPACK with the
large S3 site of thrombin are strong and so we introduced relatively
small amino acids at P3.
As a result, we generated the series of analogs depicted in Table
1. The 3D structure of compound 10 bound to FVIIa/sTF revealed
that the side chain of
D-Thr fits well into the small S3 site of
In the case of compound 10, the X-ray structure suggested that
a methionine side chain occupies a small pocket at the S2 site of
FVIIa/sTF (Fig. 4), similar to other FVIIa selective inhibitors.21 The
S2 site of thrombin does not have a pocket corresponding to the
pocket in the S2 site of FVIIa/sTF to accept the methionine side
chain because Leu99 extends into the S2 site. So the modification
of P2 from proline to a larger methionine led to a decrease in the
inhibition activity of thrombin, likely caused by the unfavorable
FVIIa/sTF (Fig. 4). The small size of the P3 moiety seemed to be an-
other key factor in the improvement of selectivity versus thrombin
and the modification of P3 to
FVIIa/TF. However, this modification caused a decrease of selectiv-
ity versus thrombin. Apparently, the -Ile side chain in P3 formed
D-Ile improved inhibition activity for
D
hydrophobic interactions with the more hydrophobic thrombin
S2 site, which suggested that reduction of hydrophobicity in P3
is important for the improvement of selectivity versus thrombin.

T. Shiraishi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4533–4537
4535
steric repulsion between the large methionine side chain and the
small S2 site of thrombin.
In order to improve the selectivity over thrombin, we opti-
mized the P4 moiety based on a comparison of the crystal struc-
ture of compound 5 bound to FVIIa/sTF and the 3D-model of
compound 10 bound to thrombin (Fig. 5). In both cases, the ben-
zylsulfonamide moiety in P4 occupies the S1 subsite. Both S1
subsites are similar in size, so we focused on the difference in
charge between the Lys192 of FVIIa and the Glu192 of thrombin.
We hypothesized that if a carboxylic acid moiety was introduced
to the benzene ring of the benzylsulfonamide moiety, the inhibi-
tion activity for FVIIa/TF would improve from a reduction of
binding affinity for thrombin. The X-ray crystal structure and
computational model suggested that the best position for the
carboxylate group was the meta position of the benzene ring
of benzylsulfonamide.
The synthesis of carboxylic acid derivatives 12a–f is shown in
Scheme 2. These compounds were synthesized using standard
methods.
Figure 3. X-ray crystal structure of compound 5 (white) bound to FVIIa/sTF32–34
(green). The Gln side chain at P2 creates strong hydrogen bond networks with
Table 2 summarizes the biological activity of the carboxylic acid
analogs. As in our model, the favorable substituent position is meta
for 12a–c. Lead compounds 5, 7, 9, and 11 led to dramatically im-
proved selectivity of 12b, 12d, 12e, and 12f over thrombin because
of the electrostatic repulsion of thrombin with Glu192. The X-ray
structure of our compound with similar molecular structure sug-
gested that the carboxylate group formed an ionic interaction with
Lys192, and the O atoms of carboxylate group are 3.8 Å.37 However,
inhibition activities for FVIIa/TF of these compounds showed better
retention than the lead compounds. In general, the contribution of
an ionic interaction to ligand-binding free energy is not significant
in a solvent-exposed region because of the free-energy penalty of
the desolvation of the charged groups,38,39 and that the side chain
of Lys192 of FVIIa/TF is fully exposed to solvent. Therefore, the
introduction of a charged group would not cause an increase in
the inhibition activity for FVIIa/TF.
Asp60, Tyr94, and Thr98. The relatively small S3 is a good fit into D-Ile at P3. The
hydrogen bond with Gly219 and hydrophobic interactions with S4 contribute to the
increase in inhibition activity.35
The inhibitors were also tested in standard clotting assays
including a prothrombin time (PT) assay and an activated partial
thromboplastin time (APTT) assay to confirm effects on blood
coagulation from the improved specificity for FVIIa/TF inhibition.
Theoretically, specific inhibition of FVIIa/TF, which blocks only
extrinsic coagulation, should result in prolongation of only PT
without affecting APTT, thus making the concentration ratio for a
2-fold prolongation of APTT and PT (2Â APTT/2Â PT) infinitely
large. The measured 2Â APTT/2Â PT ratio of compound 12e was
3.8,40 which is higher than that of the thrombin selective inhibitor
argatroban41 (0.4, in-house data), and the FXa selective inhibitor
DX-9065a42 (2.2, in-house data).
