Efforts toward oral bioavailability in factor VIIa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.04.018

Efforts toward developing orally bioavailable factor VIIa inhibitors starting from parenteral lead compound 1 are described. SAR resulted in improved physicochemical properties, leading to enhanced oral absorption in rat.

Full text of DOI:10.1016/j.bmcl.2006.04.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3829–3832
Efforts toward oral bioavailability in factor VIIa inhibitors
Dange Vijaykumar,* Roopa Rai, Michael Shaghafi, Tony Ton, Steve Torkelson,
Ellen M. Leahy, Jennifer R. Riggs, Huiyong Hu, Paul A. Sprengeler, William D. Shrader,
Colin O’Bryan, Ronnell Cabuslay, Ellen Sanford, Erik Gjerstadt, Liang Liu,
Juthamas Sukbuntherng and Wendy B. Young
Celera, 180 Kimball Way, South San Francisco, CA 94979, USA
Received 20 December 2005; revised 11 April 2006; accepted 11 April 2006
Available online 2 May 2006
Abstract—Efforts toward developing orally bioavailable factor VIIa inhibitors starting from parenteral lead compound 1 are
described. SAR resulted in improved physicochemical properties, leading to enhanced oral absorption in rat.
ꢀ 2006 Elsevier Ltd. All rights reserved.
There is increasing interest in developing oral anti-coag-
ulants with improved therapeutic efficacy over the exist-
ing standard of care.1 Our approach toward developing
an anticoagulant is the direct inhibition of factor VIIa
(fVIIa), a key enzyme in the coagulation cascade.2,3 In
this communication, we disclose our efforts to improve
the oral bioavailability of our lead fVIIa inhibitors by
modifying its physicochemical properties.
a
S1
S2
Lys192
+
Lys60a
+
NH3
–
CO2
O–
Asp
189
NH2
–
Asp60
O
CO2
+H3N
O
N
N
–O
NH2
H2
N
H
O
His57
O
HO
HO
NH
1
OH
His57
We have reported the development of potent and selec-
tive small-molecule inhibitors of the factor VIIa/tissue-
factor (fVIIa/TF) complex.3 An example, compound 1,
is efficacious after bolus intravenous (iv) dosing in a ba-
boon model of arterial thrombosis.3 Expectedly, com-
Ser195
b
fVIIa Selectivity against
fVIIa5
Ki(μM)
2xPT6
pounds similar to
1
with amidine and diacid
PSA
(oA2)
Thrombin
100,000
fXa
Trypsin
10,000
MW
532
(μM)
1.9
functionalities had limited oral absorption when dosed
in rats. We recognized the need to modify the properties
of this class of compounds to lead us toward oral bio-
availability.4 Of concern was the charged nature of the
amidine and diacid functionalities. Combined with the
urea, these lead to a high polar surface area (PSA) and
molecular weight (MW).
0.001
3,600
248
Figure 1. (a) Representation of key interactions of 1 with fVIIa. (b)
In vitro profile and calculated physicochemical parameters for
compound 1.
Asp189 in the S1 pocket. A prodruging approach
(as the hydroxy amidine) would be employed to neutral-
ize the charge and help improve absorption. The success
of this approach has been demonstrated in the develop-
ment of the thrombin inhibitor Ximelagatran.7 The
amidine, in conjunction with the imidazole NH and
the 2-phenolic OH, is integral to the binding of this class
of inhibitors to trypsin-like enzymes.8 In addition, the
distal 20-phenol offers further fVIIa potency and
We undertook an analysis of the role of each part of
compound 1 in the potency and selectivity profile, as
outlined in Figure 1a. This analysis is based on a crystal
structure of 1 in fVIIa.3 The amidine interacts with
Keywords: Factor VIIa inhibitor; Oral bioavailability; Parenteral lead;
Selectivity; Serine protease; Polar surface area.
*
Corresponding author. E-mail: dvijayku@hotmail.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.018

3830
D. Vijaykumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3829–3832
selectivity against other trypsin-like enzymes.9 There-
fore, our plan was to preserve these interactions, while
exploring the possibility of replacing the succinic acid
and urea substituents. The goal was to considerably
reduce the MW and PSA.
synthesized analogs suffered from rapid clearance. After
iv dosing in rat, compound 1 has a mean residence time
(MRT) of greater than 1 h,3 while the analogs 2–5 had
MRTs of less than 20 min (data not shown). Therefore,
while removing the succinic acid from compound 1
allowed us to considerably reduce the PSA and molecu-
lar weight, we were left with the need for further
optimization.
