Factor VIIa inhibitors: Gaining selectivity within the trypsin family

Article abstract of DOI:10.1016/j.bmcl.2005.12.040

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3- yl)-benzimidazole (1) scaff

Full text of DOI:10.1016/j.bmcl.2005.12.040

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1596–1600
Factor VIIa inhibitors: Gaining selectivity within the trypsin family
William D. Shrader,a,* Aleksandr Kolesnikov,a Jana Burgess-Henry,b Roopa Rai,a
John Hendrix,c Huiyong Hu,a Steve Torkelson,a Tony Ton,a Wendy B. Young,a
Bradley A. Katz,a Christine Yu,a Jie Tang,a Ronnel Cabuslay,a Ellen Sanford,a
James W. Janca and Paul A. Sprengelera
aCelera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
bDepartment of Chemistry, California State University, Chico, Chico, CA 95929, USA
cDepartment of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA
Received 20 October 2005; revised 7 December 2005; accepted 7 December 2005
Available online 18 January 2006
Abstract—Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challeng-
ing. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaf-
fold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a
unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa
lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.
Ó 2005 Elsevier Ltd. All rights reserved.
The development of novel antithrombotic agents for the
treatment of coagulation disorders is an active area of
research in the pharmaceutical industry. The enzymes
(Factors IIa, Xa, VIIa, IXa, and XIa) that comprise
the extrinsic and intrinsic pathways of coagulation, lead-
ing to the formation of a blood clot, are trypsin-family
serine proteases.1 Preclinical models of thrombosis in
several species have suggested that a selective inhibitor
of the coagulation proteases earlier in the cascade (Fac-
tors VIIa and IXa) may have a greater therapeutic/safe-
ty index than inhibition of proteases later in the cascade
(Factors Xa and IIa).2–4 Based on this pharmacology
guidance, we chose to develop potent and selective
inhibitors of factor VIIa-tissue factor complex (fVIIa)
as an effective strategy for treatment of coagulation
disorders.
we report on the further development of our 5-ami-
dino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole 1 scaf-
fold to achieve increased potency for factor VIIa and
high selectivity against trypsin and the late coagulation
pathway proteases; fIIa, fXa (see Fig. 1).
Our efforts toward developing a selective fVIIa inhibitor
began with the broad spectrum trypsin-family protease
inhibitor, 1. The potency of 1 is mediated by a unique
network of hydrogen bonds to the catalytic Ser-195,
common to all proteases in this family. This protease-in-
hibitor binding paradigm is observed at high resolution
in a large set of crystal structures (>400 structures).7–10
Compound 1 was chosen for further optimization to
obtain a highly selective fVIIa compound due to its
initial potency for fVIIa (Ki = 0.074 lM), high solubili-
ty, and excellent parenteral pharmacokinetic profile.11
We have previously described the development of active
site small-molecule inhibitors which interact with both
fVIIa and factor Xa (fXa).5 Within the trypsin family
of coagulation proteases, developing highly selective
inhibitors of Factor VIIa has proved difficult.6 Herein,
HO2C
CO2H
Ki(µM)
fVIIa
fIIa
fXa
0.074
210
2.4
N
HN
Trypsin
6.7
NH OH
Keywords: Factor VIIa; Trypsin; Factor Xa; Thrombin; fIIa; Selectiv-
ity; Suzuki; Amidine; Lysine-192; Crystallography; Inhibitor.
