Development of a novel tricyclic class of potent and selective FIXa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2015.07.078

Using structure based drug design, a novel class of potent coagulation factor IXa (FIXa) inhibitors was designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds were evaluated in rat IV/PO pharmacokinetic (PK) studies and demonstrated desirable oral PK profiles. Finally, the pharmacodynamics (PD) of this class of molecules were evaluated in thrombin generation assay (TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a FIXa inhibitor.

Full text of DOI:10.1016/j.bmcl.2015.07.078

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5437–5443
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a novel tricyclic class of potent and selective FIXa
inhibitors
a,
⇑
b
c
a
a
b
Dongfang Meng a , Patrick Andre , Thomas J. Bateman , Richard Berger , Yi-Heng Chen , Kunal Desai ,
e
d
a
d
a
Sunita Dewnani , Kenneth Ellsworth , Daming Feng , Wayne M. Geissler , Liangqin Guo , Alan Hruza ,
Tianying Jian , Hong Li , Joe Metzger , Dann L. Parker , Paul Reichert , Edward C. Sherer ,
Cameron J. Smith , Lisa M. Sonatore , Richard Tschirret-Guth , Jane Wu , Jiayi Xu , Ting Zhang ,
Louis-Charles Campeau , Robert Orr , Marc Poirier , Jamie McCabe-Dunn , Kazuto Araki ,
a
a
b
a
e
f
a
d
c
a
a
a
g
g
g
g
h
h
h
h
a
Teruyuki Nishimura , Isao Sakurada , Tomokazu Hirabayashi , Harold B. Wood
a
Department of Discovery Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Cardiometabolic Disease, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Pharmacokinetics, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Structural Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Chemistry Modeling and Informatics, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Department of Process Chemistry, Merck Research Laboratories, Merck & Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
b
c
d
e
f
g
h
Discovery Research, Mochida Pharmaceutical Co., LTD, 7, Yotsuya 1-Chome, Shinjuku-ku, Tokyo 160-8515, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Using structure based drug design, a novel class of potent coagulation factor IXa (FIXa) inhibitors was
designed and synthesized. High selectivity over FXa inhibition was achieved. Selected compounds were
evaluated in rat IV/PO pharmacokinetic (PK) studies and demonstrated desirable oral PK profiles. Finally,
the pharmacodynamics (PD) of this class of molecules were evaluated in thrombin generation assay
Received 21 May 2015
Revised 20 July 2015
Accepted 23 July 2015
Available online 31 July 2015
(TGA) in Corn Trypsin Inhibitor (CTI) citrated human plasma and demonstrated characteristics of a
FIXa inhibitor.
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
Benzimidazole
Quninazolinone
FIXa inhibitor
TGA
Structure based drug design
1
2
Recently, apixiban and rivaroxiban (Factor Xa inhibitors) and
Herein, we continue the discussion on further SAR studies on
the cyclic series, leading to potent and selective FIXa inhibitors
with desirable PK profiles and demonstrated efficacy in PD
assay. While we will discuss SAR to achieve selectivity over
FXa, it should be noted these cyclic analogs were essentially
devoid of activity against the other enzymes in the coagulation
cascade.
3
dabigatran (direct thrombin (factor IIa) inhibitor) have been intro-
duced to the market as novel anticoagulant therapies for stroke
prevention in atrial fibrillation (SPAF). Inhibition of Factors which
resides on the intrinsic pathway is hypothesized to be an attractive
4
target for SPAF with a potentially diminished bleeding risk. Inhi-
bition of FIXa, right above Factor Xa on the ‘intrinsic’ pathway of
the coagulation propagation cascade, may lead to better safety
margin clinically with similar efficacy.5
Several groups published their finding on potent and selective
FIXa inhibitors. Previously, we have reported our early hit-to-lead
The final steps of general syntheses of analogs of compound 1
are described in Scheme 1. Starting from acid 3, Curtius rearrange-
ment and deprotection gave amine 4. Amide coupling with tail
piece 5 afforded amide 6, which was subjected to chiral separation
to give enantiomerically pure FIXa inhibitor 7. Analogs shown in
Tables 1–4 were synthesized using similar approaches described
–7
8
efforts leading to 2 classes of FIXa inhibitors (1 and 2 in Fig. 1) with
9
moderate potency and selectivity over the FXa, as well as the SAR
of the acyclic lead series (1).10
above.
9,10
One of the distinguishing features of the cyclic series is the
novel pyrazolopyridine moiety. To explore the SAR at P1, the
syntheses of different P1 moieties are shown in Schemes 2–4.