The succinic acid at the 5-position improves fVIIa
potency by interacting with Lys192, while enhancing
thrombin selectivity through negative interactions with
Glu192. In our earliest reports of fVIIa inhibitors, the
50-acidic group played a very important role in potency
and selectivity gains.2a More importantly, we have uti-
lized this position as a handle to modulate pharmacoki-
netic properties.10 With the discovery of the distal urea
functionality,3 we reasoned that the 5-acidic group
may be expendable. Numerous 5-substituted analogs
were synthesized and evaluated, of which, selected
examples are shown in Table 1. We noted that replacing
the succinic acid with small non-polar substituents such
as –CH3, –F, and –H had minimal effect on the in vitro
parameters.5 The fVIIa potency was unaffected by these
changes and the selectivity profile remained impressive.
Indeed, the crystal structure of 1 in fVIIa had revealed
numerous other points of interaction with the enzyme,
which are most likely maintained in this new set of com-
pounds. It was gratifying that the in vitro efficacy, as
measured by the 2 · PT6 assay, did not change. However,
as suspected, the pharmacokinetic profile of the newly
Next, we examined the role of the urea functionality on
the distal aryl ring. As depicted in Figure 1a, it makes
specific contacts with His57, Asp60, and Lys60a.
Among numerous analogs made, we found that amides
which lost the interaction with Asp60 but preserved the
‘CH2–NHC(O)’ part of the urea were reasonably well
tolerated and retained desirable in vitro characteristics.
Selected examples (6–10) of analogs replacing the urea
are shown in Table 2.
These include alkyl amides (7 and 10), amides incorpo-
rating polar groups (6 and 8) as well as a disubstituted
urea 9. These analogs were prepared with an indole
replacing the benzimidazole ring. Based on previous
experience with this class of compounds,2 we were con-
fident that this change would be well tolerated, while
providing another opportunity to lower the PSA. The
loss of the Asp60 interaction in the amide series is
reflected in an overall loss of selectivity, particularly
Table 1. SAR at the 50-position
NH
R
N
H2N
O
N
H
HO
HO
HN
NH2
Compound
R
fVIIa Ki5 (lM)
2 · PT6 (lM)
Selectivity against
PSAa (ꢁA2)
MW
Thrombin
fXa
Trypsin
1
2
3
4
5
CH(COOH)CH2COOH
CH2COOH
0.001
0.014
0.004
0.005
0.014
1.9
—
100,000
10,700
10,900
11,700
6923
3600
235
303
404
207
11,333
328
584
250
213
175
175
175
532
475
431
435
417
CH3
F
2.0
2.5
2.3
744
276
H
a Polar surface area.
Table 2. SAR to replace the urea
NH
H2N
N
R
H
HO
HN
HO
O
Compound
R
fVIIa Ki (lM)
2 · PT (lM)
Selectivity against
PSA (ꢁA2)
MRTa (min)
Thrombin
fXa
Trypsin
6
7
8
CH2OH
CH3
CH2CH2CH2N(Me)2
0.003
0.12
0.016
0.9
—
1233
1250
3375
60
6
400
142
181
157
135
138
117
—
0.9
37
50
9
0.017
0.013
2.0
1.4
3117
7222
28
194
237
147
135
—
89
N
O
10
CH2(CH2)3CH3
107
a Mean residence time.

D. Vijaykumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3829–3832
3831
against fXa. Selected compounds were dosed iv (intrave-
a, b, c
O
H
nous) to male Sprague–Dawley rats and the pharmaco-
kinetic parameters compared. The MRTs are listed in
Table 2. Our SAR leads us to the hexanoyl amide analog
10 which had the best combination of lowered PSA and
MW as well as in vitro potency, efficacy, and selectivity.
In addition, this lead offered pharmacokinetic parame-
ters which warranted further evaluation. Since we were
aware, based on our previous study,11 that even simple
amidino compounds (without distal ring substituents)
in this series had poor oral absorption, we decided to
convert amidino group in 10 to a neutral hydroxyami-
dine in order to improve chances of oral absorption.
