Cycloalkanediamine derivatives as novel blood coagulation factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.05.068

This paper describes the synthesis of orally available potent fXa inhibitors 2 and 3 by modification of the piperazine part of lead compound 1. Carbonyl derivative 3 showed potent fXa activity but not sulfonyl derivative 2. Among the compounds synthesized, cyclohexane derivatives 3g and 3h and cycloheptane derivative 3j had potent anticoagulant activity as well as anti-fXa activity. Synthetic study of the optical isomers of 3g demonstrated that (-)-3g had more potent activity.

Full text of DOI:10.1016/j.bmcl.2007.05.068

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4683–4688
Cycloalkanediamine derivatives as novel blood coagulation
factor Xa inhibitors
Tsutomu Nagata,a,* Toshiharu Yoshino,a Noriyasu Haginoya,a Kenji Yoshikawa,a
Yumiko Isobe,b Taketoshi Furugohrib and Hideyuki Kannoa
aMedicinal Chemistry Research Laboratory, Daiichi Pharmaceutical Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku,
Tokyo 134-8630, Japan
bNew Product Research Laboratories 2, Daiichi Pharmaceutical Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
Received 28 December 2006; revised 27 April 2007; accepted 22 May 2007
Available online 25 May 2007
Abstract—This paper describes the synthesis of orally available potent fXa inhibitors 2 and 3 by modification of the piperazine part
of lead compound 1. Carbonyl derivative 3 showed potent fXa activity but not sulfonyl derivative 2. Among the compounds syn-
thesized, cyclohexane derivatives 3g and 3h and cycloheptane derivative 3j had potent anticoagulant activity as well as anti-fXa
activity. Synthetic study of the optical isomers of 3g demonstrated that (À)-3g had more potent activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The extrinsic and intrinsic coagulation systems converge
at the activation of factor X to Xa. Activated factor X
(fXa) has an important role in conversion of prothrom-
bin (fII) to thrombin (fIIa), which produces blood clots.1
Thus, fXa is a key enzyme in the coagulation cascade
and also an attractive target enzyme for the therapy of
thrombosis and related diseases.
OH
O
H2N
NH
N
NH
O
Figure 1. Structure of DX-9065a.
groups. The study showed that 5-chloroindole and 5-
methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine groups
were suitable sub-structures for the S1 and aryl (S4)
binding sites of fXa,4,5 respectively (Fig. 2).6 However,
the oral bioavailability of compound 1 was not
satisfactory.
A decade ago, we reported a low-molecular selective fXa
inhibitor DX-9065a (Fig. 1) having two amidino
groups.2 DX-9065a showed potent fXa inhibitory activ-
ity in vitro and ex vivo, while it had poor oral bioavail-
ability (10% in monkeys) probably due to its strong
basic amidino groups. As a result, DX-9065a has only
been used as an injectable formulation in clinical studies.
Therefore, we examined the synthesis of orally available
potent fXa inhibitors by modification of the piperazine
part of compound 1. Figure 3 shows the drug design
Many researchers have attempted synthesis of fXa
inhibitors with improved oral bioavailability.3a Zeneca’s
researchers have reported epoch-making non-amidino
fXa inhibitors, 1-[(6-chloro-2-naphthyl)sulfonyl]-4-[(1-
pyridin-4-ylpiperidin-4-yl)carbonyl]piperazine.3b We also
previously synthesized a variety of non-amidino deriva-
tives, and reported compound 1 having 5-chloroindole
and 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine
Cavity Ref 5
Aryl (S4) Binding Site
S
Cl
O
N
O
S
N
N
N
N
H
S1 Binding Site
O
Cation hole Ref 5
Figure 2. Binding mode of fXa and compound 1.
Keywords: Factor Xa inhibitors; Cycloalkanediamine; Non-amidine;
Anticoagulant.
*
Corresponding author. E-mail: nagatso1@daiichipharm.co.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.068

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4683–4688
New ring formation in this part
O
O
S
S
H
N
H
N
N
N
H
O
S
O
N
N
N
HN
N
N
X
Cl
Cl
1
fXa IC50 = 5.0 nM
2: X = -SO2-
3: X = -(C=O)-
Deletion of this ethylene moiety
Figure 3. Drug design.
of cycloalkanediamine derivatives. Sulfonyl derivative 2
was first synthesized based on the drug design, though it
did not have potent activity. In the next step, we
attempted the synthesis of carbonyl derivative 3, which
showed highly potent activity. In this paper, we describe
the synthesis and pharmacological properties of cycloal-
kanediamine derivatives 2 and 3.
