Stereoselective synthesis and biological evaluation of 3,4-diaminocyclohexanecarboxylic acid derivatives as factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.07.031

There have been few reports on synthetic methods for cis-1,2-diaminocyclohexane bearing a third ring substituent. Starting from 3-cyclohexenecarboxylic acid, we developed efficient methods for synthesizing the 3,4-diaminocyclohexanecarboxylic acid derivat

Full text of DOI:10.1016/j.bmcl.2008.07.031

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4587–4592
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stereoselective synthesis and biological evaluation of
3,4-diaminocyclohexanecarboxylic acid derivatives as factor Xa inhibitors
b
b
b
b
Tsutomu Nagata a, , Masatoshi Nagamochi , Shozo Kobayashi , Satoshi Komoriya , Toshiharu Yoshino ,
*
Hideyuki Kanno c
a Medicinal Chemistry Research Laboratories I, DAIICHI SANKYO CO., LTD., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Medicinal Chemistry Research Laboratories II, DAIICHI SANKYO CO., LTD., 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
c DAIICHI SANKYO RD ASSOCIE CO., LTD., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
There have been few reports on synthetic methods for cis-1,2-diaminocyclohexane bearing a third ring
substituent. Starting from 3-cyclohexenecarboxylic acid, we developed efficient methods for synthesizing
the 3,4-diaminocyclohexanecarboxylic acid derivatives 2–5. We also evaluated their anti-Xa and antico-
agulant activities. Among the compounds, acid 2a and amide 2b exhibited the most potent in vitro anti-
fXa activity, indicating that the position and stereochemistry of a polar functional group on the cyclohex-
ane ring greatly affected the in vitro anti-fXa activity.
Received 29 March 2008
Revised 27 June 2008
Accepted 10 July 2008
Available online 15 July 2008
Keywords:
Factor Xa inhibitors
Ó 2008 Elsevier Ltd. All rights reserved.
3,4-Diaminocyclohexanecarboxylic acid
Stereoselective synthesis
The extrinsic and intrinsic coagulation systems converge at the
activation of factor X to Xa. Activated factor X (fXa) has an impor-
tant role in the conversion of prothrombin to thrombin, which pro-
duces blood clots.1 Thus, fXa is a key enzyme in the coagulation
cascade and is also an attractive target enzyme for the therapy of
thrombosis and related diseases.
diols. Although we expected that the electrophilic reagent could
exhibit a certain preference for attack anti to the methyl ester
group, no stereoselectivity was observed in this oxidation step.
The mixture was subsequently treated with 2,2-dimethoxypro-
pane and separated by silica gel chromatography to give acetonide
7 (41%) and its stereoisomer 8 (35%), respectively. Acidic hydrolysis
of the acetonide 7 afforded the diol, which was successively mesy-
lated and treated with sodium azide to give azide 9. Reduction and
deprotection were followed by acylation with commercially avail-
able 5-chloroindole-2-carboxylic acid to give a separable 1:1 mix-
ture of amides 10a and 10b, showing no regioselectivity in this
acylation step. Amides 12a and 12b were also prepared from 8
by the procedures described above. Finally, amides 10a, 10b, 12a,
and 12b were condensed with thiazolopyridinecarboxylic acid, fol-
lowed by ester hydrolysis to give the carboxylic acids 2a–5a.
Next, we examined a regio- and stereoselective synthesis of 2–5
for further evaluation. Our strategy is summarized in Figure 2. In
In the previous paper,2 we reported that the synthesis of cis-1,2-
diaminocyclohexane derivative (À)-1 has a potent inhibitory activ-
ity for blood coagulation factor Xa (Fig. 1). However, compound
(À)-1 showed poor oral bioavailability (F = 6.1%) in monkeys. The
reason for such poor bioavailability of (À)-1 can be explained by
its low remaining rate (46%) in a human liver microsome test. Con-
sequently, we speculated that the introduction of a polar func-
tional group into the chemical structure would decrease the
lipophilicity, and hence improve the metabolic stability. Based on
this hypothesis, we planned the synthesis and pharmacological
evaluation of four regio- and stereoisomers 2–5 as racemates, with
a carboxylic acid group or dimethylcarbomoyl group on the central
cyclohexane ring (Fig. 1).