B
Br
OMEM
O
O
OMEM
OH
12
H
f
13
O
OMEM
NHBoc
CN
OH
NHBoc
16
b, d, e
Br
Br
g, h, i
OMEM
14
15
5
Scheme 1. Synthesis of amidine 5. Reagents and conditions: (a)
MgCl2, (HCHO)n, NEt3, CH3CN, reflux; (b) MEMCl, Hunig’s base,
DCM, rt; (c) bis-pinacolato diborane, Pd(II), dioxane, reflux, K2CO3;
(d) diborane, THF reflux; (e) (Boc)2, NEt3 THF; (f) Pd(PPh3)4, 2 N
K2CO3, toluene, reflux; (g) 3,4-diamino-benzamidine, DMF, 1,4-
benzoquinone, 60 ꢁC; (h) 4 M HCl in dioxane, rt; (i) KOCN, MeOH rt.
Compound 11, the hydroxyamidine prodrug of 10,
was dosed orally at 10 mg/kg to male Sprague–Dawley
rats with jugular vein and portal vein cannulae.12
Monitoring the blood levels of both prodrug 11 and
the parent compound 10 revealed that while the oral
bioavailability of 11 was 11%, that of the parent
was negligible13 (Table 3).
OMEM
NC
I
Scheme 1 outlines the synthesis of benzimidazole com-
pound 5. The commercially available bromo phenol 12
was selectively ortho formylated with paraformaldehyde
and magnesium chloride as reported previously.14 The
bromo salicylaldehyde intermediate thus obtained was
protected as its MEM ether and the aryl bromide con-
verted to the pinacol boronate ester 13 using bis-(pina-
colato)diboron with PdCl2(dppf) as a catalyst.15 Aryl
bromide 14 was obtained from 15 in three steps via phe-
nol protection as the MEM ether, reduction of the nitrile
followed by protection of the benzyl amine as its Boc
derivative. Suzuki coupling of 13 with aryl bromide 14
furnished the biaryl aldehyde 16. Oxidative condensa-
tion of the aldehyde 16 with 3,4-diamino-benzamidine,9
removal of protecting groups (MEM ethers and Boc)
followed by treatment of resultant benzyl amine with
potassium cyanate gave urea 5. Other benzimidazole
derivatives 2–4 were prepared in an analogous manner
a
16
NHMs
OMEM
17
18
NHBoc
N
b
c, d
N
H
NHBoc
MEMO
MEMO
19
N
e
N
H
HO
R
NHCOC5H11
HO
20
N
H2N
N
H
starting from
salicylaldehyde.
the
appropriately
substituted
HO
NHCOC5H11
R1 = OH 11
R1 = H 10
HO
f
To prepare indole derivative 10 and hydroxy amidine11
(Scheme 2), aldehyde 16 was converted to alkyne
17 using dimethyl-1-diazo-2-oxopropylphosphonate.16
Palladium-mediated coupling of the alkyne 17 with
N-(4-cyano-2-iodo-phenyl)-methanesulfonamide 1817 fol-
lowed by alkaline hydrolysis of the mesylate gave
Scheme 2. Synthesis of amidine 10 and hydroxyamidine 11. Reagents
and conditions: (a) (1-diazo-2-oxo-propyl)-phosphonic acid dimethyl
ester, K2CO3, MeOH, rt; (b) i—Pd(PPh3)2Cl2, CuI, NEt3, THF, 60 ꢁC;
ii—50% aq NaOH, MeOH 60 ꢁC; (c) 4 M HCl in dioxane; (d) hexanoyl
chloride in DCM and NEt3, rt; (e) NH2OH, EtOH, reflux; (f) i—Ac2O,
AcOH, rt; ii—Pd(OH)2 on C, MeOH, atm of H2.
Table 3. Pharmacokinetics, bioconversion, and oral bioavailability of prodrug 11 following iv and oral administration in rats
PK parameter12 (po 10 mg/kg, n = 3)
Prodrug (11)
Parent (10)
PVCa
JVCb
1.73
PVCa
JVCb
Cmax (lM)
Tmax (min)
1.31
31
0.014
40
0.012
30
30
144
54
AUC (lM min)
Terminal t1/2 (min)
Oral absorption (%)
Oral bioavailability (%)
122
49
0.88
26
0.73
37
9
11
—
Negligible
a PVC, blood samples collected from portal vein catheter.
b JVC, blood samples collected from jugular vein catheter.