⇑
Corresponding author. Tel.: +1 732 594 3169; fax: +1 732 594 2840.
E-mail address: dongfang-meng@merck.com (D. Meng).
http://dx.doi.org/10.1016/j.bmcl.2015.07.078
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

5
438
D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
N
N
OH
O
F
N
H
N
HN
Me
N
N
NH
O
Me
N
Me
N
Me
2
hFIXa Ki 2 mM
hFXa Ki >62.5 mM
1
hFIXa Ki 9 mM
hFXa Ki >62.5 mM
Figure 1. Hits of interest from high throughput screening campaign, acyclic 1 and cyclic 2.
Cl
Ar
c
O
O
Cl
5
R
R
a,b
Ar
Ar
NH2
OH
X
R'
H
H
4
3
R'
X
R'
X
R H
R H
N
Ar
R"
d
Ar
N
Ar
H
H
O
R"
O
6
7
Scheme 1. Reagents and conditions: (a) DPPA, TEA, Toluene, BnOH or t-BuOH; (b) Pd/C or TFA; (c) HOBT, EDC, DMF, rt; (d) Chiral separation on.
Table 2
Table 1
Cl
Cl
R H
N
R H
N
N
Ar
N
N
N
N
H
N
N
N
O
O
X
X
R
X
Compd
hFIXa K
i
(nM)
i
hFXa K (nM)
Tricyclic
X
Compd hFIXa K
i
hFXa K
i
(
nM)
(nM)
Me
Cl
Cl
53
59
10.2
13.8
2145
1006
OH
OH
OH
Cl 52
12.1
2259
Cl
H
60
61
12.5
6.9
258
662
N
N
NH
Cl 53
10.2
6.9
2145
662
N
N
H
62
63
64
65
2.1
3.0
6.6
3.1
605
378
NH
O
H
N
N
Ph
H
H
54
55
NH
F
Cl
Cl
246
N
Ph
5.9
2036
28890
Cl
CF3
N
NH
1682
Cl 56
267.6
11.7
Cl
Cl
66
67
4.5
4.2
1553
1110
HN
NH
F
F
Cl 57
10710
4227
N
NH
NH
HOOC
HOOC
Cl
Cl
68
69
10.5
5.0
1540
743
F
N
N
N
N
H
58
2.7

D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
5439
Table 3
Table 4
Cl
R H
N
N
Het
H
N
X
N
O
N
X
Het
O
Y
R
R/Het
X
Compd
hFIXa K
i
(nM)
i
hFXa K (nM)
R/Het
X/Y
Compd
hFIXa K
i
(nM)
i
hFXa K (nM)
N
N
H
62
2.1
1.8
605
N
N
N
N
H
N
N
Cl/C
73
34.8
—
N
N
N
N
N
N
Cl
Cl
70
71
590
Me
N
N
Cl/C
Cl/C
Cl/C
Cl/C
74
75
76
77
6.3
8.0
8.4
5.1
1748
1416
3283
1082
N
Me
Me
10.2
2145
N
N
N
N
N
N
H
72
10.5
518
CF3
N
N
COOH
CN
N
N
In Scheme 2, the pyrazolopyridine synthesis is described. Start-
ing from ketone 8, Pd-catalyzed -arylation with pyridine halide 9
led to 10. Compound 10 was first condensed with hydroxylamine
O
N
N
a
Cl/C
78
1.2
193
NH2
NOH
to form oxime 11, which was then converted to 12 following
1
1
N
N
Cl/C
Cl/C
Cl/C
Cl/C
Cl/C
Cl/C
79
80
81
82
83
84
2.4
2.9
0.8
2.4
1.6
5.5
397
known procedure. The unmasked ketone 13 was treated with
desired Grignard reagent to form alcohol 14, then further con-
verted to amide 15 through a Ritter reaction. Hydrolysis of 15 gave
amine 16.
N
N
N
N
>5,000
337
COOH
COOH
In Scheme 3, a tricyclic imidazole synthesis is described. Con-
densation of cyanoester 17 and chloride 18 gave nitrile 19, which
was reduced to an amine and cyclized in situ to form lactam 20.
A routine saponification and Curtis rearrangement generated 22,
which was coupled with aryl bromide 23, to give intermediate
N
N
N
N
NH
N
N
N
507
N
N
2
4. Reduction of nitro group under acidic conditions yielded imida-
zole 25, which after deprotection, gave amine 26.