general, 1-substituted-3,4-epoxycyclohexanes undergo
a ring
opening with nucleophiles to give trans-diaxial products with high
regio- and stereoselectivity (Fürst-Plattner rule).4 Therefore, to
take advantage of this rule, we employed cis- and trans-3,4-epoxy-
cyclohexanecarboxylates A and D as synthetic precursors. Treat-
In the synthesis of 2–5, there have been few reports on syn-
thetic methods for cis-1,2-diaminocyclohexane bearing a third ring
substituent.3 Therefore, we first examined the conventional syn-
thetic method described in Scheme 1.
ment with sodium azide would afford azides
B and E,
Commercially available ester 6 was oxidized utilizing microen-
capsulated osmium (VIII) oxide (MC.OsO4) to give a mixture of cis-
respectively, which could be subsequently converted to diamino
derivatives C and F in several steps. Compounds C and F have
not only the desired relative stereochemistry for compounds 2–5,
but also independently removal protecting groups on each func-
tional group (the azido functionality serves as an amine protecting
* Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nagata.tsutomu.ni@daiichisankyo.co.jp (T. Nagata).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.031

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4587–4592
R
R
R
*
N
H
*
N
HN
H
HN
Cl
*
O
*
S
2
3
N
H
N
R
HN
N
N
H
O
*
N
H
*
(-)-1
N
H
HN
HN
*
fXa IC50 = 16 nM
*
5
4
a: R = -COOH
b: R = -CONMe
2
(All compounds were racemates)
Figure 1. 3,4-Diaminocyclohexanecarboxylic acid derivatives.
COOMe
COOMe
COOMe
(a), (b)
+
O
O
O
O
6
7
8
R1
COOMe
N3
(c), (d), (e)
R2
(f), (g), (h)
Cl
7
H2N
N3
HN
N
H
9
O
10a: R1 =
10b: R1 = H
R2 = H
R2 =
COOMe
COOMe
R1
COOMe
N3
R2
Cl
(c), (d), (e)
(f), (g), (h)
8
N3
H2N
HN
N
H
11
O
12a: R1 =
12b: R1 = H
R2 = H
R2 =
COOMe
COOMe
O
12a
(i), (j)
2a
S
Li
12b
10a
10b
O
3a
4a
5a
+
N
N
Scheme 1. Reagents and conditions: (a) MC.OsO4, 4-methylmorpholine 4-oxide, CH3CN, 60 °C, 19 h (91%); (b) 2,2-dimethoxypropane, pyridinium p-toluenesulfonate, CH2Cl2,
rt, 1d (7: 41%, 8: 35%); (c) p-toluenesulfonic acid, MeOH/THF, rt, 13 h (91–97%); (d) MsCl, Et3N CH2Cl2, À78 °C ? 0 °C, 2 h (77–88%); (e) NaN3, DMF, 100 °C, 1d (82–85%); (f) H2,
Pd-C, Boc2O, THF, rt, 1d, (46–68%); (g) HCl/EtOH, CH2Cl2, 1d; (h) 5-chloroindole-2-carboxylic acid, WSCI, HOBt, 4-methylmorpholine, DMF, rt, 1d (10a: 22%, 10b: 20%; 12a:
22%, 12b: 23%); (i) WSCI, HOBt, DMF, rt, 1-4d (41–52%); (j) LiOH, THF/H2O, 1–21 h (76–82%).
group). Finally, the selective deprotection and acylation of C and F
would readily afford target compounds 2–5.
The synthetic pathway of key intermediate 18 (compound C,
R = ethyl) is detailed in Scheme 2. Commercially available acid 13
was treated with I2-KI to afford 4-iodo-6-oxabicyclo[3.2.1]octan-
7-one (14).5 The conversion of 14 to cis-epoxide 15 was accom-
plished by ethanolysis and successive treatment under an alkaline
condition (1.2 equivalents of NaOH). In this reaction, an excess or
lower amount of NaOH resulted in a decrease in the yield of 15.
cis-Epoxide 15 was treated with sodium azide in the presence of

T. Nagata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4587–4592
4589
COOR
COOR
COOR
4
OH
1
O
ROOC
2
ROOC
2, 3
Boc
N
3
N3
N3-
3
H
N3
N3
O
OH
C
B
A
COOR
COOR
N3
COOR
OH
1
O
4
2
ROOC
ROOC
N3-
N3
4, 5
HO
3
N3
HN
O
4
Boc
D
E
F
Figure 2. Summary of regio- and stereoselective synthesis of 2–5.