H2N
Distal Ring,
Resides in S1'-Pocket
1
*
Corresponding author. Tel.: +1 650 866 6539; fax: +1 650 866
6655; e-mail: bill.shrader@celera.com
Figure 1. The potency for 1 versus fVIIa, fXa, fIIa, and trypsin.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.040

W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1596–1600
1597
Compound 1, while possessing good selectivity for IIa,
had suboptimal selectivity for fXa and trypsin. Our ini-
tial goal was to gain 1000-fold selectivity for fVIIa ver-
sus fIIa, fXa, and trypsin (Table 1). From
crystallography and modeling analysis, further improve-
ments in selectivity against fXa and trypsin were envi-
Table 1. SAR data for selected compounds 1, 9–58
CO2H
HO2C
N
R2
H2N
HN
NH OH
9-58
Compound
R2
Selectivity ratios versus fVIIa
IIa Xa
2,838 32
fVIIa Ki (lM)
Trypsin
1
9
Phenyl
0.074
0.004
0.0054
0.006
0.010
0.012
0.013
0.009
0.014
0.025
0.021
0.022
0.022
0.027
0.029
0.033
0.036
0.038
0.054
0.064
0.066
0.068
0.076
0.077
0.088
0.11
91
4400
2500
8500
1789
2960
1077
398
421
192
216
268
25
2-Hydroxy-5-fluorophenyl
2-Hydroxy-5-chlorophenyl
2-Hydroxy-5-nitrophenyl
2-Hydroxy-5-aminophenyl
2-Hydroxy-5-cyanophenyl
2-Hydroxyphenyl
>55,500
27,800
25,000
15,800
12,000
20,800
17,000
10,700
6000
5744
9545
5455
33,300
29,700
5512
3333
3947
2778
1719
2273
9706
11,800
1458
1705
5636
1182
5917
3750
6923
1154
1100
1000
714
1800
722
1450
505
848
458
216
171
112
86
42
109
43
62
73
128
61
28
63
24
24
17
21
41
17
51
16
30
15
54
25
17
35
20
31
13
24
39
18
11
7
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
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
2-Hydroxy-3-bromo-5-chlorophenyl
2-Hydroxy-3,5-dichlorophenyl
2-Hydroxy-4,6-dichlorophenyl
3-(Hydroxymethyl)phenyl
3-Nitrophenyl
2-Nitrophenyl
3,5-Dichlorophenyl
3,5-Dimethylphenyl
3-Acetylphenyl
98
1077
197
250
108
102
103
59
3-Aminophenyl
3-Methylphenyl
N-(3-Methylphenyl)acetamide
2-Thiomethylphenyll
3-Chlorophenyl
3,5-Difluorophenyl
3-Isopropylphenyl
3-Cyanophenyl
115
40
71
99
3-Hydroxyphenyl
5-Chlorothiophene
3-Acetamidylphenyl
3-(Difluoromethoxy)phenyl
2-Methoxyphenyl
10
0.11
0.12
118
51
0.12
0.13
55
34
3-Chloro-4-fluorophenyl
2-Methoxyphenyl
0.13
0.13
123
33
5-(Hydroxymethyl)thiophene
2-Fluorophenyl
0.135
0.21
0.25
43
39
2,3,5-Trichlorophenyl
2,5-Dichlorophenyl
2,3-Dichlorophenyl
3,4-Phenyldioxolone
2-Methoxy-5-cyanophenyl
2-Methoxy-5-fluorophenyl
2-Aminophenyl
600
556
35
44
0.27
0.28
536
540
20
54
0.28
0.33
455
357
52
41
0.42
0.42
4-Methylphenyl
4-Chlorophenyl
310
341
8
0.44
0.50
2
16
2-Methylphenyl
3-Pyridyl
200
209
12
14
13
9
0.55
0.73
31
16
2-(Hydroxymethyl)phenyl
3-(Aminomethyl)phenyl
4-Hydroxyphenyl
205
192
0.78
0.88
18
10
170
67
14
3
4-Methoxyphenyl
2-Acetylphenyl
2.25
4.0
1
28
38
>24
16
3
H
4-tert-Butylphenyl
6.4
1
16
9
4
1
Data shown are factor VIIa Ki and fold-selective ratios (anti-target Ki/fVIIa Ki) for coagulation factors IIa, Xa, and trypsin.16,17

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W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1596–1600
sioned via modifications of the distal biaryl ring. The
distal aryl ring of 1 resides within the S10-pocket of tryp-
sin-family proteases. Due to the structural variation
amongst the trypsin-family proteases in the S10-pocket,
we chose to gain further selectivity for fVIIa by explor-
ing this region. This strategy was executed by generating
a varied set of distal substituted biaryl compounds via a
Suzuki-based synthesis strategy.
From the set of 50 compounds generated via this
Suzuki-based synthesis strategy, fVIIa potencies ranging
from 4 nM (9) to 16 lM (58) and varying selectivities
were observed. This range of activity illustrated that
simple substitutions on the distal ring of 1 can effectively
sample the structural variations within the S10-pocket.
