Factor VIIa inhibitors: Chemical optimization, preclinical pharmacokinetics, pharmacodynamics, and efficacy in an arterial baboon thrombosis model

Article abstract of DOI:10.1016/j.bmcl.2005.12.059

Highly selective and potent factor VIIa-tissue factor (fVIIa · TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating

Full text of DOI:10.1016/j.bmcl.2005.12.059

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2037–2041
Factor VIIa inhibitors: Chemical optimization,
preclinical pharmacokinetics, pharmacodynamics, and efficacy
in an arterial baboon thrombosis model
Wendy B. Young,a,* Joyce Mordenti,a Steven Torkelson,a William D. Shrader,a
Aleksandr Kolesnikov,a Roopa Rai,a Liang Liu,a Huiyong Hu,a Ellen M. Leahy,a
Michael J. Green,a Paul A. Sprengeler,a Bradley A. Katz,a Christine Yu,a James W. Janc,a
Kyle C. Elrod,a Ulla M. Marzecb,ꢀ and Stephen R. Hansonb,ꢀ
aCelera Genomics, 180 Kimball Way, South San Francisco, CA 94080, USA
bEmory University, Atlanta, GA 30322, USA
Received 5 October 2005; revised 13 December 2005; accepted 15 December 2005
Available online 18 January 2006
Abstract—Highly selective and potent factor VIIa–tissue factor (fVIIaÆTF) complex inhibitors were generated through structure-
based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demon-
strating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition
in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentra-
tion–response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenoxꢀ) in the
baboon model are also presented.
ꢁ 2006 Elsevier Ltd. All rights reserved.
The activated factor VIIa–tissue factor (fVIIaÆTF) com-
plex has been shown to play a key role in blood coagu-
lation. Accordingly, much research has focused on the
generation of small molecule inhibitors of the fVIIaÆTF
complex as effective and potentially safer anti-thrombot-
ic agents.1 We have previously reported on the develop-
ment of small molecule inhibitors of the fVIIaÆTF
complex.1a,1b Herein, we describe the continued optimi-
zation of the 2-[5-(5-carbamimidoyl-1H-benzoimidazol-
To continue in the optimization of our fVIIaÆTF inhib-
itor series, we realized that the next goal was to improve
the ex vivo efficacy as monitored by the PT (prothrom-
bin time) assay.2 While working toward this goal, we
also aimed to maintain high selectivity (>1000-fold)
for fVIIa versus related enzymes such as factor Xa
(fXa) and factor IIa (fIIa).3 Although compound 1 dem-
onstrated excellent selectivity and potency for the puri-
fied enzyme system (fVIIaÆTF Ki = 0.004 lM), the
ex vivo efficacy in human plasma was rather poor
(2· PT = 10.2 lM). As it is generally well accepted that
lowering binding to serum albumin will improve the re-
sults in the PT assay, we aimed to generate more polar
and water-soluble analogs to decrease protein binding.
Additionally, further increases in potency for fVIIaÆTF
would lead to improved ex vivo efficacy.
2-yl)-50-fluoro-6,20-dihydroxy-biphenyl-3-yl]
succinic
acid scaffold (1)1b (Table 1). The preclinical pharmaco-
kinetics, pharmacodynamics, and in vivo efficacy of lead
analog 9 in a baboon arterial thrombosis model are the
focus of this communication.
A crystal structure of 1 in fVIIaÆTF revealed that the
trajectory off the para position to the C-ring hydroxyl
offers a large pocket that provides access to Lys60A
and Asp60, as well as the backbone of His57.1b It was
anticipated that we could improve binding to fVIIa by
incorporating moieties that bind to such residues. In this
process, incorporation of increasingly polar moieties
Keywords: Factor VIIa; fVIIa; Tissue factor; TF; Coagulation; Anti-
thrombotic; Baboon thrombosis model; Pharmacokinetics; Pharma-
codynamics; Serine protease; Thrombin; Factor Xa.
*
Corresponding author. E-mail: wendy.young@yahoo.com
Present address: Oregon Health and Science University, OGI School
of Science and Engineering, 20000 NW Walker Road, Beaverton, OR
97006, USA.