converted into mesylate, followed by treatment with sodium azide
to afford the desired key intermediate 186 (39%). In this final step,
epimer 196 was unexpectedly obtained as a by-product (8%), sug-
gesting neighboring-group (tert-Boc) participation in forming the
oxazolidine derivative, as shown in Figure 3.7
O
O
COOEt
COOH
(a)
(b)
O
I
The synthetic pathway of compound 26 (compound F,
R = methyl) is described in Scheme 3. Acid 13 was esterified with
benzylbromide to afford benzyl ester 20. Treatment of ester 20
with m-CPBA afforded a 2:1 mixture of epoxides 21 and 22. This
mixture was separated by silica gel chromatography to give the de-
sired trans-epoxide 21. Epoxidation of methyl 3-cyclohexenecarb-
oxylate was also used instead of 20 to produce a mixture of its
cis- and trans-epoxide.8 However, the isolation of trans-epoxide
from the resulting mixture was unsuccessful on a large scale be-
cause of closed Rf values. Epoxide 21 was treated with sodium
azide by the procedures described above, affording azide 23 as
the sole product in a quantitative yield. Azide 23 was reduced by
the use of triphenylphosphine and successively protected by using
Boc2O to afford compound 24 in a good yield. Benzyl ester 24 was
cleaved by hydrogenolysis, and then esterified with TMSCHN2, fur-
nishing methyl ester 25. Ester 25 was converted into mesylate, fol-
lowed by a treatment of sodium azide to afford the desired key
intermediate 26.
The obtained intermediates 18 and 26 were reduced over Pd-C
to yield amines 27 and 30, followed by selective acylation and
deprotection to afford the acids 2a–5a. Dimethylcarbamoyl ana-
logues 2b–5b were derived from 2a–5a using amide coupling, as
shown in Scheme 4.
The synthesized compounds were evaluated for in vitro anti-fXa
activity (IC50) and anticoagulant activity (PTCT2: the concentration
required to double the prothrombin time). The metabolic stability
was estimated by the remaining rate of the compounds after 5 min
of incubation with human liver microsomes. The results are shown
in Table 1.
13
14
15
COOEt
COOEt
(c)
(d)
Boc
N
H
N3
OH
OH
16
17
COOEt
COOEt
(e)
Boc
Boc
+
N
H
N
H
N3
N3
18
19
(undesired)
(desired)
Scheme 2. Reagents and conditions: (a) KI, I2, NaHCO3, rt, 3 h (92%); (b) 1.2 equiv
2 N—NaOH, EtOH, rt, 3–7 h (68%); (c) 1.5 equiv NaN3, 1.5 equiv NH4Cl, DMF, 75 °C,
12 h (93%); (d) H2, Pd-C, Boc2O, EtOAc, rt, 12 h (quant.); (e) MsCl, Et3N CH2Cl2,
À78 °C ? 0 °C ? rt, 2 h; then NaN3, DMF, 75 °C, 12 h (18: 39%, 19: 7.7%).
ammonium chloride to afford azide 16 as a single isomer. In this
ring-opening reaction, the addition of ammonium chloride was
very effective in preventing the hydrolysis of the ester group. Cat-
alytic hydrogenation of 16 in the presence of Boc2O gave Boc pro-
tected amino alcohol 17 in a quantitative yield. Alcohol 17 was
Carboxylic acids 2a–5a showed significantly different anti-fXa
activities, indicating that the position and stereochemistry of a car-
boxylic group on cyclohexane ring greatly affected the in vitro anti-
fXa activity. Among them, acid 2a was the most potent compound
and was twofold more active than unsubstituted compound (À)-1.
In contrast, the regioisomer 3a showed a 15-fold decreased inhibi-
tion compared with that of 2a. With respect to stereoisomers, com-
pound 4a, which was stereoisomer of 2a, also exhibited 10-fold
weaker potency in vitro. However, its regioisomer 5a recovered
anti-fXa activity the same as compound (À)-1.
O
O
N+
O
H
O
N3-
Replacement of the carboxyl group with a dimethylcarbamoyl
group did not affect the anti-fXa activity and showed almost the
same anti-fXa inhibitory activity as the corresponding carboxylic
acids. However, focusing on the anticoagulant activity, dimethyl-
Figure 3. Neighboring-group participation.