N
NH
2
N
N
In Scheme 4, a tricyclic indole synthesis is described. A modified
Fisher indole synthesis was employed to condense ketone 27 and
hydrazine 28, to give intermediate 29, which was converted to
an amine through either deprotection or Curtius rearrangement.
Alkylation and deprotection gave substituted indole amine 30.
In Scheme 5, a reversed indole core synthesis is described. Con-
densation of 31 and ethyl acrylate gave 33, which was converted to
lactam with formalin and ammonium acetate, then the nitro group
reduced to an amine and cyclized in situ to form lactam 34. A N–C
coupling with aryl iodide 35 generated 36, which was converted to
bromide 37. The bromide can be converted to phosphonium salt 38
or nitrile 41, then cyclized with the lactam to form indoles 39 or 42
257
N
OH
N
N
5315
N
CF
3
O
N
N
N
H/C
H/C
H/N
85
86
87
2.6
0.6
2.5
1035
234
—
NH2
O
N
N
N
N
N
N
OH
O
N
N
(
X = CN). C3 activation of the indole can be done with electrophiles
OH
O
at this stage to, for example, generate an amide or ester 42. Reduc-
tion of nitro group under acidic condition yields amine 43.
In Scheme 6, derivitization of the reversed indole C-3 position is
described with a fully functionalized quaternary amine. Iodoniza-
tion gave a handle for multiple couplings, especially Suzuki. Some
heteryl cycles, for example tetrazole 47, can be made from 46
N
N
N
N
Cl/C
Cl/C
88
89
8.6
0.6
1053
409
OH
O
OH
N
(
R = CN). Electrophilic reactions also work on 44 to form aldehyde
or ketones 48, which can be either reduced to alcohol 49 or con-
verted to oxime 50. A trifluoromethyl group can also be introduced
through a novel photo reaction to form 51.
i
account the assay conditions. The apparent K in each case was
used to compare FIXa and FXa for selectivity.
FIXa and FXa inhibitory activities (IC50) were measured by the
cleavage of a fluorescent peptide. The cleaved fluorophore was
detected with a fluorescence detector.12 As competitive inhibitors,
their apparent Kis were derived from measured IC50s taking into
Previously, we reported the X-ray crystal structure of a pyra-
zolopyridine compound with human Factor IXa, this result and
all the following X-rays proved the absolute stereo chemistry at
the quaternary center is as drown.
9

5
440
D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
R
a
HO
R
O
O
N
N
O
O
R
c
N
b
+
N
9
Cl
N
N
O
d
O
O
O
O
O
R
12
1
0
8
11
R1
R1
R1
O
NH2
NHAc
g
OH
f
e
N
N
N
N
N
N
N
R
R
N
R
R
1
5
16
1
4
1
3
2 2
Scheme 2. Reagents and conditions: (a) Pd(OAc) , 2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane, NaHMDS in THF, dioxane, 100 °C, 3 h; (b) HONH ,
KOAc, MeOH, 70 °C; (c) p-toluenesulfonyl chloride, TEA, DCM, rt; (d) HCl, rt, overnight; (e) RMgBr, THF; (f) MeCN, H
2
SO
4
; (g) HCl, 95 °C.
O
O
CO Et
Cl
OEt
2
R'
HN
R'
HN
R'
d
1
8
a
b
c
O
R'
NC
CO Et
2
NC
CO Et
2
9
1
7
CO
2
Et
CO H
2
1
20
N
21
NO2
R
R
NO2
N
O
O
R
R
g
f
N
N
HN
R'
Br
N
2
e
3
R'
2
NH2
R'
NHCbz
NHCbz
R'
NHCbz
6
2
5
2
2
24
Scheme 3. Reagents and conditions: (a) NaOEt, EtOH; (b) PtO
g) Pd(OH) /C, H , EtOH, rt, 97%.
2
, H
2
, MeOH/AcOH; (c) LiOH; (d) DPPA, DIEA, BnOH; (e) Xantphos, Pd
2
2 2 3
(dba) , Cs CO , toluene, 85 °C; (f) AcOH, Fe;
(
2
2
O
NHNH
2
H
N
O
HO
R
2
R
b, c
N
2
8
a
R
X
R'
X
R'
2
7
29
X
3
0
R'
X = NH-P, or COOR"
Scheme 4. Reagents and conditions: (a) Zn(OTf)
2
2 3
, EtOH, 50 °C; (b) tert-butyl acrylate, Cs CO ; (c) TFA, DCM.