ꢀ
0960-894X/$ - see front matter ꢁ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.059

2038
W. B. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2037–2041
Table 1. SAR exploring the para position on the C-ring phenol
CO2H
HO2C
OH
B
N
H2N
HN
C
NH
OH
A
R
Compound
R
VIIaÆTF Ki (lM)
Selectivity for fVIIaÆTF
versus
2· PT (lM)
Xaa
IIab
55,500
1
2
3
4
5
6
7
8
9
F
CO2H
0.004
0.043
0.012
0.013
0.014
0.012
0.044
0.035
0.002
1800
300
10.2
8.2
6.2
5.4
3.6
6.4
4.9
2.9
1.9
3500
12,500
11,500
10,700
12,500
3400
CH2CO2H
CH2CH2CO2H
CONH2
125
350
1100
280
CH2CONH2
NHCONH2
CH2NH2
270
510
4200
>100,000
CH2NHCONH2
3600
a fXa Ki (lM)/fVIIaÆTF Ki (lM) = fold selectivity.
b fIIa Ki (lM)/fVIIaÆTF Ki (lM) = fold selectivity.
would also improve the ex vivo coagulation efficacy. To-
ward this end, we generated a series of analogs as depict-
ed in Table 1. Based upon our X-ray structures, the
succinic acid moiety extends into solvent, and the chiral-
ity was predicted to have minimal effect on binding
potency. All analogs were generated and evaluated as
a racemic mixture.
A crystal structure of 9 in fVIIaÆTF (Fig. 1)6 revealed
that the urea was indeed making contact with His57.
A hydrogen bond was observed between the His57 car-
bonyl and the proximal aminomethyl NH of the inhibi-
tor. The Lys60A donates an H-bond to the urea
carbonyl perhaps contributing to the strength of the
His57 interaction. This may account in part for the
selectivity of the urea since fVIIa is the only enzyme with
lysine in this position. The conformation of the carbonyl
of His57 is also a likely contributor since in many en-
zymes it is involved in an intra-enzyme hydrogen bond
or, as in trypsin, is pointed away from the ideal vector
required for this interaction. The distal nitrogen of the
urea is also involved in a hydrogen bond with Asp60.
Analogs 2–4 were generated to potentially interact with
Lys60A (or Lys192) via a salt bridge. While we did not
improve binding to the protein with these analogs, as
compared to 1, it was encouraging to see that the ex vivo
clotting values were improving. Similarly, extension of
an amide into this region, as in analogs 5–6, as well as
the urea 7 and benzyl amine 8 did not offer any binding
advantage, but the 2· PTs were again improved by the
increased polarity of these analogs. Next, we extended
the urea out by one methylene unit, producing 9, the
most potent compound in the series. Compound 9 was
two times more active than 1 and had an improved
2· PT of 1.9 lM.
The pharmacokinetic properties of 9 were evaluated
after intravenous (IV) administration to C57BL/6 fe-
male mice, male Sprague–Dawley rats, male New Zea-
land White rabbits, male Beagle dogs, and male
Compound 9 was evaluated against additional serine
proteases, as well as a panel of biochemical assays in or-
der to obtain a more complete selectivity profile. The
compound was highly selective (>1000-fold) versus fXa,
fIIa, trypsin, plasmin, uPa, aPC, tPa, and, hepsin and
showed moderate selectivity (P100-fold) against factors
IXa and XIa. It was essentially equipotent toward
plasma-kallikrein. The 2· aPTT of analog 9 in human
plasma is 0.8 lM. The potency of this analog in this assay
is attributed to its plasma kallikrein activity. Analog
9 was tested for inhibition against five cytochrome P450
isozymes (1A2, 2C9, 2C19, 2D6, and 3A4), and inhibition
was <40% at 10 lM. Additionally, analog 9 showed
no alerts in a preliminary screen against 26 primary
molecular targets4 and was negative in the Ames test.5
Figure 1. Crystal structure of analog 9 in fVIIaÆTF complex.

W. B. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2037–2041
2039
baboons.7 The pharmacokinetic parameters for plasma
2.0
clearance (CL), volume of distribution at steady state
(Vss), and mean residence time (MRT) are provided in
Table 2.
1.5
1.0
0.5
0.0
Preclinical pharmacokinetic data were extrapolated to
humans using an allometric equation.8 The allometric
coefficient (a) and exponent (b) for each pharmacokinet-
ic parameter are provided in Table 2, and the allometric
relationship for CL is illustrated in Figure 2. From this
analysis, plasma CL in a 70 kg human is predicted to be
147 mL/min, Vss is predicted to be 71 L (or ꢀ1 L/kg),
and MRT is predicted to be 491 min (or ꢀ8.2 h), which
suggests once-a-day dosing in humans is possible.
