Anthranilamide-based N,N-dialkylbenzamidines as potent and orally bioavailable factor Xa inhibitors: P4 SAR

Article abstract of DOI:10.1016/j.bmcl.2009.02.114

Anthranilamide-based benzamidine compound 4 and its N-substituted analogs were designed and examined as factor Xa inhibitors using substituted benzamidines as unconventional S4 binding element. A group of N,N-dialkylbenzamidines (11, 17 and 24) have been

Full text of DOI:10.1016/j.bmcl.2009.02.114

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2186–2189
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Anthranilamide-based N,N-dialkylbenzamidines as potent and orally
bioavailable factor Xa inhibitors: P4 SAR
à
Penglie Zhang , Liang Bao , Jingmei Fan, Zhaozhong J. Jia ,§, Uma Sinha §, Paul W. Wong –, Gary Park ||,
*
Athiwat Hutchaleelaha §, Robert M. Scarborough §, Bing-Yan Zhu à
Millennium Pharmaceuticals, Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Anthranilamide-based benzamidine compound 4 and its N-substituted analogs were designed and exam-
ined as factor Xa inhibitors using substituted benzamidines as unconventional S4 binding element. A
group of N,N-dialkylbenzamidines (11, 17 and 24) have been discovered as potent factor Xa inhibitors
with strong anticoagulant activity and promising oral PK profiles.
Received 13 January 2009
Revised 25 February 2009
Accepted 26 February 2009
Available online 3 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Factor Xa inhibitor
Anticoagulant
Anthranilamide
Benzamidine
N,N-Dialkylbenzamidine
Activated by both the extrinsic and the intrinsic pathways, the
serine protease factor Xa (fXa) plays a pivotal role in the blood
coagulation cascade. In the prothrombinase complex comprised
of fXa, factor Va and Ca2+ assembled on the platelet surface, fXa
catalyzes the conversion of prothrombin to thrombin, the final en-
zyme responsible for fibrin clot formation in this cascade.1 FXa is a
particularly attractive target for anticoagulant therapy for the
treatment of severe cardiovascular diseases.2 Selective inhibition
of fXa provides antithrombotic effects by diminishing the ampli-
fied generation of thrombin without affecting existing thrombin
levels. Clinical findings have confirmed the potential of fXa inhibi-
tion for producing excellent antithrombotic efficacy with minimal
bleeding risk when compared to direct thrombin inhibitors.3,4
Small molecule, reversible and orally bioavailable fXa inhibitors
have been pursued extensively for a decade. We have discovered a
series of neutral and mono-basic anthranilamide-based fXa inhib-
itors as exemplified by compounds 1, 2 and 3 (Fig. 1), wherein the
chloropyridine ring interacts with the S1 specificity pocket.5,6
Anthranilamides 1, 2 and 3 are potent and selective fXa inhibitors,
with inhibitory IC50 values of 6.7 nM, 1.5 nM and 4.7 nM, respec-
tively.5,7 The biphenyl group of compound 1, the cyclized guani-
dine group of compound 2 and the oxazolidin-2-imine group of
compound 3 are thought to bind to the highly aromatic fXa S4
pocket either through hydrophobic or non-specific p-cation inter-
actions.6c,f Though orally bioavailable (F 35% in rat), compound 1
shows very poor in vitro anticoagulant activity in our human plas-
ma-based thrombin generation assay (2 Â TG >5
pounds and with increased P4 hydrophilicity provides
M and 0.56 M, respec-
l
M).5a,8 Com-
2
3
improved functional activity (2 Â TG 0.58
l
l
tively), possibly due to their lower human plasma protein binding.
The guanidine moiety’s high basicity in compound 2 (calculated
9
pKa ꢀ14) likely leads to its low oral absorption (F <1% in rat)
O
HN
O
O
HN
O
N
Cl
O
S
NH
N
H2N O
N
N
H
* Corresponding author at present address: Portola Pharmaceuticals, Inc., 270
East Grand Ave, Ste 22, South San Francisco, CA 94080. Tel.: +1 650 246 7073; fax:
+1 650 246 7776.