2. The 1-benzenesulfonyl group was removed in the
course of the condensation.
Cycloalkanediamine 4 was acylated with commercially
available 5-chloroindole-2-carboxylic acid to give mono-
amide 8, which was successively condensed with
thiazolopyridinecarboxylic acid 7 to provide carbonyl
derivative 3. However, sometimes the yield of mono-
amide 8 from cycloalkanediamine 4 (especially in the
acylation of trans-diamine) was significantly low because
of the production of diacylated compound 9. In that case,
mono-amide 8 was synthesized via mono-Boc protected
derivative 10 in a better yield.
Scheme 1 shows the synthetic pathway of the key inter-
mediate cycloalkanediamines. The cycloalkanediamines
were synthesized from commercially available dicarbox-
ylic acids, which were converted to the corresponding
cycloalkanediamines via
a Curtius rearrangement
(Route A). Cycloalkanediamines were also synthesized
from commercially available diols or their precursor
cycloalkenes (Route B). Oxidation of the cycloalkenes
to cis- or trans-diols was carried out by use of OsO4/
NMO (Route B-1) or H2O2 (Route B-2). The diols were
mesylated to give di-mesylated compounds, which were
successively treated with sodium azide and reduced over
Pd–C to yield cycloalkanediamines.
Compounds 2 and 3 were also prepared via mono-amide
11, which was prepared from cycloalkanediamine 10
using a method similar to that of mono-amide 8.
Since these cycloalkanediamines are racemates, we have
synthesized optical isomers to study pharmacological
properties. We selected a compound (3g) and synthe-
sized its optical isomers. The synthetic pathway of the
optical isomers (+)-(1S,2R)-3g and (À)-(1R,2S)-3g is
outlined in Scheme 3. Optical resolution8 of racemic
cis-cyclohexanediamine 12 afforded optical isomers 13
and 14. Optical isomers 13 and 14 were condensed with
5-chloroindole-2-carboxylic acid to give (+)-(1S,2R)-3g
and (À)-(1R,2S)-3g, respectively. The absolute configu-
ration of (À)-(1R,2S)-3g was determined by X-ray
The synthetic pathway of sulfonyl derivative 2 and car-
bonyl derivative 3 is outlined in Scheme 2. Treatment
of cycloalkanediamine
4 with (1-benzenesulfonyl-
5-chloroindol-2-yl)sulfonyl chloride4 (5) afforded
sulfonamide 6. Compound 6 was condensed with 5-methyl-
4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylic
acid lithium salt7 (7) to give the desired sulfonyl derivative
Route A
Curtius
rearragement
H2N
HOOC
NH2
cis- or trans-
cycloalkanediamine
COOH
cis- or trans
dicarboxylic acid
Route B-1
Route B-2
1) NaN3
OsO4
HO
2) H2, Pd-C
MsCl
MsCl
cis
-
Ms
O
Ms
H2N
H2N
cycloalkanediamine
O
OH
cis-diol
NH2
NH2
1) NaN3
2) H2, Pd-C
H2O2
trans-
cycloalkanediamine
Ms
O
HO
O
OH
Ms
trans-diol
Scheme 1. Synthesis of cycloalkanediamine spacers.

T. Nagata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4683–4688
4685
a sealed tube for 2 days to afford a mixture of diastereo-
mers 16a and 16b, which were easily separated by silica
gel chromatography to give pure 16a and 16b.10 Com-
pound 16a was treated with 10% Pd–C and HCOONH4,
and successively protected using Boc2O to afford com-
pound 17. Compound 17 was mesylated and then trea-
ted with sodium azide to provide azide 18. Azide 18
was catalytically hydrogenated and then acylated with
thiazolopyridinecarboxylic acid 7 to give compound
19, which was successively treated with acid to yield
intermediate 14.
Cl
(a)
(c)
(b)
H2N
H2N
2
HN
NH2
(d)
S
N
O
O
SO2Ph
4
6
Cl
(b)
(c), (e)
H2N
3
Boc
N
HN
N
H
H
NH2
8
O
Table 1 shows in vitro anti-fXa activity of sulfonyl
derivatives 2a–f. The activity of all these compounds
was less potent than that of compound 1 (IC50:
5.0 nM). However, it is noted that cis-isomers were more
potent than trans-isomers and that cis-cyclopropane
derivative 2a showed the most potent activity.