0
10
20
30
40
50
60
70
Time (min)
The anti-thrombotic and anti-hemostatic effects of
analog 9 were investigated in a baboon thrombosis
model.9 The ratio of the baboon-to-human prothrom-
bin time (PT) for analog 9 was 1.05, indicating that it
is near equipotent in both species. At single IV bolus
doses of 0.03, 0.1, and 0.5 mg/kg (Fig. 3), analog 9
inhibited platelet deposition at 1 h post dose by
14%, 61%, and 87%, respectively, versus control ani-
mals (Table 3). Fibrin deposition was progressively re-
duced by increasing doses of 9, similar to the results
for platelet deposition.
Figure 3. Effects of saline (control), analog 9, heparin, and enoxaparin
on platelet deposition in a baboon thrombosis model. Key: d = saline
control (n = 12); h = enoxaparin (n = 3); s = heparin (n = 3); analog
9: Ç = 0.03 mg/kg (n = 3); j = 0.1 mg/kg (n = 4); m = 0.5 mg/kg
(n = 3).
plus 50 U/kg/h IV infusion) and enoxaparin (2 mg/kg
subcutaneous administration) were also evaluated in this
model and found to decrease platelet deposition by 77%
and 62%, respectively, and to reduce fibrin deposition by
81% and 79%, respectively. These standard doses of hep-
arin and enoxaparin increased the BT values by 1.9- and
2.1-fold over control values, respectively.
Concurrently, PT values were prolonged 1.10-, 1.10-,
and 1.13-fold over control values, respectively, while
the bleeding time (BT) was slightly prolonged only at
the highest dose (from 3.0 to 5.2 min) (Table 3). In com-
parison, standard doses of heparin (50 U/kg IV bolus
Pharmacodynamic modeling10 was used to determine
the dose (milligrams per kilogram) and plasma concen-
tration (micromolar) of analog 9 that were required to
produce 50% of the maximum effect on platelets at
60 min post dose, that is, ED50 and EC50, respectively.
From this evaluation, the ED50 was 0.06 mg/kg, and
the EC50 was 0.17 lM (Fig. 4).
Table 2. Pharmacokinetics of 9 in preclinical species
Species
Weight (kg) CL (mL/min) Vss (mL) MRT (min)
Mouse
Rat
0.025
0.30
2.43
0.103
0.81
6.10
2.43
55.2
1080
23.5
69.0
Rabbit
Dog
Baboon
182
To the best of our knowledge, this is the first report of a
single IV bolus injection of a small molecule fVIIaÆTF
inhibitor to show complete inhibition of thrombus for-
mation in a baboon model. Analog 9 appears to be
approximately 50-fold more efficacious per weight basis
than a previously studied small molecule inhibitor to
fVIIaÆTF, which required a IV bolus plus continuous
IV infusion during the study period, using the same
thrombosis model.1d There are reports of other selective
small molecule fVIIaÆTF inhibitors interrupting throm-
bosis in non-primate animal models, but these studies
also required continuous infusion of drug.1p
11.3
11.5
30.5
26.5
6750
5854
224
232
Allometric
Equation
a
b
2.84
0.93
285
102
1.3
0.37
1000
100
10
Predicted
Human
Baboon
Dog
Rabbit
All analogs were generated from compound 10 as out-
lined in Scheme 1. Starting from the commercially avail-
able 4-iodoanisole, the succinate moiety was inserted via
a Heck reaction with subsequent catalytic hydrogena-
tion to give 11. This intermediate was then prepared
for magnesium-directed ortho formylation by removing
the methyl ether followed by re-esterification of the acid
functions to produce 12. After formylation, bromina-
tion, and boron ester formation, 13 underwent a Suzuki
reaction with 14 to give product 15. The remaining alde-
hyde group was the site for an oxidative-cyclization
Rat
1
Mouse
0.1
0.01
0.01
0.1
1
10
100
Body Weight (kg)
Figure 2. Interspecies scaling of 9 in mouse, rat, rabbit, dog, and
baboon to predict the clearance in human.8

2040
W. B. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2037–2041
Table 3. Inhibition of platelet and fibrin deposition, prothrombin time (PT), and bleeding times (BT) in baboon thrombosis model
Inhibitor (n/group) Dose and route
Platelet deposition SDa Fibrin deposition SDa PT SDa (sec) BT SDb (min)
platelets · 109
mg fibrin/graft
(% reduction vs control) (% reduction vs control)
Saline (12)
0 (control) IV
0.03 mg/kg IV
0.1 mg/kg IV
0.5 mg/kg IV
1.97 0.58
1.77 0.71
10.3 0.8
11.3 0.7
11.3 0.5
11.6 0.6
NA
3.0 0.8
3.2 0.6
3.8 1.0
5.2 1.8*
5.7 1.3*
6.3 5.0*
Analog 9 (3)
Analog 9 (4)
Analog 9 (3)
Heparinc(3)
Enoxaparind (3)
1.68 0.24 (14)
0.77 0.47* (61)
0.25 0.27* (87)
1.41 0.30 (20)
0.63 0.13* (64)
0.25 0.24* (86)
0.34 0.28* (81)
0.38 0.39* (79)
50 U/kg bolus + 50 U/kg/h IV 0.45 0.27* (77)
2 mg/kg SC
0.74 0.43* (62)
NA
NA = not available.