HN
Cl
O
HN
Cl
1
2
E-mail address: zjia@portola.com (Z.J. Jia).
Present address: ChemoCentryx, Inc., Mountain View, CA 94043, USA.
Present address: Genentech, Inc., South San Francisco, CA 94080, USA.
Present address: Portola Pharmaceuticals, Inc., South San Francisco, CA 94080,
N
HN
Cl
à
NH
O
O
N
§
HN
Cl
USA.
3
–
Present address: Berkeley HeartLab, Inc., Alameda, CA 94501, USA.
Present address: Rigel Pharmaceuticals, Inc., South San Francisco, CA 94080, USA.
||
Figure 1. Anthranilamide-based fXa inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.114

P. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2186–2189
2187
Table 2
and high clearance, while compound 3 displays good oral pharma-
N,N-Disubstituted benzamidines 15–22 as fXa inhibitors
cokinetic (PK) profile (F 44%, t1/2 8.5 h, CL 18.3 mL/min/kg, Vd
13.6 L/kg in rat).
From our SAR explorations, we learned that a variety of posi-
tively charged groups are potent P4 motifs possibly due to p-cation
interactions. Additionally, modulation of S4 binding elements pro-
vides a promising strategy to improve anticoagulant activity and
oral PK properties.
HN
R1
O
HN
O
N
R2
N
HN
Cl
15-22
Compd
NR1R2
fXa IC50 (nM)
15
16
17
18
19
20
21
22
1-azetidinyl
8
8
3
7
12
256
38
714
Compared to compound 2, the corresponding benzamidine ana-
log 4 (Table 1) has lower basicity (calculated pKa ꢀ10), leading us
to examine the benzamidine groups as the P4 motifs in our anthra-
nilamide scaffold for fXa inhibition. For proof of concept, we de-
signed compound 4 bearing a primary benzamidine as the P4
group, though this moiety had been well-know for its strong PK lia-
bility in the fXa inhibitor discovery arena. Not surprisingly, com-
pound 4 suffers poor oral absorption (F <5% in rat). To our
delight, it displays potent fXa inhibitory activity (IC50 4 nM) and
excellent selectivity over other serine proteases, with IC50 values
1-pyrrolidinyl
1-piperidinyl
4-morpholinyl
4-thiomorpholinyl
4-(1,1-dioxo)thiomorpholinyl
1-piperazinyl
4-Me-1-piperazinyl
more potent than the azetidine 15, pyrrolidine 16, morpholine 18
and thiomorpholine 19 analogs. Oxidation of the thiomorpholine
to the corresponding sulfone 20 decreases the fXa potency signifi-
cantly. The lower activity of compounds 21 and 22 suggests the
second basic nitrogen atom in the ring structure detrimental to
binding in the fXa S4 pocket.
>10
lM for thrombin, trypsin, tissue plasminogen activator (t-
PA), activated protein C (aPC), plasmin and IC50 value of 4.9
lM
for kallikrein. Unlike the early fXa inhibitors bearing the amidine
group to ionically interact with Asp189 in the fXa S1 pocket, we be-
lieved that the amidine group in compound 4 interacts with the
A group of P4 cyclized amidine moieties have also been studied
in this anthranilamide-based fXa inhibitor series (Table 3). Imidaz-
oline 23 and tetrahydro-pyrimidine 26 are approximately equipo-
tent fXa inhibitors. N-Methylation profoundly increases the fXa
potency, especially in the case of N-methylimidazoline 24 (IC50
20 nM). Replacement of the methyl substituent by a bulkier ethyl
group decreases the inhibitory potency by threefold in compound
25.