10
Cl
O
H
N
N
H
HN
O
N
H
O
Cl
9
Table 2 shows the in vitro anti-fXa activity of carbonyl
derivatives 3a–l. Cyclohexane and cycloheptane deriva-
tives 3g, 3h, and 3j exhibited potent activity, while
3- to 5- and 8-membered ring derivatives 3a–f and 3k,l
showed less potent activity. Thus, the ring size signifi-
cantly impacted on the potency. With respect to config-
uration, trans-5- to 7-membered ring derivatives 3f, 3h,
and 3j were more potent than the corresponding cis-iso-
mers 3e, 3g, and 3i. However, the order of potency
between cis- and trans-isomers was reversed in 3- and
4-membered ring compounds.
(a), (f)
(c)
2
3
S
(b), (e)
N
10
H
N
NH2
N
11
Scheme 2. Reagents: (a) (1-benzenesulfonyl-5-chloroindol-2-yl)sulfo-
nyl chloride (5), TEA, DCM; (b) 5-methyl-4,5,6,7-tetrahydrothiazol-
o[5,4-c]pyridine-2-carboxylic acid lithium salt (7), WSCI, HOBt,
DMF; (c) 5-chloroindole-2-carboxylic acid, WSCI, HOBt, DMF; (d)
(Boc)2O, DCM or BocON, TEA, DCM; (e) HCl/EtOH or TFA,
DCM; (f) 1 N-NaOH, THF, EtOH.
Comparing sulfonyl derivatives 2a–f with carbonyl
derivatives 3a–l, there seems to be a distinct structure–
activity relationship in the ring size and cis-/trans-
configuration. In the sulfonyl derivatives, cis-cyclopro-
pane derivative 2a was the most potent, while
crystallographic analysis.9 Intermediate 14 was alterna-
tively prepared from epoxide 15 as follows. Epoxide 15
was treated with (R)-a-methylbenzylamine at 160 ꢁC in
O
O
R
O
(a)
S
S
S
N
S
S
N
+
R
H
N
H
N
NH2
N
N
NH2
H
N
N
NH2
N
14
(b)
13
12
(b)
Cl
Cl
O
O
S
R
S
S
N
N
R
S
H
H
N
HN
N
N
HN
N
N
N
H
H
O
O
(+)-(1S,2R)-3g
(-)-(1R,2S)-3g
O
(c)
(h), (i)
(f), (g)
(d) (e)
(j)
S
N
14
HO
N3
HO
HO
H
O
N
HN
N
HN
16a
38%
HN
HN
HN
Boc
+
Boc
Boc
15
19
18
16b
Ph
17
Ph
42%
Scheme 3. Reagents and condition: (a) optical resolution, CHIRAL-PACK AD; (b) 5-chloroindole-2-carboxylic acid, WSCI, HOBt, DMF (76% for
(+)-3g, 66% for (À)-3g); (c) (R)-a-methylbenzylamine, sealed tube, 160 ꢁC; (d) 10% Pd–C, HCOONH4, MeOH; (e) Boc2O, DCM (59%, two steps); (f)
MsCl, pyridine (94%); (g) NaN3, DMF (47%); (h) H2, 10% Pd–C; (i) 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-carboxylic acid lithium salt
(7), WSCI, HOBt, DMF (44%, two steps); (j) HCl/EtOH (quant.).

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4683–4688
Table 1. Sulfonyl derivatives
Table 2. Carbonyl derivatives
R1
=
R1
=
R2 =
R2 =
H
N
H
N
S
S
*
*
*
*
N
N
Cl
Cl
N
N
Compound Structure
Stereochemistry Anti-fXa
a
IC50 (nM)
Compound Structure
Stereochemistry Anti-fXa
a
IC50 (nM)
O
O
R1
N
H
2a
cis
270
R1
N
H
3a
cis
187
R2
R2
HN
HN
S
2b
O
O
trans
>10,000
3b
3c
3d
trans
>10,000
O
O
O
O
O
O
2c
2d
cis
3200
R1
N
H
R1
N
H
cis
265
R2
R2
HN
HN
HN
HN
S
S
trans
>10,000
O
O
O
trans
2000
2e
2f
cis
4400
R1
N
H
R1
3e
3f
cis
118
86
N
H
R2
HN
R2
O
O
trans
>10,000
trans
O
a The method for measuring anti-fXa activity was described in Ref. 11.