Firstly, as shown in Table 1, we conducted SAR studies on the S1
selectivity (Table 1, compound 55). Generally, replacement with
an indole without substitution on the nitrogen afforded analogs
with modest activity, but substitutions, especially with HBDs and
HBAs, significantly improve potency (Table 1, compounds 56–58),
and will be discussed later.
Table 2 shows a selected list of tolerated groups at the quater-
nary center after a thorough investigation of both alkyl and aryl
substituents. Small alcohols gave essentially the same potency
and selectivity as a methyl group (Table 2, compounds 53, 59
and 60). Introducing larger alkyl groups resulted in a loss of activ-
ity. Aromatic rings can be introduced at the quaternary position to
improve both potency and selectivity. Meta-substituted phenyl
groups are tolerated and usually improve selectivity over Factor
Xa (Table 2, compounds 61–66). The para-position tolerated only
small groups. A 4,5-disubstituted benzene offers no potency
moiety of the molecule. The P1 site of FIXa protein structure is a
narrow pocket that has several polar groups in the backbone. In
general, fused aromatic rings were preferred binders to this region.
Heteroaryls with hydrogen bond donors (HBDs) or hydrogen bond
acceptors (HBAs) were investigated to pick up polar interactions
with the P1 backbone. Results are shown in Table 1. Investigation
of the substitution pattern on the pyrazolopyridines revealed that
the phenol is not critical for FIXa potency and FXa selectivity, espe-
cially when the methyl group is moved from the 7- to 6-position
Table 1, compounds 52 and 53). In addition, changing the quater-
nary methyl to other alkyl and aromatic rings is also tolerated
Table 1, compounds 54), and will be discussed further. Borrowing
from our knowledge of the S1 SAR in the acyclic series (1), resulted
in cyclic benzimidazoles with comparable potency, but improved
(
(

D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
5441
Me
OH
Ph
NO2
Ph
NO2
3
+
1
2
Ph
CO Et
2
b
d
HN
c
Ph
NO2
a
N
NO2
O
Me
CO Et
OH
O
2
3
3
3
4
3
36
I
3
5
P X-
Me
Ph
3
Me
Me
Br
O
e
f
g
Ph
N
N
N
NO2 Me
N
NO2
NH
2
O
NO
2
Ph
39
Ph
Ph
3
7
38
4
0
j, X = CONH2
h
k, X = CO Me
2
X
Me
X
CN
Ph
NO2
i
N
g
N
N
O
NH2
NO2
Ph
Ph
4
1
4
3
4
2
Scheme 5. Reagents and conditions: (a) Et
3
N, MeCN; (b) formalin, NH
4
OAc, EtOH/H
2
O, 70 °C; (c) CuI, diamine, Cs
2
CO
Cl
3
, dioxane; (d) CBr
4
, PPh
3
, DCM; (e) PPh
3
, PhMe, 100 °C;
(
f) LDA, PhMe; (g) Zn, AcOH; (h) KCN, 19-c-6, MeCN; (i) LDA, THF, À10–50 °C; (j) chlorosulfonyl isocyanate, CH
2
2
; (k) phosgene, CH
2
Cl2, then MeOH, TEA.
N
N
N
HN
I
R1
Me
Me
Me
Me
c
N
O
N
O
O
b
N
O
N
H
Ar
N
H
a
Ar
N
H
Ar
Ph
HO
Ph
Ph
4
5
46
47
O
R2
R2
Me
Me
e
N
d
N
O
N
O
N
H
Ar
Ph
4
4
N
Ar
Ph
N
H
Ar
4
8
49 Ph
H
g
f
HON
CF3
R2
Me
Me
N
O
N
O
N
H
Ar
Ph
50
N
H
Ar
Ph
5
1
Scheme 6. Reagents and conditions: (a) NIS, DCM, À10 °C; (b) boronic acid/ester, chloro[(tri-tert-butylphosphine)-2-(2-aminobiphenyl)] palladium(II), tripotassium
phosphate, THF, 50 °C; (c) (CH SiN dibutyltin (IV) oxide, toluene; (d) POCl DMF/DMA/TFA; (e) NaBH MeOH; (f) HONH pyridine; (g) CF I, Tris[2-(4,6-
difluorophenyl)pyridinato-C ,N]iridium(III), K CO , light.
