Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.06.098

The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC₅₀ of 7 nM and EC_2×PT of 1.7 μM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.

Full text of DOI:10.1016/j.bmcl.2011.06.098

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7516–7521
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arylsulfonamidopiperidone derivatives as a novel class of factor Xa inhibitors
⇑
Yan Shi , Stephen P. O’Connor, Doree Sitkoff, Jing Zhang , Mengxiao Shi, Sharon N. Bisaha, Ying Wang,
Chi Li, Zheming Ruan, R. Michael Lawrence, Herbert E. Klei, Kevin Kish, Eddie C.-K. Liu, Steve M. Seiler,
Liang Schweizer, Thomas E. Steinbacher, William A. Schumacher, Jeffrey A. Robl, John E. Macor,
Karnail S. Atwal, Philip D. Stein
Research and Development, Bristol-Myers Squibb Company, PO Box 5400, Princeton, NJ 08543-5400, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and
selective FXa inhibitors is reported. The arylsulfonamide–valerolactam scaffold was derived based on
the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The
SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC50 of 7 nM
Received 3 June 2011
Accepted 22 June 2011
Available online 28 June 2011
and EC2ÂPT of 1.7 lM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide–val-
erolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4
pockets.
Keywords:
Factor Xa inhibitor
Bioisostere
Ó 2011 Elsevier Ltd. All rights reserved.
Valerolactam
Arylsulfonamide
Antithrombotic agent
X-ray crystal structure
Thromboembolic events are a leading cause of mortality world-
wide.1 Inhibition of the trypsin-like serine protease factor Xa (FXa)
has emerged as a key point of intervention in the blood coagulation
cascade for the development of antithrombotic agents for some
time.2 Selective factor Xa inhibitors may effectively block coagula-
tion, since factor Xa is positioned at the start of the common
pathway of the extrinsic and intrinsic coagulation systems.3
Currently, rivaroxaban4 and apixaban5 have been approved in the
EU for the prevention of venous thromboembolism (VTE) in adults
undergoing elective knee or hip replacement surgery; edoxaban6
has been approved in Japan for prevention of venous thromboem-
bolic events in patients undergoing major orthopedic surgery;
other oral and direct factor Xa inhibitors such as betrixaban7 and
darexaban (YM150)8 are in late stages of clinical development.
Factor Xa contains a deep S1 and a box-like S4 recognition site
at the enzyme’s active site. Potent FXa inhibitors generally require
both an S1 and an S4 binding element which are connected
through L-shaped or other ‘bent’ scaffolds.2 We have previously
reported several series of caprolactam based FXa inhibitors
containing a ketene aminal 1,9 a cyanoguanidine 2,10 and an aroyl-
guanidine 311 as linkers of the P1 and P4 pharmacophores. To iden-
tify suitable backups for these initial series, we have focused on
NC
HN
N
NC
HN
CN
O
O
N
N
N
H
N
H
N
N
O
O
1
O
2
O
IC50 = 30 nM
EC2xPT = 19 µM
IC50 = 12 nM
EC2xPT = 30 µM
O
O
N
N
N
O
O
O
S
N
N
N
H
O
O
N
HN
N
H
N
O
O
4
3
IC50 = 1.1 µM
O
IC50 = 7 nM
EC2xPT = 2.5 µM
⇑
Corresponding author. Tel.: +1 609 818 4124; fax: +1 609 818 3450.
E-mail address: yan.shi@bms.com (Y. Shi).
Current address: Hoffman-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110,
USA.
Scheme 1. The ketene aminal 1, cyanoguanidine 2, aroylguanidine 3 and benzo-
furan-5-sulfonamide 4.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.098

Y. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7516–7521
7517
O
O
O
NH2
SO2Cl
a, b
c
S
N
N
N
O
O
O
O
5
6
4
O
O
O
O
d
S
N
N
N
N
H2N
N
Ar
O
O
7
8-23
Scheme 2. The synthesis of 4 and 8–23. Reagents and conditions: (a) NaNO2, HCl/HOAc, À10 °C to À5 °C. (b) SO2, CuCl, 29% yield for 2 steps. (c) 7, EtOAc/NaHCO3 (sat.), 83%
yield. (d) Arylsulfonyl chloride, EtOAc/NaHCO3 (sat.), 65–93% yield.