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4587–4592
COOBn
COOBn
COOBn
(a)
(b)
13
+
O
O
20
21
22
(desired)
(undesired)
COOBn
COOBn
N3
(c)
(d)
21
HO
HO
HN
Boc
23
24
COOMe
COOMe
(e)
(f)
N3
HO
HN
HN
Boc
Boc
25
26
Scheme 3. Reagents and conditions: (a) BnBr, Et3N, DMF, rt, 12 h (83%); (b) mCPBA, CH2Cl2, 0 °C, 2 h (21: 55%, 22: 28%); (c) NaN3, NH4Cl, DMF, 70 °C, 24 h (quantitative yield);
(d) PPh3, THF/H2O, rt, 20 h; then Boc2O, rt, 2 h (93%); (e) H2, 10% Pd-C, EtOAc, rt, 20 h; then TMSCHN2, MeOH/toluene, rt, 0.5 h (92%); (f) MsCl, Et3N CH2Cl2, À78 °C
(0.5 h) ? 0 °C (0.5 h); then NaN3, DMF, 70 °C, 12 h (30%).
COOEt
COOEt
COOEt
(b)
Cl
(a)
(c), (d), (e)
(f)
Boc
N
2a
3a
Boc
Boc
2b
H
N
H
N
HN
H
N
N3
NH2
27
H
O
18
28
COOEt
(c), (b), (e)
(d)
(f)
Boc
N
N
N
3b
H
HN
29
S
O
COOMe
COOMe
COOMe
O
(d)
(a)
(c), (b), (e)
(f)
S
4a
5a
4b
5b
N
N3
H2N
H
N
HN
N
HN
HN
Boc
Boc
Boc
(b)
26
30
31
COOMe
O
H
(c), (d), (e)
N
(f)
N
H
HN
Boc
32
Cl
Scheme 4. Reagents and conditions: (a) H2, 10%Pd-C, EtOH/EtOAc or EtOAc, rt (95%-quantitative yield); (b) 5-chloroindole-2-carboxylic acid, WSCI, HOBt, triethylamine,
CH2Cl2 or DMF, rt (36–76%); (c); saturated HCl/EtOH, EtOH, rt or 4 N-HCl/dioxane, MeOH, rt (quantitative yield); (d) 5-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine-2-
carboxylic acid lithium salt, WSCI, HOBt, triethylamine, DMF, rt (56–78%); (e) 1 N-NaOH, EtOH/THF, rt or LiOH, THF/H2O, rt (78–86%); (f) Me2NH/HCl, WSCI, HOBt,
triethylamine, CHCl3 or DMF, rt (39–83%).

T. Nagata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4587–4592
4591
Table 1
Acids 2a, 5a, and amide 2b were further evaluated for their oral
activity in rats. Their anti-fXa activities in rat plasma after oral
administration are shown in Table 2. Disappointingly, acids 2a,
5a, and amide 2b were all found to have less oral activity in rats
than compound (À)-1. Since these compounds were more meta-
bolically stable than (À)-1, we reasoned that the introduction of
polar functionalities might decrease the membrane permeability
in rats.
Starting from 3-cyclohexenecarboxylic acid, we developed the
regio- and stereoselective synthesis of 3,4-diaminocyclohexane-
carboxylic acid derivatives 2–5 and performed a biological evalua-
tion on them as factor Xa inhibitors. SAR for the anti-fXa study of
this series revealed that the position and stereochemistry of the
polar functional group was critical to the in vitro anti-fXa activity.
Among the compounds, acid 2a and amide 2b exhibited the most
potent anti-fXa activity. Furthermore, we also revealed that the
dimethylcarbamoyl group could have better functionality for po-
tent in vitro anticoagulant activity. In addition, acids 2a–5a and
amide 2b with low lipophilicity showed improved metabolic sta-
bility compared with compound (À)-1, as we expected. However,
these low lipophilic compounds also resulted in low oral activity
probably due to low permeability. Therefore, to obtain more potent
fXa inhibitors with improved oral bioavailability, we need to bal-
ance the lipophilicity for both metabolic stability and permeability.
Further modifications of these compounds will be reported in due
course.
Introduction of a carboxylic group or dimethylcarbamoyl group on the cyclohexane
ring
R1
R2
Cl
O
S
N
H
N
HN
N
N
H
O
Compound R1
R2
Anti-fXa PTCT2 in PTCT2 Remaining
IC50
human
in rat
ratec (%)
a
(nM)
plasmab plasma
(
lM)
ab
(
lM)
(À)-1
H
H
H
16
2.9
9.2
2.8
46
2a
7.5
2.8
0.9
100
COOH
2b
3a
3b
4a
CONMe2
H
5.1
1.8
63
97
36
98
H
H
110
>20
>20
>20
>20
>20
15
COOH
CONMe2 840
H
H
75
88
COOH
4b
5a
CONMe2
6.8
19
43
98
19
H
H
16
CONMe2 12
8
3.0
3.8
COOH
5b
1.2
References and notes
a
The method for measuring anti-fXa activity is described in Ref. 9.