All the compounds listed in Tables 1–3 have excellent enzyme
selectivity profile toward fXa as shown by compound 4. Based on
their fXa inhibitory activity and structural diversity, compounds
11, 17 and 24 were selected for further in vitro and in vivo studies
(Table 4). Their measured human plasma protein binding ranges
between 73% and 89%.12 All three compounds show good in vitro
highly aromatic S4 pocket through
p-cation interactions. Based
on this hypothesis, we rationalized that this benzamidine group
could be N-substituted without compromising the fXa binding
affinity,10 whereas the benzamidine group interacting with the
fXa S1 pocket is generally not tolerated for any substitution due
to negative steric interactions. Further rationale for us to explore
N-substituted benzamidines as S4 binding element was that these
substitutions could provide significantly improved oral absorption
from the primary benzamidine analog, previously reported in the
platelet GP IIb–IIIa antagonist literature.11
For the P4 SAR exploration around compound 4, we first exam-
ined N-substitution of the benzamidine using a variety of moieties
(Table 1). Benzamidines substituted by a hydroxyl, methoxy, ami-
no, dimethylamino, methyl and ethyl group (5–10) are less potent
than compound 4. Increasing the bulkiness of the alkyl group re-
duces fXa potency. Interestingly, the N,N-dialkylated benzamidine
compounds 11–14 are more potent than the N-monoalkylated ana-
logs 9–10. Smaller methyl group is preferred over ethyl, and the
N,N-dimethylbenzamidine 11 (IC50 3 nM) is the most potent fXa
inhibitor among all the compounds studied.
anticoagulant activity (2 Â TG 0.36–1.1
l
M). The cLogD values9
of these compounds are 0.17, 0.94 and 0.22, respectively at pH
7.4. For comparison, compounds 1–3 (Fig. 1) have cLogD values
of 3.78, À1.26 and 0.80, respectively at pH 7.4. Compared with
compound 11 (IC50 3 nM; 2 Â TG 0.54
lM), compound 17 (IC50
3 nM; 2 Â TG 0.36
lM) has similar anti-fXa potency and slightly
stronger in vitro anticoagulant activity. Dosed at 0.4 mg/kg IV
and 6 mg/kg PO in Sprague–Dawley rat, all three compounds dem-
onstrated promising PK profiles. Compound 11 (F 31%; CL 16.8 mL/
min/kg; Vd 6.3 L/kg) gives significantly better oral bioavailability
(F), lower clearance (CL) and lower volume of distribution (Vd) than
compound 17 (F 15%; CL 32.2 mL/min/kg; Vd 16.7 L/kg). Thus com-
pound 11 was chosen over compound 17 as the lead among this
class of fXa inhibitors. The PK profiles of compounds 11 and 24
To further investigate the P4 N,N-disubstituted benzamidines,
we synthesized compounds 15–22 using various cyclic amine star-
ing materials (Table 2). Piperidine compound 17 (IC50 3 nM) is
Table 1
Benzamidine 4 and its N-substituted analogs as fXa inhibitors
HN
R1
O
HN
O
N
R2
N
Table 3
HN
Cl
4-14
Cyclic benzamidines 23–27 as fXa inhibitors
N
O
HN
O
Compd
NR1R2
fXa IC50 (nM)
n
4
5
6
7
8
NH2
4
N
R
NHOH
NHOMe
NHNH2
NHNMe2
NHMe
NHEt
35
290
25
30
23
93
3
6
9
10
N
HN
Cl
23-27
Compd
n
R
fXa IC50 (nM)
9
23
24
25
26
27
1
1
1
2
2
H
Me
Et
316
20
64
10
11
12
13
14
NMe2
NMeEt
NMePr
NEt2
H
Me
226
78

2188
P. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2186–2189
Table 4
Biological and animal pharmacokinetic data for the leading fXa inhibitors
Compd
11
17
HN
24
HN
O
HN
O
O
HN
O
O
HN
O
N
N
N
N
N
N
N
HN
Cl
HN
Cl
HN
Cl
fXa IC50 (nM)
3
3
20
2 Â TG (
l
M)
0.54
73
0.36
89
1.1
82
human plasma protein binding (%)
cLogD (pH 7.4)
0.17
0.94
0.22
Rat PK
F (%)
31
18
22
t1/2 (h)
CL (mL/min/kg)
Vd (L/kg)
4.3
16.8
6.3
6.0
32.2
16.7
>6.0
7.1
3.7
Dog PK
F (%)
t1/2 (h)
CL (mL/min/kg)
Vd (L/kg)
69
3.5
45.7
13.7
nd
nd
nd
nd
50
2.2
49.6
9.2
were further assessed in beagle dog (dosed at 0.1 mg/kg IV and
0.5 mg/kg PO). They both were well absorbed with oral bioavail-
ability values of 69% and 50%, respectively.