Overall, this initial strategy generated 21 compounds
more potent than the initial unsubstituted base scaffold
1
(Table 1). Nevertheless, the majority of the
Synthesis of the distal-arene-substituted analogs of 1 be-
gan via a Heck coupling of dimethyl fumarate with com-
mercially available 4-iodophenol 2 (Scheme 1). The
subsequent olefin was reduced with hydrogen and Pearl-
man’s catalyst, followed by selective ortho-formylation
of 3 with paraformaldehyde and MgCl2 using the condi-
tions of Hofslokken et al.12 The resulting salicylalde-
hyde was brominated with N-bromosuccinimide in
DMF, followed by protection of the phenol with Mem-
Cl to afford aryl ether 4. The distal-arene diversity was
installed by a Suzuki mediated coupling of aryl bromide
4 with selected boronic acids (Scheme 1, reaction h).
Alternatively, when the corresponding boronic acid
was not readily available, the aryl bromide 4 was con-
verted to the boronic acid pinacol ester 5 by reaction
of 4 with bis(pinicolato)diboron and PdCl2(dppf) as a
catalyst.13 (Scheme 1, reactions f and g) The benzimid-
azole ring system was constructed via a 1,4-benzoqui-
none mediated oxidative cyclization of the aryl
aldehyde 6 with 3,4-diaminobenzamidine hydrochloride
7. To provide the desired fVIIa inhibitor, the phenol was
deprotected with methanolic HCl, followed by treat-
ment with refluxing 1 N aqueous HCl to hydrolyze the
methyl esters to the corresponding carboxylic acids. Uti-
lizing this synthetic strategy, compounds 1 and 9–58
were obtained. All compounds were purified by prepara-
tive reverse-phase HPLC, and isolated as their corre-
sponding HCl salts after lyophilization. All
compounds were synthesized and characterized in the
inhibition assays as a racemic mixture.
compounds surveyed within this set had comparable
selectivity to 1, with Factor Xa and trypsin selectivities
below our set criteria. However, compounds 9–14,
containing the ortho-phenol on the distal aryl ring (R2,
Table 1), demonstrated significantly improved selectivity
and potency over all other compounds within this diver-
sity set. The simple conversion of the 2-aryl hydrogen of
base scaffold 1, to a 2-hydroxy (compound 14), provides
a 5-fold increase in potency and a 10-fold increase in
selectivity. Increasing the acidity of this distal phenol
by the incorporation of a fluorine para to the phenol
provided another 5-fold increase in potency, resulting
in the fVIIa inhibitor 9 (fVIIa Ki = 4 nM), which signif-
icantly surpassed our initial selectivity criteria and yield-
ed a selectivity of >55,500-fold against fIIa, 1800-fold
selectivity against fXa and 4400-fold selectivity against
trypsin. To highlight the importance of the 2-hydroxy
for selectively targeting fVIIa, capping of the hydroxy
in 9 as the corresponding methyl ether 46 resulted in a
100-fold loss in potency. Additional substitutions to
the 2-hydroxy distal aryl ring, at sites other than the
5-position, caused fXa and trypsin selectivity to decrease
(15–17).
In an effort to explain the enhanced selectivity and
potency derived from the distal 2-hyroxyphenyl on com-
pounds 9–14, we developed a hypothesis based on a un-
ique fVIIa structural change observed by Banner and
coworkers in fVIIa/sTF X-ray structures.14 In this re-
port, Banner describes a unique Lys-192, Gly-193 main
h (50-90%)
CO2Me
CO2Me
CO2Me
Br
HO2C
MeO2C
MeO2C
MeO2C
I
CO2H
a,b
c,d,e
70%
f
g
H
O
H
O
O
B
H
O
90%
75%
40-70%
R
O
OMem
OH
OH
OMem
OMem
2
3
4
5
6
CO2H
CO2Me
NH HCl
HO2C
MeO2C
NH2
H2N
90%
i
NH2
N
N
7
H2N
HN
H2N
HN
j,k
R
R
NH OH
NH
OMem
95%
9-58
8
Scheme 1. Reagents and conditions: (a) Pd(OAc)2, (o-Tol)3P, TEA, dimethyl fumarate; (b) MeOH, 50 psi H2, Pd(OH)2 on carbon (Pearlman’s
catalyst); (c) MgCl2, anhyd paraformaldehyde, TEA, CH3CN, reflux, 4 h; (d) NBS, DMF; 0 °C (e) 2-methoxyethoxymethyl chloride (MemCl), TEA,
CH2Cl2; (f) bis(pinicolato)diborane, PdCl2(dppf), KOAc, DMF; (g) Pd(PPh3)4, aq K2CO3, DME, MeOH, ArBr or ArI, 85 °C; (h) Pd(PPh3)4, aq
K2CO3, DME, MeOH, ArB(OH)2; (i) 3,4-diaminobenzamidineÆHCl, 1,4-benzoquinone (1 equiv), MeOH, reflux 6 h; (j) 4 N HCl in dioxane/MeOH
1 h; (k) aq 1 N HCl reflux, 1 h.