a 60 min after initiation of thrombosis.
b 30 min after initiation of thrombosis.
c American Pharmaceutical Partners, Schaunburg, IL.
d Lovenoxꢀ (Sanofi-Aventis SA).
* p versus control <0.05.
CO2Me
100
80
CO2Me
I
MeO2C
MeO2C
a, b
e, f
c, d
90%
83%
95%
OMe
10
OMe
11
OH
12
60
Sigmoid Emax Conc-Response Model
CN
40
20
0
CO2Me
Br
Emax
EC50
γ
99%
CO2Me
MeO2C
14
Br
MeO2C
CN
0.17µM
OMEM
i
H
2.2
H
O
g, h
O
OH
OMEM
15
OMEM
~40%
0
0.1
0.2
0.3
0.4
0.5
0.6
13
Plasma Concentration (µM)
Figure 4. Pharmacodynamic model showing the plasma concentration
of 9 versus reduction in platelet deposition in a baboon thrombosis
model.10
CO2H
HO2C
CN
N
l, m
40%
j, k
HN
9
OH
HO
NH
16
60%
H2N
reaction to install the 5-amidinobenzimidazole moiety
followed by the removal of the protecting groups. Final-
ly, catalytic reduction of the nitrile group gave the cor-
responding amine, which was reacted with potassium
cyanate to afford the crude product, which was purified
by preparative HPLC to yield 9 as a racemic mixture.
Scheme 1. Reagents and conditions: (a) dimethylfumarate, PdCl2,
NEt3, MeCN; (b) Pd(OH)2/H2, MeOH; (c) HBr, reflux; (d) SOCl2,
MeOH; (e) paraformaldehyde, MgCl2, TEA, MeCN; (f) N-bromosuc-
cinimide, DMF; (g) MEM chloride, DIPEA, CH2Cl2; (h) KOAc,
bis(pinacolato)diboron, dichloro[1,10-bis-(diphenylphosphino)-ferro-
cene]palladiumII, dioxane; (i) Pd(PPh3)4, Na2CO3, toluene; (j) 3,4-
diaminobenzamidine–HCl, O2, EtOH; (k) 2 M HCl in dioxane then
1 M HCl at reflux; (l) H2/Pd(OH)2, 0.5 M HCl; (m) KOCN, water.
In summary, highly potent and selective inhibitors of the
fVIIaÆTF complex were generated by structure-based
design. The pharmacokinetics after IV dosing of lead
analog 9 were evaluated in mouse, rat, rabbit, dog, mon-
key, and baboon. Allometric scaling predicts it could be
suitable for once-a-day dosing in human. Compound 9
was found to be efficacious in an arterial baboon throm-
bosis model with an ED50 of 0.06 mg/kg and an EC50 of
0.17 lM after IV bolus dosing. At 0.5 mg/kg, it was
more efficacious than standard doses of heparin and
enoxaparin. Additionally, 9 did not impair hemostasis
at the lower doses and was comparable to heparin and
enoxaparin at the 0.5 mg/kg dose. These encouraging
in vivo data provide additional support to our desire
to develop fVIIaÆTF inhibitors for improved anticoagu-
lant therapy. Further advances in this chemical series
will be reported in due time.