In summary, we investigated a variety of N-substituted ben-
zamidine moieties as the S4 binding motifs in our anthranila-
mide-based fXa inhibitor scaffold. Through this exploration, a
group of N,N-dialkylbenzamidine compounds (11, 17 and 24) have
been discovered as highly potent and selective fXa inhibitors.
These compounds are moderately protein bound in human plasma
and demonstrate good anticoagulant activity in our human plas-
ma-based thrombin generation assay. More importantly, these
compounds display good oral PK profiles in rat and dog. Compound
11, N-(5-chloropyridin-2-yl)-2-(4-(N,N-dimethylcarbamimidoyl)-
benzamido)benzamide (fXa Ki 1.4 nM),14 has been chosen as the
lead from this class of N,N-dialkylbenzamidine fXa inhibitors for
further modifications.
The synthesis of compound 4 and its analogs is described in
Scheme 1.13 Isatoic anhydride 28 was reacted with 2-amino-5-
chloropyridine and potassium t-butoxide to afford compound 29.
Coupling of aniline 29 with 4-cyanobenzoyl chloride furnished
benzonitrile 30. It was converted to methyl thioimidate 31 by
treatment with excess hydrogen sulfide gas, followed by S-methyl-
ation using methyl iodide. Compounds 4–27 were prepared from
methyl thioimidate 31 using various amines in a parallel synthesis
fashion. The yield of this final step was improved by treating the
free amines with acetic acid prior to their addition to the methyl
thioimidate, to suppress the generation of benzonitrile 30 from
31 promoted by basicity.
References and notes
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23, 17.
O
NC
2. (a) Schaffer, L. W.; Davidson, J. T.; Vlasuk, G. P.; Dunwiddie, C. T.; Siegl, P. K. S.
Arterioscler. Thromb. 1992, 12, 879; (b) Zhu, B.-Y.; Scarborough, R. M. Annu. Rep.
Med. Chem. 2000, 35, 83; (c) Linkins, L.-A.; Julian, J. A.; Rischke, J.; Hirsh, J.;
Weitz, J. I. Thromb. Res. 2002, 105, 241; (d) Walenga, J. M.; Jeske, W. P.;
Hoppensteadt, D.; Fareed, J. Curr. Opin. Invest. Drugs 2003, 4, 272; (e) Samama,
M. M. Thromb. Res. 2002, 106, V267; (f) Kaiser, B. Cell. Mol. Life Sci. 2002, 59, 189.
3. (a) McBride, B. F. J. Clin. Pharmacol. 2005, 45, 1004; (b) Saiah, E.; Soares, C. S.
Curr. Top. Med. Chem. 2005, 5, 1677.
HN
O
H2N
O
HN
O
c
a,b
O
N
N
O
HN
Cl
HN
Cl
28
30
29
HN
O
HN
O
HN
O
f
d, e
4. (a) Roehrig, S.; Straub, A.; Pohlmann, J.; Lampe, T.; Pererstorfer, J.; Schlemmer,
K.-H.; Reinemer, P.; Perzborn, E. J. Med. Chem. 2005, 48, 5900; (b) Perzborn, E.;
Strassburger, J.; Wilmen, A.; Pohlmann, J.; Roehrig, S.; Schlemmer, K.-H.;
Straub, A. J. Thromb. Haemostasis 2005, 3, 514; (c) Eriksson, B. L.; Borris, L.; Dahl,
O. E.; Haas, S.; Huisman, M. V.; Kakkar, A. K. J. Thromb. Haemostasis 2006, 4, 121;
(d) Quan, M. L.; Lam, P. Y. S.; Han, Q.; Pinto, D. J. P.; He, M. Y.; Li, R.; Ellis, C. D.;
Clark, C. G.; Teleha, C. A.; Sun, J.-H.; Alexander, R. S.; Bai, S.; Luettegn, J. M.;