O
O
a
b, c
N
BOC
N
H2N
O
N
H
OH
O
O
24
25
O
O
O
O
O
O
R1
d
S
N
S
N
N
N
N
OH
O
R2
O
Cl
Cl
27-36, 40
26
O
O
O
O
O
O
e
S
N
S
N
N
N
N
N
O
O
O
O
NH2
HN
Cl
Cl
34, 35
37, 38
O
O
O
O
O
O
S
N
S
N
e
N
N
N
N
O
O
N
Cl
NH
Cl
BOC
36
39
j,
e
g
f
h
43
i
k
41
42
44
45
46
Scheme 3. The synthesis of 27–46. Reagents and conditions: (a) MeOH, HCl, 0 °C to rt, 96%. (b) 6-Chloronaphthalene-2-sulfonyl chloride, EtOAc/NaHCO3 (sat.). (c) LiOH.2H2O,
THF-H2O, 85% yield for 2 steps. (d) HNR1R2, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrchloride (EDCI), Et3N, CH2Cl2, 25–90% yield. (e) TFA, CH2Cl2, 60–98% yield.
(f) Acetic anhydride, Et3N, CH2Cl2, 85% yield. (g) TMSNCO, CH2Cl2, 93% yield. (h) (1) Ethyl 2-chloro-2-oxoacetate, Et3N, CH2Cl2; (2) NH4OH, 32% for 2 steps. (i) Methylsulfonyl
chloride, Et3N, CH2Cl2, 63% yield. (j) 1-(tert-Butoxycarbonyl)piperidine-4-carboxylic acid, EDCI, Et3N, CH2Cl2, 91% yield. (k) 3-Amino-1H-pyrazole-4-carboxylic acid, Et3N,
CH2Cl2, 23% yield.
preparing structurally diverse compounds that would address
potential issues in the parent series. To this end, targets were
designed to maintain the key interactions in the S1 and S4 pockets
of FXa by modifying the central scaffold and the linker region to
eliminate the possibility of generating an aniline-type metabolite.
Inspection of the X-ray crystal structures of 210 and a compound
closely related to 311 bound to FXa suggested that the benzofuran-
guanidine moiety may be replaced with an arylsufonamide,12
whilst maintaining the proper projection of the aryl group into
the deep S1 pocket. To test this hypothesis, we synthesized three

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Y. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7516–7521
Table 1
Table 2
SAR of the aromatic binding element
SAR of the amide pharmacophore
O
O
O
O
O
O
R1
S
N
S
N
N
N
N
N
Ar
O
O
R2
Cl
19, 27-33, 37-40
4, 8-23
Compound
NR1R2
IC50 (nM)
EC2ÂPT
12
(lM)
a
b
a
b
Compound
Ar
IC50 (nM)
EC2ÂPT
(
lM)
19
N
N
32
4
1110
–
O
27
28
29
130
129
337
38
8
9
9750
–
–
Br
N
N
N
>50
>50
11750
S
N
Cl
Cl
10
11
61
11
–
S
30
31
32
33
58
121
26
40
–
N
N
N
N
N
S
1015
S
12
13
2794
122
–
S
O
N
674
34
1957
–
S
Cl
Cl
14
15
60
20
–
37
209
17
S
NH2
1948
N
S
38
39
1330
22
–
NH2
NH
16
17
1708
2218
–
–
N
N
3.5
40
13
4.1
N
Cl
O
a
Cl
Cl
IC50 values are measured against human factor Xa utilizing the cleavage of a
18
19
1010
32
–
synthetic substrate S-2222.
b
Concentration of inhibitor required to double the prothrombin based clotting
time in human plasma; data are the average of two independent determinations
12
benzofuran-5-sulfonamide compounds with different ring sizes in
the central lactam core. While the 7-membered caprolactam-con-
taining compound is not active, both 5- and 6-membered lactam
compounds showed weak activity against human FXa, with the
20
21
19
32
11
50
Br
Cl
N
N
H
benzofuran sulfonamidopiperidine-2-one
4 having an IC50 of
1.1 M (Scheme 1). Based on this lead, we initiated SAR studies
l
S
to further improve the potency. This communication describes
the in vitro SAR of this novel series of sulfonamidopiperidine-2-
one based FXa inhibitors.13
22
23
51
14
29
13
S
N
Cl
Cl
Compounds 4 and 8–23 were synthesized by the reaction of
various aryl- or alkyl-sulfonyl chlorides and the valerolactam
amine 7 in 65–93% yields as shown in Scheme 2. For example,
the 2-methylbenzofuran-5-amine 5 was treated with NaNO2/HCl/
HOAc and the corresponding diazonium was sulfonylated with
SO2/CuCl to give 2-methylbenzofuran-5-sulfonyl chloride 6 in
29% yield. Further reaction of 6 with lactam amine 7 afforded the
corresponding sulfonamides 4 in 83% yield.