W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1596–1600
1599
8. Verner, E.; Katz, B. A.; Spencer, J. R.; Allen, D.; Hataye,
J.; Hruzewicz, W.; Hui, H. C.; Kolesnikov, A.; Li, Y.;
Luong, C.; Martelli, A.; Radika, K.; Rai, R.; She, M.;
Shrader, W.; Sprengeler, P. A.; Trapp, S.; Wang, J.;
Young, W. B.; Mackman, R. L. J. Med. Chem. 2001,
44(17), 2753.
9. Katz, B. A.; Spencer, J. R.; Elrod, K.; Luong, C.;
Mackman, R. L.; Rice, M.; Sprengeler, P. A.; Allen, D.;
Janc, J. J. Am. Chem. Soc. 2002, 124(39), 11657.
10. Katz, B. A.; Elrod, K.; Verner, E.; Mackman, R. L.;
Luong, C.; Shrader, W. D.; Sendzik, M.; Spencer, J. R.;
Sprengeler, P. A.; Kolesnikov, A.; Tai, V. W.; Hui, H. C.;
Breitenbucher, J. G.; Allen, D.; Janc, J. W. J. Mol. Biol.
2003, 329(1), 93.
11. Kolesnikov, A.; Rai, R.; Young, W.B.; Torkelson, S.;
Shrader, W.D.; Leahy, E.M.; Katz, B.A.; Sprengeler,
P.A.; Liu, L.; Mordenti, J.; Gjerstad, E.; Janc, J. Abstracts
of Papers, 229th ACS National Meeting, March 13–17,
2005; MEDI-250.
chain amide which is capable of a 180°-rotation due to a
hydrogen bond to Gln-143. This rotation repositions the
Lys-192 main chain carbonyl into the oxy-anion hole,
removing one of the essential catalytic features of the ac-
tive site. This main chain amide rotation is not likely in
the other human trypsin-family proteases since they do
not have an equivalent residue corresponding to the
Gln-143 of fVIIa. This unique fVIIa rearrangement pro-
vides the basis for a potential fVIIa selective interaction.
When compound 9 is modeled in the active site of fVIIa,
the distal 2-hydroxyphenyl is in a favorable orientation
to form a hydrogen bond between the rotated main
chain carbonyl of Lys-192 and the hydrogen of the phe-
nol.15 This potential hydrogen bond we hypothesize is
the basis for our observed increase in potency and selec-
tivity with compounds 9–14.
12. Hofslokken, N. U.; Skattebol, J. Acta Chem. Scand. 1999,
53(4), 258.
13. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem.
1995, 60(23), 7508.
14. Sichler, K.; Banner, D. W.; D’Arcy, A.; Hopfner, K. P.;
Huber, R.; Bode, W.; Kresse, G. B.; Kopetzki, E.;
Brandstetter, H. J. Mol. Biol. 2002, 322(3), 591.
In conclusion, a systematic SAR study on the distal ring
of 1 revealed that the scaffolds which incorporate a 2-hy-
droxy on the terminal aryl ring display significantly en-
hanced potency and selectivity. Accordingly, these
analogs are candidates for further development and
the results of such work will be disclosed in due time.
15. Shrader, W. D.; Costerison, J.; Hendrix, J.; Hu, H.;
Kolesnikov, A.; Kumar, V.; Leahy, E.; Rai, R.; Shaghafi,
M.; Ton, T.; Torkelson, S.; Wesson, K.; Young, W.B.;
Katz, B.A.; Sprengeler, P.A.; Yu, C.; Cabuslay, R.;
Gjerstad, E.; Janc, J.; Sanford, E. Abstracts of Papers,
229th ACS National Meeting, San Diego, CA, March 13-
17, 2005, MEDI-251.