Knabb, R. M.; Wong, P. C.; Wexler, R. R. J. Med. Chem. 2005, 48, 1729; (e) Wong,
P. C.; Crain, E. J.; Watson, C. A.; Wexler, R. R.; Lam, P. Y. S.; Quan, M. L.; Knabb, R.
M. J. Thromb. Thrombolysis 2007, 24, 43; (f) Pinto, D. J. P.; Orwat, M. J.; Koch, S.;
Rossi, K. A.; Alexander, R. S.; Smallwood, A.; Wong, P. C.; Rendina, A. R.;
Luettgen, J. M.; Knabb, R. M.; He, K.; Xin, B.; Wexler, R. R.; Lam, P. Y. S. J. Med.
Chem. 2007, 50, 5339; (g) Furugohri, T.; Isobe, K.; Honda, Y.; Kamisato-
Matsumoto, C.; Sugiyama, N.; Nagahara, T.; Morishima, Y.; Shibano, T. J.
Thromb. Haemostasis 2008, 6, 1542; (h) Turpie, A. G.; Bauer, K. A.; Erilsson, B. I.;
Lassen, M. R. Arch. Intern. Med. 2002, 162, 1833.
H2N
HN
MeS
O
N
N
4
31
HN
Cl
HN
Cl
g
h
HN
O
R1
N
HN
O
N
O
HN
O
R2
n
N
N
R
n= 1 or 2
HN
Cl
5-22
N
HN
Cl
23-27
Scheme 1. Reagents and conditions: (a) 2-amino-5-chloropyridine (1 equiv), KOBut
(2 equiv), rt, THF, 10 min; (b) H2O, 65% for two steps; (c) 4-cyanobenzoyl chloride
(1 equiv), THF, rt, 30 min, 95%; (d) H2S (g), pyridine, Et3 N, rt, overnight; (e) MeI
(2 equiv), acetone, reflux, 30 min; (f) NH4OAc (2 equiv), MeOH, reflux, 10 min, 65%
for three steps; (g) NHR1R2 (2 equiv), HOAc (3 equiv), MeOH, reflux, 60–85%; (h)
H2 N(CH2)nCH2 NHR (2 equiv), HOAc (5 equiv), MeOH, reflux, 60–80%.
5. (a) Zhang, P.; Bao, L.; Zuckett, J. F.; Goldman, E. A.; Jia, Z. J.; Arfsten, A.; Edwards,
S.; Sinha, U.; Hutchaleelaha, A.; Park, G.; Lambing, J. L.; Hollenbach, S. J.;
Scarborough, R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett. 2004, 14, 983; (b) Zhang,
P.; Bao, L.; Zuckett, J. F.; Jia, Z. J.; Woolfrey, J.; Arfsten, A.; Edwards, S.; Sinha, U.;
Hutchaleelaha, A.; Lambing, J. L.; Hollenbach, S. J.; Scarborough, R. M.; Zhu, B.-
Y. Bioorg. Med. Chem. Lett. 2004, 14, 989.

P. Zhang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2186–2189
2189
6. Several other laboratories have also investigated anthranilamide-derived fXa
inhibitors: (a) Yee, Y. K.; Tebbe, A. L.; Linebarger, J. H.; Beight, D. W.; Craft, T. J.;
Gifford-Moore, D.; Goodson, T., Jr.; Herron, D. K.; Klimkowski, V. J.; Kyle, J. A.;
Sawyer, J. S.; Smith, G. F.; Tinsley, J. M.; Towner, R. D.; Weir, L.; Wiley, M. R. J.