S
a
IC50 values are measured against human factor Xa utilizing the cleavage of a
synthetic substrate S-2222.
Concentration of inhibitor required to double the prothrombin based clotting
time in human plasma; data are the average of two independent determinations.
b

Y. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7516–7521
7519
a
F174
b
w1
Y99
G216
C220
C191
W215
w2
S214
D189
Y228
Figure 1. (a) X-ray crystal structure of 40 bound in FXa. The direct hydrogen bond between the ligand’s lactam carbonyl and Gly216 backbone amide NH (3.3 Å) and the
interaction between the ligand’s lactam carbonyl and Ser214 backbone carbonyl via water w2 (2.8 Å, 2.6 Å) are highlighted, in addition to the contact between one of the
sulfonamide oxygens and FXa’s Cys220 sulfur (3.7 Å). Conserved P4 water w1 is also shown. (b) The overlay of cyanoguanidine–caprolactam 2 (orange carbons) with 40
(magenta carbons) bound in FXa. Figures were created using PyMol (http://www.pymol.org).
Synthesis of compounds 27–46 are outlined in Scheme 3. The
lactam acid 24 was synthesized following Friedinger’s protocol.14
Simultaneous esterification and deprotection afforded the interme-
diate ester 25, which was reacted with the 6-chloronaphthalene-2-
sulfonyl chloride to give the sulfonamide. Further hydrolysis with
lithium hydroxide provided the common intermediate acid 26.
EDC mediated amide formation furnished the compounds 27–36
and 40. Deprotection of the BOC group in 34–36 afforded com-
pounds 37–39. Further derivatization of the amine function in 39
provided compounds 41–46.
group. Selected analogs are shown in Table 2. Similar to the thio-
urea and cyanoguanidine series,10 the replacement of the pyrroli-
dine with non-cyclic amines led to significant losses in potency
(data not shown). Substitutions at the C-2 and C-3 positions on
the pyrrolidine ring were tolerated with the C-2 substituted analogs
slightly more potent than the C-3 substituted ones. For example,
compound 27 with a 2-methylpyrrolidine has an IC50 of 130 nM.
The 2-(3-pyridyl) substituted pyrrolidine 28 (IC50 = 129 nM) is
slightly more potent than the 3-substituted analog 29 (IC50
=
337 nM), although both show diminished EC2ÂPT (>50 M). Modest
l
Early efforts in this study focused on analogs of 4 where the aryl
group was varied. Selected analogs are shown in Table 1 to illus-
trate the SAR. The monocyclic arylsulfonamide compounds are
generally weak FXa inhibitors (compounds 8 and 9), with the 5-
changes to the ring size (thiazolidine, piperidine, morpholine and 4-
methylpiperazine) also provided compounds less active than 19.
While the thiazolidine compound 30 (IC50 = 58 nM) is only slightly
less potent than 19, the piperidine compound 31 (IC50 = 121 nM) is
about 4-fold less potent than 19, and compounds with a morpho-
line (32, IC50 = 674 nM) and 4-methylpiperazine (33, IC50 = 1957
nM) are significantly less active.
Among the substituted piperidine analogs 37–40, the 3-amino-
methyl substituted piperidine compound 37 (IC50 = 209 nM) is
only slightly less active than the unsubstituted piperidine com-
pound 31, but it is about 6-fold more potent than its 4-substituted
analog 38 (IC50 = 1330 nM). Finally, replacement of the pyrrolidine
group in 19 with a 3,7-diazabicyclo[3.3.1]nonane or a cytisine pro-
vide the bicyclic analogs 39 and 40, both of them are more potent
chloro-2-yl-thiophene analog 9 having an IC50 of 11.8 lM. Adding
an ethylene spacer between the thiophene and sulfonyl group in-
creases activity dramatically. Both compounds 10 and 11 are sig-
nificantly more potent than 9. The (E)-2-(5-chlorothiophen-2-
yl)ethylene derivative 10 (IC50 = 61 nM) is 192-fold more potent
than 9, and 16-fold more potent than its 5-methylthiophene analog
11 (IC50 = 1015 nM). An activity difference between chloro- and
methyl-substitution is also found in the benzothiophene series.