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D. Vijaykumar et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3829–3832
Marzec, U. M.; Hanson, S. R. Bioorg. Med. Chem. Lett.
2006, 16, 2037.
triaryl compound 19. Removal of protecting groups
(MEM and Boc) with 4 M HCl in dioxane, followed
by treatment of the resulting benzyl amine with hexa-
noyl chloride, generated amide 20. Hydroxyamidine
prodrug 11 was generated by heating amide 20 with
aqueous hydroxylamine in ethanol. To prepare amidine
10, hydroxyamidine 11 was acylated and reduced under
hydrogenation conditions. Other indoles (6–9) were pre-
pared in an analogous manner.
4. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P.
J. Adv. Drug Delivery Rev. (Netherlands) 2001, 46, 3.
5. Inhibition assays for fXa 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. The trypsin and fVIIa assays were
performed and analyzed as in the above reference with the
following additional details. FVIIa (Enzyme Research) was
incubated at 7 nM and CH3SO2–D-CHA–But-Arg–pNA
(Centerchem) was used as the substrate. The buffer for the
fVIIa assay was supplemented with 11 nM relipidated tissue
factor and 5 mM CaCl2. Trypsin (Athens Research Insti-
tute) was incubated at 10 nM with variable concentrations
of 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).
We have described SAR that has allowed us to evolve
from compound 1, which is suitable for development
as a parenteral anticoagulant agent, to lead compound
10, which may be explored as an orally administered
anticoagulant. We demonstrated that it is possible to
considerably reduce MW and PSA of 1, while maintain-
ing suitable in vitro characteristics. The strategy of mit-
igating the charge of the amidine in the form of a
prodrug was successful in improving oral bioavailability
of the prodrug. Our studies with the hydroxyamidine
prodrug 11 revealed that it is not reduced to the parent
10 in vivo in rat. Efforts directed at exploring other pro-
drugs which will be cleaved in vivo and offer improved
absorption will be the subject of future publications.
6. Bajaj, S. P.; Joist, J. H. Semin. Thromb. Hemost. 1999, 25,
407.
7. Eriksson, U. G.; Bredberg, U.; Hoffmann, K.; Thuresson,
´
A.; Gabrielsson, M.; Ericsson, H.; Ahnoff, M.; Gislen, K.;
Fager, G.; Gustafsson, D. Drug Metab. Dispos. 2003, 31,
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8. 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,
E.; Young, W. J. Mol. Biol. 2001, 307, 1451.
9. Shrader, W. D.; Kolesnikov, A.; Burgess-Henry, J.; Rai,
R.; Hu, H.; Torkelson, S.; Young, W. B.; Sprengeler, P.
A.; Yu, C.; Katz, B.; Cabuslay, R.; Gjerstad, E.; Janc, J.
Bioorg. Med. Chem. Lett. 2006, 16, 1596.
Acknowledgments
The authors thank Michael J. Green, Robert Booth,
Peter Young, Jim Yee, James Janc, and Joyce Mordenti
for scientific input and support.
10. 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, San Diego, CA,
United States, March 13–17, 2005; MEDI-250.
11. Riggs, J. R.; Kolesnikov, A.; Hendrix, J.; Young, W. B.;
Shrader, W. D.; Vijaykumar, D.; Stephens, R.; Liu, L.;
Pan, L.; Mordenti, J.; Green, M. J.; Sukbuntherng, J.
Bioorg. Med. Chem. Lett. 2006, 16, 2224.
12. Plasma concentrations of prodrugs and parent compounds
were determined by LC/MS/MS. Pharmacokinetic data
were analyzed by WinNonlin-Pro (Pharsight Corp.), using
compartmental and non-compartmental analysis for iv
and po data, respectively. Oral absorption (Abs) and
bioavailability (F) in rats were evaluated in portal vein
(pv) and jugular vein (jv) cannulated animals and
calculated from dose-normalized area-under-the-curve
(AUC) values as follows: Abs = AUCpo,pv/AUCiv,jv and
F = AUCpo,jv/AUCiv,jv. Prodrug conversion was calculat-
ed by dividing the dose-normalized AUC of the parent
after iv prodrug administration by the AUC of the parent
after iv parent administration.
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