Med. Chem. 2000, 43, 873; (b) Shrader, W. D.; Young, W. B.; Sprengler, P. A.;
Sangalang, J. C.; Elrod, K.; Carr, G. Bioorg. Med. Chem. Lett. 2001, 11, 1801; (c)
Alder, M.; Kochanny, M. J.; Ye, B.; Rumennik, G.; Light, D. R.; Biancalana, S.;
Whitlow, M. Biochemistry 2002, 41, 15514; (d) Chou, Y.-L.; Davey, D. D.; Eagen,
K. A.; Griedel, B. D.; Karanjawala, R.; Philips, G. B.; Sacchi, K. L.; Shaw, K. J.; Wu,
S. C.; Lentz, D.; Liang, A. M.; Trin, L.; Morrissey, M. M.; Kochanny, M. J. Bioorg.
Med. Chem. Lett. 2003, 13, 507; (e) Kochanny, M. J.; Alder, M.; Ewing, J.; Griedel,
B. D.; Ho, E.; Karanjawala, R.; Lee, W.; Lentz, D.; Liang, A. M.; Morrissey, M. M.;
Phillips, G. B.; Post, J.; Sacchi, K. L.; Sakata, S. T.; Subramanyam, B.; Vergona, R.;
Walters, J.; White, K. A.; Whitlow, M.; Ye, B.; Zhao, Z.; Shaw, K. J. Bioorg. Med.
Chem. 2007, 15, 2127; (f) Ye, B.; Arniaz, D. O.; Chuo, Y.-L.; Griedel, B. D.;
Karanjawala, R.; Lee, W.; Morrissey, M. M.; Sacchi, K. L.; Sakata, S. T.; Shaw, K. J.;
Wu, S. C.; Zhao, Z.; Adler, M.; Cheeseman, S.; Dole, W. P.; Ewing, J.; Fitch, R.;
Lentz, D.; Liang, A.; Light, D.; Morser, J.; Post, J.; Rumennik, G.; Subramanyam,
B.; Sullivan, M. E.; Vergona, R.; Walters, J.; Wang, Y.-X.; White, K. A.; Whitlow,
M.; Kochanny, M. J. J. Med. Chem. 2007, 50, 2967; (g) Mendel, D.; Marquart, A.
L.; Joseph, S.; Waid, P.; Yee, Y. K.; Tebbe, A. L.; Ratz, A. M.; Herron, D. K.;
Goodson, T.; Masters, J. J.; Franciskovich, J. B.; Tinsley, J. M.; Wiley, M. R.; Weir,
L. C.; Kyle, J. A.; Klimkowski, V. J.; Smith, G. F.; Towner, R. D.; Froelich, L. L.;
Buben, J.; Craft, T. J. Bioorg. Med. Chem. Lett. 2007, 17, 4832; (h) Corte, J. R.; Fang,
T.; Pinto, D. J. P.; Han, W.; Hu, Z.; Jiang, X.-J.; Li, Y.-L.; Gauuan, J. F.; Hadden, M.;
Orton, D.; Rendina, A. R.; Luettgen, J. M.; Wong, P. C.; He, K.; Morin, P. E.; Chang,
C.-W.; Cheney, D. L.; Knabb, R. M.; Wexler, R. R.; Lam, P. Y. S. Bioorg. Med. Chem.
Lett. 2008, 18, 2845.
7. The fXa IC50 values were determined by the method described in: Sinha, U.; Ku,
P.; Malinowski, J.; Zhu, B. Y.; Scarborough, R. M.; Marlowe, C. K.; Wong, P. W.;
Lin, P. H.; Hollenbach, S. J. Eur. J. Pharmacol. 2000, 395, 51.
8. For description of our human plasma-based thrombin generation assay, see:
Sinha, U.; Lin, P. H.; Edwards, S. T.; Wong, P. W.; Zhu, B.; Scarborough, R. M.; Su,
T.; Jia, Z. J.; Song, Y.; Zhang, P.; Clizbe, L.; Park, G.; Reed, A.; Hollenbach, S. J.;
Malinowski, J.; Arfsten, A. E. Arterioscler. Thromb. Vasc. Biol. 2003, 23, 1098.
9. The pKa and cLogD values were calculated using the program commercially
available from the ACD Labs.
10. The strategy of using N,N-disubstituted benzamidines for P4 motifs was also
investigated at the same time in the other series of fXa inhibitors we had: (a)
Jia, Z. J.; Wu, Y.; Huang, W.; Zhang, P.; Song, Y.; Woolfrey, J.; Sinha, U.; Arfsten,
A. E.; Edwards, S. T.; Hutchaleelaha, A.; Hollenbach, S. J.; Lambing, J. L.;
Scarborough, R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett. 2004, 14, 1229; (b) Jia, Z.