The 5-chlorobenzo[b]thiophene-2-sulfonamide 14 (IC50 = 60 nM)
is 46-fold more active than unsubstituted benzo[b]thiophene-2-
sulfonamide 12 (IC50 = 2792 nM), and is 32-fold more potent than
FXa inhibitors than 19 with FXa IC50s of 22 nM (EC2ÂPT = 3.5
and 13 nM (EC2ÂPT = 4.1 M), respectively.
lM)
its
5-methylbenzo[b]thiophene-2-sulfonamide
analog
15
l
(IC50 = 1948 nM). This is consistent with the previously noted pref-
erence for chloro- versus methyl-substituted P1 groups in FXa and
can be explained by the stronger hydrophobic nature of chloro-
To elucidate the binding mode of this arylsulfonamide–valero-
lactam series, an X-ray crystal structure of compound 40 bound
to FXa was determined at a resolution of 2.42 Å. The resulting com-
plex is shown in Figure 1.17 As expected, the arylsulfonamide
creates the needed L-shaped turn seen in many FXa inhibitors,
allowing the 6-chloro-2-naphthyl group to fit deep into the S1
pocket where the chlorine atom fills a key hydrophobic interaction
site above Tyr228 (3.4 Å from Cl to C-OH). The cytisine occupies the
hydrophobic S4 pocket with the piperidine ring bound by Tyr99,
Trp215 and Phe174. One of the sulfonyl oxygens contacts the disul-
fide bond between Cys191 and Cys220, and the valerolactam
carbonyl forms an elongated hydrogen bond with NH of Gly216
(3.3 Å); the valerolactam carbonyl also interacts with Ser214 back-
bone carbonyl via a crystallographic water (2.8 Å and 2.6 Å between
the water–valerolactam C@O and the water–Ser214 C@O, respec-
tively). The structure of 40 in FXa has many features in common
with the previously reported X-ray crystal complex structures
versus methyl-substituted aromatic rings, Cl-p interactions, and
by enhanced electrostatic interactions with FXa’s Asp189 due to
Cl-induced polarization of the P1 aryl ring.15
Replacement of the 2-methyl-2-yl-benzofuran group in 4 with a
2-naphthyl group provides 16, which is slightly less active
(IC50 = 1708 nM). A survey of chloro-substitution on the 2-naph-
thyl group leads to the 6-chloro-2-naphthyl compound 19, which
has an IC50 of 32 nM (EC2ÂPT = 12 l
M).16 The 6-bromo-2-naphthyl
compound 20 (IC50 = 19 nM) has similar FXa activity to 19. Other
chloro-substituted bicyclic aryl sulfonamides 21–23 also showed
good anti-FXa activity, with compound 23 having an IC50 of
14 nM (EC2ÂPT = 13 lM).
With an optimized P1 pharmacophore and central lactam ring in
hand, we next turned our attention to the SAR of the P4 binding

7520
Y. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7516–7521
Table 3
SAR of the 3,7-diazabicyclo[3.3.1]nonane P4 pharmacophore
O
O
O
S
N
N
N
H
O
Cl
N
R1
39, 41-46
a
b
Compound
R1
IC50 (nM)
EC2ÂPT
3.5
(l
M)
Pcc nm/s)
<15
39
H
22
11
O
O
41
42
2.5
5
73
14
<15
NH2
O
43
44
32
10
3.2
9
<15
<15
NH2
O
O
S
O
O
45
46
13
7
2
<15
<15
NH
O
1.7
NH
H2N
N
Figure 2. The i.v. dosing of 40 in rat.
a
IC50 values are measured against human factor Xa utilizing the cleavage of a
synthetic substrate S-2222.
pounds 42 and 44 showed improved IC50 values, they are less
active in EC2ÂPT. The more polar oxalamide compound 43 is less
potent in IC50 but maintains equipotent EC2ÂPT. Further improve-
ments in potency are seen in compounds 45 and 46, with
b
Concentration of inhibitor required to double the prothrombin based clotting
time in human plasma; data are the average of two independent determinations.
c
Caco-2 cell permeability (apical to basolateral) measured at 3 lM of compound.
compound 46 having an IC50 of 7 nM and EC2ÂPT of 1.7 lM.