R1
N
H
trans-cycloheptane derivative 3j was the most potent
among the carbonyl derivatives. It is likely that confor-
mational differences, including difference in the distance
and bond angle between –NH–SO2– and –NH–CO–,
may affect potency.
3g
3h
cis
41
13
R2
trans
O
O
O
R1
N
H
Table 3 shows the anti-fIIa (anti-thrombin) activity and
anticoagulant activity (PTCT2) of carbonyl derivatives
3e–h,j. All the compounds listed in Table 3 showed high
selectivity for fXa versus fIIa. cis-Cyclohexane deriva-
tive 3g had the poorest selectivity, though it was still
more than 100-fold. Although derivative 3g was not
the most potent fXa inhibitor among the compounds
tested, it showed the best anticoagulant activity. In con-
trast, derivatives 3h and 3j had more potent anti-fXa
activity and exhibited somewhat less potent anticoagu-
lant activity.
3i
3j
cis
175
R2
HN
HN
trans
11.3
O
R1
N
H
3k
3l
cis
>1400
R2
O
trans
>10,000
a The method for measuring anti-fXa activity was described in Ref. 11.
Study of the optical isomers of 3g demonstrated that
(À)-(1R,2S)-3g had potent anti-fXa activity, while (+)-
(1S,2R)-3g showed 47-fold less potent activity than the
counterpart (À)-isomer. The former compound (À)-
(1R,2S)-3g also showed potent anticoagulant activity
(2.9 lM).
Table 3. Anti-fXa and anti-fIIa activities and anticoagulant activity
Compound
Anti-fXa
a
Anti-fIIa
b
PTCT2
in human
plasmac
(lM)
PTCT2
in rat
IC50
(nM)
IC50
(nM)
plasmac
(lM)
3e
118
86
27,000
36,000
4500
9.6
16
17.2
30.1
12
Table 4 shows ex vivo anti-fXa and anticoagulant activ-
ity in rats. Derivative 3g exhibited over 80% inhibition
for fXa from 0.5 to 4 h after oral administration of
30 mg/kg. Under the same conditions, derivative 3g also
showed a potent prolongation effect (1.17- to 1.23-fold)
on prothrombin time (PT). These data indicate that
derivative 3g has good oral availability as well as high
potency in vivo. On the other hand, derivatives 3f, 3h,
and 3j showed less potent activity than 3g.
3f
3g
41
4.4
>20
2.9
6.2
11.7
(+)-3g
(À)-3g
3h
753
16
>100
2600
>20
9.2
13
11.3
29,000
>100,000
9.5
14.1
3j
a The methods for measuring anti-fXa activitie was described in
Ref. 11.
b The methods for measuring anti-fIIa activitie was described in
Ref. 12.
c Anticoagulant activities in human and rat plasma were evaluated by
the plasma clotting time doubling concentration (PTCT2).13
Table 5 shows the ex vivo anti-fXa and anticoagulant
activity of optically active compound (À)-3g. Com-

T. Nagata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4683–4688
Table 4. Ex vivo anti-fXa and anticoagulant activities
4687
Compound
At 30 mg/kg (po) to rats
Anti-fXa activitya (%)
Prolongation effect of PTa (fold)
0.5 h
1 h
2 h
4 h
0.5 h
1 h
2 h
4 h
3f
3g
3h
3j
78.6 2.3
80.3 1.1
84.9 2.1
79.0 0.6
80.7 1.0
81.5 1.8
78.3 3.0
66.0 0.90
78.4 3.0
81.7 1.8
65.7 5.3
59.0 2.50
80.6 3.0
85.6 0.8
57.0 8.7
49.0 3.0
1.12 0.01
1.20 0.02
1.13 0.02
1.05 0.01
1.14 0.02
1.17 0.02
1.07 0.00
1.02 0.00
1.26 0.03
1.21 0.02
1.07 0.01
1.00 0.01
1.16 0.02
1.23 0.03
1.04 0.00
1.00 0.01
a The methods for measuring ex vivo anti-fXa and anticoagulant activities were described in Ref. 14. Values are expressed as means SE from four
rats.
Table 5. Ex vivo anti-fXa and anticoagulant activity for compound (À)-3g
Compound
At 30 mg/kg (po) to rats
Anti-fXa activitya (%)
Prolongation effect of PTa (fold)
1 h
3 h
99.5 1.0
6 h
24 h
1.7 1.7
1 h
3 h
6 h
24 h
(À)-3g
97.0 0.6
90.7 1.9
1.56 0.01
1.55 0.01
1.36 0.01
1.02 0.01
a The methods for measuring the ex vivo anti-fXa and anticoagulant activities were described in Ref. 14. Values are expressed as means SE from
four rats.
pound (À)-3g showed excellent oral anti-fXa activity
and anticoagulant activity in rats over 6 h after oral
administration.