J.; Su, T.; Zuckett, J. F.; Wu, Y.; Goldman, E. A.; Li, W.; Zhang, P.; Clizbe, L. A.;
Song, Y.; Bauer, S. M.; Huang, W.; Woolfrey, J.; Sinha, U.; Arfsten, A. E.;
Hutchaleelaha, A.; Hollenbach, S. J.; Lambing, J. L.; Scarborough, R. M.; Zhu, B.-
Y. Bioorg. Med. Chem. Lett. 2004, 14, 2073.
11. (a) Scarborough, R. M.; Gretler, D. D. J. Med. Chem. 2000, 43, 3453; (b) Okumura,
K.; Shimazaki, T.; Aoki, Y.; Yamashita, H.; Tanaka, E.; Banba, S.; Yazawa, K.;
Kibayashi, K.; Banno, H. J. Med. Chem. 1998, 41, 4036; (c) Hayashi, Y.; Katada, J.;
Harada, T.; Tachiki, A.; Iijima, K.; Takiguchi, Y.; Muramatsu, M.; Miyazaki, H.;
Asari, T.; Okazaki, T.; Sato, Y.; Yasuda, E.; Yano, M.; Uno, I.; Ojima, I. J. Med.
Chem. 1998, 41, 2345.
12. Human plasma protein binding, expressed as unbound fraction, was
determined by this protocol: The compound stock solutions were diluted in
1.0 M HEPES (pH 7.4) to yield a 1.0 mM working solution. The working solution
was added to plasma samples (EDTA was used as anticoagulant) in a ratio of 1/
100 yielding a final concentration of 10
incubated at 37 °C for 30 min. At the conclusion of the incubation 3 aliquots
(450 L) each were added to a centrifugal filter device fitted to a 96 well plate.
lM. The mixture was gently mixed and
l
Standards were prepared in protein free human plasma and transferred to the
centrifugal filter device fitted to the same plate. The plates were centrifuged for
25 min at 32 °C in a centrifuge. 15
round bottom 96 well plate and 15
mL) as internal standard was added followed by 60
l
l
L each of the filtrate was transferred to a
L of acetonitrile including KN1022 (1 g/
L of DI water. Plates were
l
l
placed on a Multi-Tube Vortexer for 30 s and vortexed. Concentrations in the
filtrate were determined by LC/MS/MS and using standard curves prepared in
ultra filtrated plasma.
13. Preparation of compound 11: (a) To a rapidly stirred solution of KOBut (224 mg,
2 mmol) in 10 mL of THF was added 2-amino-5-chloropyridine (128 mg,
1 mmol). After 10 min, isatoic anhydride 28 (163 mg, 1 mmol) was added in
three portions. Stirring was allowed to continue for 10 more minutes and the
mixture became gelatinous. The reaction was quenched with 10 mL of water,
and the resulting slurry was filtered and air-dried to give compound 29
(170 mg) as a pale yellow solid. (b) Compound 29 (123 mg) suspended in 5 mL
of THF was treated with 4-cyanobenzoyl chloride (83 mg) at rt for 30 min. The
mixture was filtered and washed with small amount of chilled methanol to
give compound 30 (145 mg) as a grey solid. (c) Hydrogen sulfide (gas) was
bubbled into a suspension of compound 30 (0.1 g) in 5 mL of pyridine and
0.5 mL of triethylamine until saturation. The mixture was stirred at rt for
overnight and the volatile was evaporated. The residue was taken up by 3 mL
of acetone and to it 0.5 mL of iodomethane was added. The resulting mixture
was refluxed for 30 min and evaporated to give a yellow solid, which was
treated with a premixed solution of 2 mL of 2 N dimethylamine in THF and
0.2 mL of acetic acid. The reaction mixture was refluxed for 10 min. The desired
product 11 was isolated using reverse phase HPLC with water–acetonitrile
(0.1% TFA) as its TFA salt (70 mg).
14. The fXa Ki value was determined by the method described in: Betz, A.; Wong, P.
W.; Sinha, U. Biochemistry 1999, 38, 14582.