Similar to the lead compounds 210 and 3,11 the arylsulfonami-
dopiperidone derivatives described above are selective FXa inhibi-
tors relative to related trypsin-like serine proteases. Table 4 shows
the selectivity profile of compound 40. Furthermore, compound 40
showed activity in rats after intravenous administration and
measurement of ex vivo clotting time.18 As shown in Figure 2,
intravenous bolus injection of 10 mg/kg of 40 led to an 1.34-fold
increase in prothrombin time (PT) after 2 h. Unfortunately, despite
their other favorable properties, compounds containing a 7-substi-
tuted-3,7-diazabicyclo[3.3.1]nonan-3-yl or a cytisine as the P4
pharmacophore generally have limited Caco-2 cell permeability
and a short metabolic half life in liver microsomes.19 Further work
is necessary to address these issues before this series can furnish
acceptable antithrombotic drug candidates.
In summary, we have discovered a novel series of FXa inhibi-
tors, which contain an arylsulfonamidopiperidone P1 and a novel
cytisine or 7-substituted-3,7-diazabicyclo[3.3.1]nonan-3-yl P4
pharmacophore. These compounds exhibit potent and highly selec-
tive anti-FXa activity. The SAR demonstrates that sulfonamidopi-
peridones are excellent bioisosteres for the cyanoguanidine–
caprolactam core in previously reported series; both anchor the
P1 and P4 pharmacophores in an L-shaped conformation. The
SAR is consistent with the binding conformation revealed by the
X-ray structure of compound 40 bound to FXa, where the
6-chloro-2-naphthyl group resides in the S1 pocket and the bulky
cytisine or 7-substituted-3,7-diazabicyclo[3.3.1]nonan-3-yl group
is bound to the S4 pocket of FXa.
Table 4
Selectivity profile of 40
Hman enzymes
Kia (nM)
Fctor Xa
4.71
Kallikrein
Thrombin
Tryptase
Activated protein C
Factor XIa
FVIIa
u-PA
Plasmin
t-PA
Chymotrypsin
6218
3280
4979
22500
13330
3684
15100
15780
1279
9359
a
Ki values are calculated from the IC50 values assuming competitive inhibition
versus the low molecular weight synthetic substrates using the relationship,
Ki = IC50/(1 + [S]/Km), where S is the substrate concentration in the assay and Km is
the Michaelis constant for that substrate. Data are the average of two independent
determinations.
between FXa and 210 and a compound closely related to 311, indicat-
ing that the linkers between the P1 and P4 groups are for the most
part interchangeable. It appears that the distal pyridone ring in 40 is
largely solvent exposed and modification of this portion of the li-
gand may provide compounds with improved physicochemical
properties without strongly impacting their FXa inhibitory activity
(IC50). Thus, further SAR investigation was concentrated on the
derivatization of the P4 NH group in compound 39 and results are
summarized in Table 3 (compounds 41–46).
Acknowledgments
Among the 7-acetyl-, 7-carboxamide, 7-oxalamide-, and 7-
methylsufonyl- substituted analogs 41–44, only the acetyl
compound 41 is more potent than 39 both in IC50 and EC2ÂPT
We thank Drs. P.T. Cheng, P.Y.S. Lam, R.R. Wexler for insightful
review of this work.
.
Although both the 7-carboxamido- and 7-methylsulfonyl-com-

Y. Shi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7516–7521
7521
10. Shi, Y.; Zhang, J.; Shi, M.; O’Connor, S. P.; Bisaha, S. N.; Li, C.; Sitkoff, D.;
Pudzianowski, A. T.; Huang, C.; Chong, S.; Klei, H.; Kish, K.; Yanchunas, J.; Liu, E.
C.-K.; Hartl, K. S.; Seiler, S. M.; Steinbacher, T. E.; Schumacher, W. A.; Atwal, K.
S.; Stein, P. D. Bioorg. Med. Chem. Lett. 2009, 19, 4034.