References and notes
1. (a) Prager, N. A.; Abendschein, D. R.; McKenzie, C. R.;
Eisenberg, P. R. Circulation 1995, 92, 962; (b) Kunitada,
S.; Nagahara, T. Curr. Pharm. Des. 1996, 2, 531.
2. Nagahara, T.; Yokoyama, Y.; Inamura, K.; Komoriya, S.;
Yamaguchi, H.; Hara, T.; Iwamoto, M. J. Med. Chem.
1994, 37, 1200.
3. (a) Al-Obeidi, F.; Ostrem, J. A. Drug Discovery Today
1998, 3, 223; (b) Faull, A. W.; Mayo, C. M.; Preston, J.
Int. Pub. No. WO 96/10022, April 4, 1996.
4. Komoriya, S.; Haginoya, N.; Kobayashi, S.; Nagata, T.;
Mochizuki, A.; Suzuki, M.; Yoshino, T.; Horino, H.;
Nagahara, T.; Suzuki, M.; Isobe, Y.; Furugoori, T.
Bioorg. Med. Chem. 2005, 13, 3927.
Table 6 shows the pharmacokinetic properties of com-
pound (À)-3g evaluated using monkeys (po and iv,
3 mg/kg) and human microsomes. For comparison, data
of compound 3f are also shown in Table 6. Compound
(À)-3g showed poor oral bioavailability (F = 6.1%)
when compared with compound 3f (F = 37%). The rea-
son for such poor bioavailability of (À)-3g can be
explained by its low remaining rate (46%), which was
significantly lower than that of 3f (89%), in the human
liver microsome test.
5. Haginoya, N.; Kobayashi, S.; Komoriya, S.; Yoshino, T.;
Suzuki, M.; Shimada, T.; Watanabe, K.; Hirokawa, Y.;
Furugoori, T.; Nagahara, T. J. Med. Chem. 2004, 47, 5167.
6. Komoriya, S.; Kobayashi, S.; Osanai, K.; Yoshino, T.;
Nagata, T.; Haginoya, N.; Nakamoto, Y.; Mochizuki, A.;
Nagahara, T.; Suzuki, M.; Shimada, T.; Watanabe, K.;
Isobe, Y.; Furugoori, T. Bioorg. Med. Chem. 2006, 14, 1309.
7. Haginoya, N.; Komoriya, S.; Osanai, K.; Yoshino, T.;
Nagata, T.; Nagamochi, M.; Muto, R.; Yamaguchi, M.;
Nagahara, T.; Kanno, H. Heterocycles 2004, 63, 1555.
8. Optical resolution: compound 12 (900 mg) was dissolved
in isopropanol (6 ml). The solution was purified in 11
portions by preparative HPLC (CHIRALPAK AD. Dai-
cel Chemical Industries, Ltd; 2.0 in diameter · 25 cm) with
In summary, we synthesized a series of sulfonyl deriva-
tives 2 and carbonyl derivatives 3, tested their pharma-
cological properties, and found that among them
carbonyl derivatives 3g, (À)-3g, 3h, and 3j had potent
anti-fXa activity. In addition, cis-/trans- and absolute
configurations affect anticoagulant activity as well as
anti-fXa activity. In the series of compounds having a
cycloalkanediamine skeleton, 5-chloroindole-2-carbonyl
group was a superior component for aryl (S4) binding
site. These results would be useful for drug design of
new fXa inhibitors.
hexane/isopropanol/diethylamine = 68:32:0.5
as
the
mobile phase at a flow rate of 6 ml/min. Compound 13:
320 mg, retention time = 24.8 min. Compound 14: 390 mg,
retention time = 33.4 min.
9. The coordinates of the complex have been deposited in the
Protein Data Bank as 2EI6.pdb.
Table 6. Pharmacokinetic profiles in monkeys and human microsomes
10. (a) Overman, L. E.; Sugai, S. J. Org. Chem. 1985, 50, 4154;
(b) Trivedi, B. K.; Padia, J. K.; Holmes, A.; Rose, S.;
Wright, D. S.; Hinton, J. P.; Pritchard, M. C.; Eden, J. M.;
Kneen, C.; Webdale, L.; Suman-Chauhan, N.; Boden, P.;
Singh, L.; Field, M. J.; Hill, D. J. Med. Chem. 1998, 41,
38.