3
)
3
3
,
3
,
4
,
2
,
3
2
2
3
advantage (Table 2, compounds 67–68). Quite a few hetero cycles
pyrido, thophino, oxadiazolo, etc.) can be used to replace phenyl,
Previously, we reported the X-ray crystal structure of a pyra-
zolopyridine compound with human Factor IXa. There is a water
9
(
exemplified as triazolo compound 69 in Table 2.
molecule between N1 and the backbone of the protein. Introducing
a small group, capable of forming a hydrogen bond with the pro-
tein, to the tricycles could replace the water and benefit from
entropy and enthalpy effects. Modifications to the 3-position of
the reverse indole supplies a perfect platform to test this assump-
tion. Parent indole 73 shows only about 35 nM affinity. Adding a
small alkyl group (Table 4, compounds 74–77), which may only
replace the water molecule without hydrogen bonding to the pro-
In general the S2–S4 SAR trends identified in the acyclic series
transferred to the tricyclic series with the following noted
exceptions. In the acyclic series, the second chlorine on the
aromatic ring boosts potency by about fivefold, but does not have
that effect in the tricyclic series (Table 3, compounds 62–70). Also,
benzimidazoles were found to be a replacement for triazoles, but
resulted in a loss of selectivity over FXa (Table 3, compounds 72).

5
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D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
tein, improves the affinity by fivefold. Groups with hydrogen bond-
ing capability further improve affinity by another 5–10 fold
(Table 4, compounds 78–83). The X-ray crystal structure of 82 sup-
ported this hypothesis (see Fig. 2). The tetrazolo group occupied
the water position, and forms a hydrogen bond with serine 195.
Alcohols without a neighboring electron withdrawing group are
not very stable, trifluoro ethyl alcohols, for example compound
8
4, are stable and maintain good potency. With these extremely
potent P1s, the Cl on the phenyl of S2–S4 unit is not required to
achieve desired affinity (Table 4, compounds 85, 86), even the phe-
nyl group itself can be replaced with a pyridine (Table 4, com-
pounds 87). The 1,3,4-triazole can also be replaced with other
triazole or triazolone, even a tetrazole (Table 4, compounds 88–
8
9). All compounds in this class maintain at least 200 fold selectiv-
ity over human Factor Xa enzyme, some even show thousand fold
selectivity. This increased selectivity was not observed in other
sub-classes with 1,3,4 triazole unit.
Based upon an understanding of the basal SAR of the cyclic lead
series, several analogs were selected to address the pharmacoki-
netic parameters. Compounds with S2–S4 1,3,4 triazole unit gener-
ally are not orally available, some bioavailable methyl 1,2,4 triazole
analogues are shown in Table 5. Compound 55 showed moderate
half-life in both rat and dog with a relatively high clearance, but
good bioavailability (Table 5). Compound 89 has lower clearance
and longer half-life (Table 5, entry 3, 4), but slightly lower
bioavailability.
Figure 3. Compound 55 being evaluated in TGA assay. (A) Compound 55 (C.55_)
and apixaban (A_) in Human CTI-citrated Plasma using FXIa as coagulation trigger;
(
B) Compound 55 (C.55_) and apixaban (A._) in Human CTI-citrated Plasma using
tissue factor (TF) as coagulation trigger.
Having demonstrated reasonable overall oral PK for this class of
molecules, we evaluated the FIXa inhibitory efficacy in TGA assay,
APTT/PT and in a rat AV shunt/cuticle bleeding model, and com-
pared with the Factor Xa inhibitor apixaban.
In TGA assay as previously described,10 see Figure 3, compound
5
5 behaves very similar to the acyclic analogues, and showed
limited activity when compared with apixaban upon increasing
concentration of Tissue Factor.
As shown in Figure 4,14 compound 55 increased APTT, which
proved their efficacy through the intrinsic pathway. At the same
time, it did not change PT at all even at high dose, which denies
Figure 2. Representation of the X-Ray crystal structures of compounds 86 with
human Factor IXa.
Figure 4. Compound 55 being evaluated in APTT/PT, and rat Av shunt/bleeding
assay. (A) Dose dependent APTT (but not PT) increase; (B) Rat AV shunt model to
check clot reduction; rat cutical bleeding assay to check bleeding liability.
Table 5
1
3
any big role of extrinsic pathway. Furthermore, it decreased clot
weight effectively in a dose dependent manor in rat AV shunt
assay, just like apixaban. At the same time, it has little effect on
bleeding time, at the doses tested. It was not possible to dose 55
higher due to solubility limitations.