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2. Bajaj,S.P.;Joist,J.H.Semin.Thromb.Hemost.1999,25,407.
3. Inhibition assays for factor Xa and thrombin were
performed as described ( Cregar, L.; Elrod, K. C.;
Putnam, D.; Moore, W. R. Arch. Biochem. Biophys.
1999, 366, 125, ) with the pH adjusted to 7.4. Factor VIIa
assay was performed and analyzed as above using 7 nM
VIIa (Enzyme Research) and using CH3SO2-D-CHA-But-
Arg-pNA (Centerchem) as the substrate. The buffer for
the factor VIIa assay was supplemented with 11 nM
relipidated tissue factor and 5 mM CaCl2. Coagulation
experiments were carried out in human plasma using a
Beckman Coulter ACL100 in accordance with the man-
ufacturer’s instructions.
6. The PDB access code for the X-ray coordinates is 2B7D.
7. Plasma concentrations of 9 were assayed by LC/MS/MS.
Pharmacokinetic data were analyzed by WinNonlin-Pro
(Pharsight Corp.), using a two-compartment model.
8. The allometric equation is written asY = aWb, where Y is
the pharmacokinetic parameter of interest, W is the body
weight, a is the y-intercept, and b is the slope obtained
from the plot of logY versus logW (as illustrated in Fig. 2
for clearance parameter). See Mordenti, J. J. Pharm. Sci.
1986, 75, 1028, for details.
9. The animal experiments were approved by the Institu-
tional Animal Care and Use Committee of Emory
University in accordance with the United States federal
guidelines. Thrombosis was initiated at a predetermined
time after drug administration by interposing a thrombo-
genic device into a chronic femoral arterio-venous (A-V)
shunt in conscious non-anticoagulated baboons. For
analog 9 and heparin, thrombosis was initiated 5 min
after IV bolus dosing (in the heparin group, an infusion
continued throughout the experiment); whereas for enox-
aparin, thrombosis was initiated 90 min after SC dosing.
The thrombogenic device consisted of a 2 cm segment of
porous expanded (poly)tetrafluoroethylene vascular graft
(ePTFE, 4 mm id) filled with relipidated tissue factor.
Blood flow was maintained at 100 mL/min, generating a
wall shear rate of 265/s. Thrombus was monitored
dynamically for 60 min by gamma camera imaging of
autologous 111-indium (111-In)-labeled platelets. Images
were acquired at 5 min intervals. Total platelet deposition
was calculated at each time point by dividing the throm-
bus radioactivity (cpm) by the blood specific activity (cpm/
mL) and multiplying by the blood platelet count (platelets/
mL). Fibrin deposition was monitored by injecting a trace
amount of 125-iodine (125-I)-labeled autologous fibrino-
gen 10 min before the start of the study, then saving the
thrombus that formed in the graft for 30 days to permit
decay of 111-In activity. The 125-I activity was measured
in a gamma well counter. 125-I activity was converted to
total deposited fibrin (mg) using values for the specific
activity of plasma-clottable fibrinogen and the plasma
fibrinogen concentration that were obtained at the time of
the study. Template bleeding times (BT) were measured on
a shaved forearm prior to dosing and at 30 min after
initiation of thrombosis. The PT and aPTT were moni-
tored throughout the study.
10. Dose (mg/kg) and plasma concentration (micromolar)
versus response data were evaluated with a sigmoid Emax
pharmacodynamic model, using WinNonlin-Pro (Phar-
sight Corp.). For plasma concentrations, the relationship
has the general form E ¼ ðEmax ꢁ CcÞ=ðCc þ EC5c0Þ, where
E = inhibition of platelet deposition (%) relative to control
at 60 min post dose, Emax = maximum effect (%), C = plas-
ma concentration of 9 at 60 min post dose, EC50 = plasma
concentration of 9 required to produce 50% of the
maximum effect, and c = sigmoidicity or shape parameter.
When dose (D) is evaluated, C is replaced by D (mg/kg) in
the equation.
4. Analog 9 was evaluated in the HitProfilingScreenTM at
MDS Pharma Services, Bothell, WA 98021.
5. Analog 9 was evaluated in the Ames Salmonella Mutage-
nicity Test with two mutant strains of Salmonella typhimu-
rium LT2 (i.e., TA98 and TA100) with and without S9
activation at MDS Pharma Services, Taipei, Taiwan.