11. In vitro anti-fXa activity was measured by using a
chromogenic substrate S-2222 (Chromogenix, Inc.) and
human fXa (Enzyme Research Laboratories). Aqueous
Compound Cl
Vdss
(mL/min/kg) (L/kg) (h)
t1/2
F
(%)
Remaining
rateb (%)
3fa
(À)-3ga
14.2
17.7
2.54
2.22
1.4
1.4
37
6.1
89
46
a Hydrogen chloride salts were used. Both compounds were adminis-
tered at a dose of 3 mg/kg po and iv (n = 3).
b The remaining rate of the two compounds after 5 min of incubation
with human liver microsomes.

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4683–4688
DMSO (5% v/v; 10 lL) or inhibitors in aqueous DMSO
the clotting time was measured. he concentration of
inhibitor required to double the clotting time (CT2) was
estimated from the concentration–response curve by a
regression analysis.
(10 lL) and 0.0625 U/mL human fXa (10 lL) were mixed
with 0.1 M Tris–0.2 M NaCl–0.2% BSA buffer (pH 7.4;
40 lL). A reaction was started by the addition of 0.75 M
S-2222 (40 lL). After the mixture was stirred for 10 s at rt,
the increase of optical densities (OD/min) was measured at
405 nm. Anti-fXa activity (inhibition %) was calculated as
follows: anti-fXa activity = 1 À [(OD/min) of sample/(OD/
min) of control]. The IC50 value was obtained by plotting
the inhibitor concentration against the anti-fXa activity.
14. Anti-fXa activity and anticoagulant activity ex vivo. Male
Wister rats were fasted overnight. Synthetic compounds
were dissolved in 0.5% (w/v) methylcellulose solution and
administered orally to rats via a stomach tube. For control
rats, 0.5% (w/v) methylcellulose solution was administered
orally. The rats were anesthetized with ravonal at several
time points when blood samples were collected in the
presence of trisodiumcitrate. After the blood samples were
centrifuged, the platelet-poor plasma samples were used
for the measurement of their anti-fXa activities or
anticoagulant activities. Anti-Xa activity: plasma (5 lL)
was mixed with 0.1 M Tris–0.2 M–NaCl–0.2% BSA buffer
(pH 7.4; 40 lL), H2O (5 lL), and 0.1 U/mL human fXa
(10 lL). A reaction was started by the addition of 0.75 M
S-2222 (40 lL). After the mixture was stirred for 10 s at rt,
the increase of optical density (OD/min) was measured at
405 nm. Anti-fXa activity (inhibition %) was calculated as
follows: anti-fXa activity = 1 À [(OD/min) of sample/(OD/
min) of control]. Anticoagulant activity was measured
with an Amelung KC-10A microcoagulometer. Plasma
(50 lL) was incubated for 1 min at 37 ꢁC in a cup. The
coagulation was started by the addition of 100 lL of
thromboplastin C Plus (0.5 U/mL) to the mixture and the
clotting time was measured. The anticoagulant activity
was evaluated by the prolongation rate of prothrombin
time versus the control.
12. In vitro anti-thrombin activity was measured by using
chromogenic substrate S-2266 (Chromogenix, Inc.) and
human thrombin (Sigma Chemical, Inc.). Aqueous
DMSO (5% v/v; 10 lL) or inhibitors in aqueous DMSO
(10 lL) and 4 U/ml human thrombin (10 lL) were mixed
with 0.1 M Tris–0.2 M–NaCl 0.2% BSA buffer (pH 7.4;
40 lL). A reaction was started by the addition of 0.50 M
S-2266 (40 lL). After the mixture was stirred for 10 s at
room temperature, the increase of optical density (OD/
min) was measured at 405 nm. Anti-thrombin activity
(inhibition percentage) was calculated as follows: anti-
thrombin activity = 1 À [(OD/min) of sample/(OD/min) of
control]. The IC50 value was obtained by plotting the
inhibitor concentration against the anti-fIIa activity.
13. Prothrombin time (PT) was measured with an Amelung
KC-10A microcoagulometer (MC Medical, Tokyo, Japan)
as follows; First, 50 lL of plasma was mixed with 50 lL of
inhibitor or 4% DMSO/saline and incubated for 1 min at
37 ꢁC. Coagulation was started by the addition of 100 lL
of thromboplastin C Plus (0.5 U/mL) to the mixture, and