IV/PO PK for selected compounds
Entry
Compd
Species
F%
T
1/2 (h)
CL (mL/min/kg)
Vdss (L/kg)
1
2
3
4
55
55
89
89
Rat
Dog
Rat
26
68
16
39
0.46
1.34
1.03
2.26
30
22
3.95
16
0.77
3.12
0.08
1.15
In conclusion, we have discovered a new class of tricyclic FIXa
inhibitors occupying the S1–S4 pockets of the FIXa active binding
Dog

D. Meng et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5437–5443
5443
site. Through structure based drug design, several highly potent
and selective FIXa inhibitors were identified, and were evaluated
in PK and PD assays. In vivo studies in animal models are promis-
ing. Additional studies to further define the efficacy/bleeding pro-
files of FIXa inhibition are on-ging and will be presented in due
course.
6. Weltermann, A.; Eichinger, S.; Bialonczyk, C.; Minar, E.; Hirschl, M.;
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Supplementary data
Supplementary data (the X-ray crystallization method and data
and the protocol for av shunt assay) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
9.
2
015.07.078.
1
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References and notes
1
.
Granger, C. B.; Alexander, J. H.; McMurray, J. J. V.; Lopes, R. D.; Hylek, E. M.;
Hanna, M.; Al-Khalidi, H. R.; Ansell, J.; Atar, D.; Avezum, A.; Bahit, C.; Diaz, R.;
Easton, J. D.; Ezekowitz, J. A.; Flaker, G.; Garcia, D.; Geraldes, M.; Gersh, B. J.;
Golitsyn, S.; Goto, S.; Hermosillo, A. G.; Hohnloser, S. H.; Horowitz, J.; Mohan,
P.; Jansky, P.; Lewis, B. S.; Lopez-Sendon, J. L.; Pais, P.; Parkhomenko, A.;
Verheugt, F. W. A.; Zhu, J.; Wallentin, L. N. Engl. J. Med 2011, 365, 981.
Patel, M. R.; Mahaffey, K. W.; Garg, J.; Pan, G.; Singer, D. E.; Hacke, W.;
Breithardt, G.; Halperin, J. L.; Hankey, G. J.; Piccini, J. P.; Becker, R. C.; Nessel, C.
C.; Paolini, J. F.; Berkowitz, S. D.; Fox, K. A. A.; Califf, R. M. N. Engl. J. Med. 2011,
11. For detailed synthetic procedures for compounds found in this report please
refer to our patents.
12. The activity of Factor IXa is measured by monitoring the cleavage of the
3 2
fluorescent peptide, CH SO -D-CHG-Gly-Arg-AFC.AcOH. (CHG = cyclohexylGly
and AFC = 7-amido-4-trifluoromethylcoumarin). 2 nM of Factor IXa is used to
cleave the amide bond between Arg and AFC, thereby releasing the AFC
fluorophore. The free AFC is detected with a fluorescence detector at an
excitation wavelength of 405 nm and emission wavelength of 510 nm. hFIXa
2
3
.
.
365, 883.
was assayed at substrate concentrations below
M
K . IC50 for hFIXa was
Connolly, S. J.; Ezekowitz, M. D.; Yusuf, S.; Eikelboom, J.; Oldgren, J.; Parekh, A.;
Pogue, J.; Reilly, P. A.; Themeles, E.; Varrone, J.; Wang, S.; Alings, M.; Xavier, D.;
Zhu, J.; Diaz, R.; Lewis, B. S.; Darius, H.; Diener, H.-C.; Joyner, C. D.; Wallentin, L.
N. Engl. J. Med. 2009, 361, 1139.
converted to K using the Cheng–Prusoff equation for competitive inhibition,
i
see: Cheng, Y.; Prusoff, W. H. Biochem Pharmacol 1973, 22, 3099.
13. PK studies were conducted in fasted rats, administering a dose of 0.5 mg/kg
bolus intravenously (IV) and 2 mg/kg orally (PO).
4
5
.
.
a) Feuerstein, G. Z.; Stern, D. Drug Discovery Today: Therap. Strat. 2005, 2, 279.
Darby, S. C.; Kan, S. W.; Spooner, R. J.; Giangrande, P. L. F.; Hill, F. G. H.; Hay, C.
R. M.; Lee, C. A.; Ludlam, C. A.; Williams, M. Blood 2007, 110, 815; Šrámek, A.;
Kriek, M.; Rosendaal, F. R. Lancet 2003, 362, 351.
14. See the Supporting information for PT, APTT, AV shunt descriptions.