Acknowledgments
The authors thank Dr. Michael J. Green for his kind
attention and useful comments regarding the prepara-
tion of the manuscript and support of this program.
16. General Inhibition Assays. Inhibitor potency measure-
ments were performed at room temperature using either a
Molecular Device’s SpectraMax 250 (absorbance assays)
or fMax (fluorescence assays) 96-well kinetic plate readers.
Reaction velocities were monitored at varying inhibitor
concentrations by following the hydrolysis of either para-
nitroanalide (A405 nm) or aminomethylcoumarin sub-
strates (ex355, em460). All substrates were added at
concentrations equal to or near their Km. All reactions
were performed in a total volume of 100 lL. Control
reactions in the absence of inhibitor were performed in
parallel. Factor VIIa: Factor VII (Enzyme Research) was
incubated at 7 nM together with recombinant soluble
human tissue factor (11 nM) with variable concentrations
of inhibitor in 50 mM Tris (pH 7.4), 150 mM NaCl,
5.0 mM CaCl2, 1.5 mM EDTA, 0.05% Tween 20, and 10%
DMSO. The reaction was initiated with the addition of
substrate, CH3SO2-D-CHA-But-Arg-pNA (Centerchem),
supplied at the Km (500 lM). The change in absorbance as
a function of time was monitored at 405 nm. Factor IIa
(Thrombin). Thrombin (Calbiochem) was incubated at
12.7 nM with variable concentrations of inhibitor in
50 mM Tris (pH 7.4), 150 mM NaCl, 5.0 mM CaCl2,
1 mM EDTA, 0.05% Tween 20, and 10% DMSO. The
reaction was initiated with the addition of substrate,
Tosyl-Gly-Pro-Lys-pNA (Centerchem), supplied at the Km
(25 lM). The change in absorbance as a function of time
was monitored at 405 nm. Factor Xa. Factor Xa (Haema-
tologic Technologies) was incubated at 2 nM with variable
concentrations of inhibitor in 50 mM Tris (pH 7.4),
150 mM NaCl, 5.0 mM CaCl2, 1.5 mM EDTA, 0.05%
Tween 20, and 10% DMSO. The reaction was initiated
with the addition of substrate, CH3OCO-D-Cha-Gly-Arg-
pNA (Centerchem), supplied at the Km (1.0 mM). The
change in absorbance as a function of time was monitored
at 405 nm. Trypsin. Trypsin (Athens Research Institute)
References and notes
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5. Young, W. B.; Kolesnikov, A.; Rai, R.; Sprengeler, P. A.;
Leahy, E. M.; Shrader, W. D.; Sangalang, J.; Burgess-
Henry, J.; Spencer, J.; Elrod, K.; Cregar, L. Bioorg. Med.
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6. (a) Groebke-Zbinden, K.; Banner, D. W.; Ackermann, J.;
D’Arcy, A.; Kirchhofer, D.; Ji, Y. H.; Tschopp, T. B.;
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7. Katz, B. A.; Elrod, K.; Luong, C.; Rice, M. J.; Mackman,
R. L.; Sprengeler, P. A.; Spencer, J.; Hataye, J.; Janc, J.;
Link, J.; Litvak, J.; Rai, R.; Rice, K.; Sideris, S.; Verner,
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W. D. Shrader et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1596–1600
was incubated at 10 nM with variable concentrations of
17. Ki apparent ðK0iÞ values were determined by a non-linear
least-squares regression fit of the experimentally derived
data to the Morrison equation for tight-binding inhibitors
as described (Kuzmic, P. et al., Anal Biochem, 2000,
281(1), 62). Enzyme and inhibitor were incubated 30-min
prior to initiation of reaction with the addition of
substrate.
inhibitor in 50 mM Tris (pH 7.4), 150 mM NaCl, 1.5 mM
EDTA, 0.05% Tween 20, and 10% DMSO. The reaction
was initiated with substrate, Tosyl-Gly-Pro-Lys-pNA
(Centerchem), supplied at the Km (25 lM). The change
in absorbance as a function of time was monitored at
405 nm.