11. Shi, Y.; Li, C.; O’Connor, S. P.; Zhang, J.; Shi, M.; Bisaha, S. N.; Wang, Y.; Sitkoff,
D.; Pudzianowski, A. T.; Huang, C.; Klei, H.; Kish, K.; Yanchunas, J., Jr.; Liu, E. C.-
K.; K.S., Hartl; Seiler, S. M.; Steinbacher, T. E.; Schumacher, W. A.; Atwal, K. S.;
Stein, P. D. Bioorg. Med. Chem. Lett. 2009, 19, 6882.
12. For other arylsulfonamide-lactam based FXa inhibitors, see: (a) Smallheer, J.
M.; Wang, S.; Laws, M. L.; Nakajima, S.; Hu, Z.; Han, W.; Jacobson, I.; Luettgen, J.
M.; Rossi, K. A.; Rendina, A. R.; Knabb, R. M.; Wexler, R. R.; Lam, P. Y.; Quan, M.
L. Bioorg. Med. Chem. Lett. 2008, 18, 2428; (b) Chan, C.; Borthwick, A. D.; Brown,
D.; Burns-Kurtis, C. L.; Campbell, M.; Chaudry, L.; Chung, C.-W.; Convery, M. A.;
Hamblin, J. N.; Johnstone, L.; Kelly, H. A.; Kleanthous, S.; Patikis, A.; Patel, C.;
Pateman, A. J.; Senger, S.; Shah, G. P.; Toomey, J. R.; Watson, N. S.; Weston, H.
E.; Whitworth, C.; Young, R. J.; Zhou, P. J. Med. Chem. 2007, 50, 1546; (c)
Kleanthous, S.; Borthwick, A. D.; Brown, D.; Burns-Kurtis, C. L.; Campbell, M.;
Chaudry, L.; Chan, C.; Clarte, M.-O.; Convery, M. A.; Harling, J. D.; Hortense, E.;
Irving, W. R.; Irvine, S.; Pateman, A. J.; Patikis, A. N.; Pinto, I. L.; Pollard, D. R.;
Roethka, T. J.; Senger, S.; Shah, G. P.; Stelman, G. J.; Toomely, J. R.; Watson, N. S.;
West, R. I.; Whittaker, C.; Zhou, P.; Young, R. J. Bioorg. Med. Chem. Lett. 2010, 20,
618.
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16. IC50s are measured against human factor Xa utilizing the cleavage of
a
synthetic substrate S-2222. The EC2ÂPT is the concentration of inhibitor
required to double the prothrombin based clotting time in human plasma.
For detailed assay methods, see Ref.15
17. The X-ray crystal structure coordinates of 40 in human Factor Xa have been
deposited in the Protein Data Bank (PDB code 3SW2).
18. Compound testing protocol: male Sprague–Dawley rats (320–390 g) were
fasted overnight and then anesthetized with sodium pentobarbital (50 mg/kg,
i.p.). The trachea was cannulated with PE-205 tubing to assure airway patency.
Catheters (PE-50) were placed in the right carotid artery for blood withdrawal
and in the left jugular vein for saline infusion (25 lL/min throughout the
experiment) and for i.v. dosing of test compound. Animals received compound
by i.v. (10 mg/kg) route in a 1 mg/mL volume of saline vehicle followed by a
0.3 mL saline flush. Arterial blood samples (0.5 mL) were withdrawn into 3.8%
Na-citrate (1/10; v/v) for ex vivo prothrombin time (PT) determination before
(0 min control), and at 30, 60, 90 and 120 min after test compound dosing. The
PT was measured using a Amelung KC4A micro coagulation analyzer (Heinrich
Amelung GmbH, Lemgo, Germany) and the standard procedure described for
Dade Thromboplastin-C reagent (Baxter Healthcare Corp., Miami, FL).
19. For example, compounds 40 and 41 have Caco-2 cell permeability (apical to
basal) of 25 and 73 nm/s at pH 6.5 respectively; both compounds have half
lives of less than 2 min in liver microsomes (human, rat, mouse, dog,
9. Shi, Y.; Zhang, J.; Stein, P. D.; Shi, M.; O’Connor, S. P.; Bisaha, S. N.; Li, C.; Atwal,
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5453.
cynomulgus monkey). Assay conditions: 0.5 lM of compound is incubated
with liver microsomal protein (1.0 mg/mL) in the presence of NAPDH
(1.0 mM), NaPi (100 mM), MgCl2 (5.0 mM) at pH 7.4 at 37 oC.