Figure 4. X-ray crystal structure of compound 10 (cyan) bound to FVIIa/sTF32-34
(green). The side chain of Met at P2 occupies a small pocket consisting of His57,
Thr98, Thr99, and Asp102.36
It is generally accepted that inhibitors containing highly basic
functions such as an amidine group are often poorly absorbed. So
not surprisingly, the oral bioavailability of 12e amounted to only
3.8% in rat.
A recent study on inhibitors with an amidine group has shown
that when the strongly basic benzamidine group was masked as an
amidoxime prodrug, oral bioavailability increased.15 Replacement
of the amidine with less basic or non-basic groups led to increased
oral bioavailability.18–20
In summary, we succeeded in target hopping in the serine pro-
tease family using crystal structures of human FVIIa/TF in complex
with peptide mimetic inhibitors. We found that L-Gln and L-Met
are suitable for the S2 site of FVIIa/TF, and that the introduction
of a carboxylic acid moiety to the meta position in benzylsulfona-
mide is effective for improving selectivity over thrombin. Further
synthetic efforts to improve pharmacokinetics are in progress. This
structural information is important in understanding the interac-
tions that contribute to specificity for FVIIa/TF and for the rational
and rapid development of specific inhibitors of FVIIa/TF.
Figure 5. Superimposition of compounds
5 (white) and 10 (cyan) onto the
thrombin structure (orange) in complex with PPACK25,26 (PPACK is not shown).

4536
T. Shiraishi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4533–4537
O
1)
R2
H
N
KN
Boc-L-Glu-OH
or
Boc-L-Met-OH
NHBoc
O
Br
NH2
O
NC
NC
EDC HCl
HOBt
2)
H2N-NH
2
CN
R2
R2
H
N
H
N
Boc-D-Ile-OH
or
Boc-D-Thr(OtBu)-OH
NH
NH2
R3
NHBoc
TFA
O
O
TFA
O
EDC HCl
HOBt
NMM
CN
CN
R2
R2
O
CO2Et
H
N
1) HCl-EtOH
2) NH3-EtOH
3) 2N NaOHaq
H
N
ClO2S
NH
NH
R3
R3
O
O
O
O
N
H
S O
NH2
Et3N
CN
CN
CO2Et
R2
H
N
12a R2=Gln, R3=Ile, R4=
o-CO2H
NH
R3
12b R2=Gln, R3=Ile, R4=
m
-CO H
O
2
O
O
12c R2=Gln, R3=Ile, R4=
12d R2=Gln, R3=Thr, R4=
p
-CO2H
N
H
S O
m
-CO2H
-CO2H
12e R2=Met, R3=Thr, R4=
m
H2N
NH
12f R2=Met, R3=Ile, R4=
m-CO2H
R4
Scheme 2. Synthetic scheme of the derivatization of P4.
Table 2
6. Haas, S.; Schellong, S. Hamostaseologie 2007, 27, 41.
In vitro inhibitory activities against serine proteases43
7. Harker, L. A.; Hanson, S. R.; Wilcox, J. N.; Kelly, A. B. Haemostasis 1996, 26, 76.
8. Himber, J.; Refino, C. J.; Burcklen, L.; Roux, S.; Kirchhofer, D. Thromb. Haemost.
2001, 85, 475.
9. Szalony, J. A.; Taite, B. B.; Girard, T. J.; Nicholson, N. S.; LaChance, R. M. J.
Thromb. 2002, 14, 113.
10. Suleymanov, O. D.; Szalony, J. A.; Salyers, A. K.; LaChance, R. M.; Parlow, J. J.;
South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003, 306,
1115.
11. Zoldhelyi, P.; McNatt, J.; Shelat, H. S.; Yamamoto, Y.; Chen, Z. Q.; Willerson, J. T.
Circulation 2000, 101, 289.
12. Kelley, R. F.; Refino, C. J.; O’Connell, M. P.; Modi, N.; Sehl, P.; Lowe, D.; Pater, C.;
Bunting, S. Blood 1997, 89, 3219.
13. Miura, M.; Seki, N.; Koike, T.; Ishihara, T.; Niimi, T.; Hirayama, F.; Shigenaga, T.;
Sakai-Moritani, Y.; Tagawa, A.; Kawasaki, T.; Sakamoto, S.; Okada, M.; Ohta, M.;
Tsukamoto, S. Bioorg. Med. Chem. 2007, 15, 160.
Compound
Ki (nM)
FIXa
Thrombin
FVIIa
FVIIa Thrombin Fxa
FXIa FXIIa
12a
12b
12c
12d
12e
12f
43
18
35
74
30
20
5490
5731
680
45,500
25,300
3850
3130 >100,000 113 >100,000 127.7
685 NT*
2483 NT*
33 NT*
318.4
19.4
17 NT*
14,700 NT*
259 >100,000 614.9
5700 >100,000 323 >100,000 843.3
920 NT*
115 >100,000 192.5
*
NT, not tested.
14. Young, W. B.; Mordenti, J.; Torkelson, S.; Shrader, W. D.; Kolesnikov, A.; Rai, R.;
Liu, L.; Hu, H.; Leahy, E. M.; Green, M. J.; Sprengeler, P. A.; Katz, B. A.; Yu, C.;
Janc, J. W.; Elrod, K. C.; Marzec, U. M.; Hanson, S. R. Bioorg. Med. Chem. Lett.
2006, 16, 2037.
15. Zbinden, K. G.; Obst-Sander, U.; Hilpert, K.; Kuhne, H.; Banner, D. W.; Bohm, H.
J.; Stahl, M.; Ackermann, J.; Alig, L.; Weber, L.; Wessel, H. P.; Riederer, M. A.;
Tschopp, T. B.; Lave, T. Bioorg. Med. Chem. Lett 2005, 15, 5344.
16. Schweitzer, B. A.; Neumann, W. L.; Rahman, H. K.; Kusturin, C. L.; Sample, K. R.;
Poda, G. I.; Kurumbail, R. G.; Stevens, A. M.; Stegeman, R. A.; Stallings, W. C.;
South, M. S. Bioorg. Med. Chem. Lett. 2005, 15, 3006.
17. Sagi, K.; Fujita, K.; Sugiki, M.; Takahashi, M.; Takehana, S.; Tashiro, K.;
Kayahara, T.; Yamanashi, M.; Fukuda, Y.; Oono, S.; Okajima, A.; Iwata, S.;
Shoji, M.; Sakurai, K. Bioorg. Med. Chem. 2005, 13, 1487.
18. Hu, H.; Kolesnikov, A.; Riggs, J. R.; Wesson, K. E.; Stephens, R.; Leahy, E. M.;
Shrader, W. D.; Sprengeler, P. A.; Green, M. J.; Sanford, E.; Nguyen, M.; Gjerstad,
E.; Cabuslay, R.; Young, W. B. Bioorg. Med. Chem. Lett. 2006, 16, 4567.
Acknowledgment
We thank Ms. F. Ford for proofreading the manuscript.
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DMSO solution of a test compound (20
fXa (5 mM, American Diagnostica Inc.), 20
1500 mM NaCl, 50 mM CaCl2), and 80 l distilled water in a 96-well assay
plate. The reaction was initiated by the addition of 20 l human FVIIa solution
l
l) was mixed with 20
ll Spectrozyme
ll buffer (500 mM Tris–HCl, pH 7.5,
l
l
(20 nM, Enzyme Research Laboratories), and absorbance at 405 nm was then
monitored to measure the initial velocity of the reaction. Percent inhibition at
each concentration was calculated from the experimental and control samples.
IC50 was calculated from a concentration–reaction activity curve of each test
compound.Human factor XIIa (FXIIa) inhibition was measured as follows: 10%
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(v/v) DMSO solution of test compound (20
(200 mM Tris–HCl, pH 8.3, 300 mM NaCl), 20
Chemical), and 40 l distilled water in a 96-well assay plate. The reaction was
initiated by the addition of 20 l human factor XIIa solution (50 nM, Enzyme
Research Laboratories), and absorbance at 405 nm was then monitored to
measure the initial velocity of the reaction. Percent inhibition at each
concentration was determined from the experimental and control samples.
IC50 was calculated from the concentration–reaction activity curve of each test
compound.
l
l) was mixed with 100
ll buffer
ll S-2302 (2 mM, Daiichi Pure
l
l