Anticoagulant activities in human and rat plasma were evaluated by the con-
b
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. Nagata, T.; Yoshino, T.; Haginoya, N.; Yoshikawa, K.; Isobe, Y.; Furugoori, T.;
Kanno, H. Bioorg. Med. Chem. Lett. 2007, 17, 4683.
3. Synthsis of 1-hydroxy-3,4-diaminocyclohexane Witiak, D. T.; Rotella, D. P.;
Wei, Y.; Filppi, J. A.; Gallucci, J. C. J. Med. Chem. 1989, 32, 214.
centration required to double the prothrombin time (PTCT2). Themethod is described
in Ref. 10.
c
The remaining rate of the compounds after 5 min of incubation with human
liver microsomes.
4. Furst, A.; Plattner, P. A. Helv. Chim. Acta 1949, 32, 275.
5. (a) Grewe, R.; Heinke, A. Chem. Ber. 1956, 89, 1978; (b) Kato, M.; Kageyama, M.;
Tanaka, R.; Kuwahara, K.; Yoshikoshi, A. J. Org. Chem. 1975, 40, 1932; (c)
Bartlett, P. A.; McQuaid, L. A. J. Am. Chem. Soc. 1984, 106, 7854.
6. Configurations of compounds 18 and 19 were established by X-ray
crystallographic analysis of their N,N-dimethylamide derivatives 33 and 34,
respectively. Crystallographic data (excluding structure factors) have been
deposited with the Cambridge Crystallographic Data Centre as supplementary
publication Nos. CCDC 637942 (33) and 637941 (34). Copies of the data can be
obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2
1EZ, UK, (fax: +44-(0)1223-336033 or e-mail:deposit@ccdc.cam.ac.uk).
Table 2
Ex vivo anti-fXa activity
Compound
Anti-fXa IC50 (nM)
Anti-fXa activity (%) at 1 h
after oral administrationa
(À)-1
2a
2b
16
78
0
39
4
7.5
5.1
16
5a
a
The methods for measuring the ex vivo anti-fXa activity are described in Ref. 11.
O
N
O
N
Boc
Boc
carbamoyl analogues exhibited enhanced anticoagulant activity. In
particular, compound 5b showed sevenfold higher potency than
the corresponding acid 5a, suggesting that the dimethylcarbamoyl
group could have better functionality for potent anticoagulant
activity. In this series of compounds, 2b showed the most potent
N
N
H
H
N3
33
N3
34
7. (a) Qiu, J.; Silverman, R. B. J. Med. Chem. 2000, 43, 706; (b) Katagiri, N.;
Matsuhashi, Y.; Kokufuda, H.; Takebayashi, M.; Kaneko, C. A. Tetrahedron Lett.
1997, 38, 1961.
8. Bellucci, G.; Marioni, F.; Marsili, A. Tetrahedron 1972, 28, 3393.
9. In vitro Anti-fXa activity was measured by using a chromogenic substrate S-
2222 (Chromogenix, Inc.) and human fXa (Enzyme Research Laboratories).
activities (fXa IC50: 5.1 nM, PTCT2: 0.9 lM) and was threefold more
potent than compound (À)-1.
As expected, the introduction of a carboxylic group on the
cyclohexane ring significantly improved the metabolic stability.
Acids 2a–5a showed excellent remaining rate ranging from 98%
to 100%, whereas compound (À)-1 had a low remaining rate of
46%. This stability could be mainly attributed to decreased lipo-
philicity. In contrast, dimethylcarbamoyl analogues 2b–5b re-
sulted in low to modest metabolic stability, ranging from 19% to
63%. Interestingly, the position and stereochemistry of a dimethyl-
carbamoyl group on cyclohexane ring is likely to affect metabolic
stability to some extent. Among the dimethylcarbamoyl analogues,
only 2b showed an improvement in the metabolic stability com-
pared with (À)-1.
Aqueous DMSO (5% V/V; 10
0.0625 U/mL human fXa (10
l
L) or inhibitors in aqueous DMSO (10
lL) and
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 L). After the mixture was stirred for 10 s at rt, the increase of optical
l
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.
10. Prothrombin time (PT) was measured with an Amelung KC-10A micro
coagulometer (MC Medical, Tokyo, Japan) as follows: first, 50
was mixed with 50 L of inhibitor or 4% DMSO/saline and incubated for 1 min
at 37 °C. Coagulation was started by the addition of 100 L of thromboplastin C
lL of plasma
l
l

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T. Nagata et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4587–4592
Plus (0.5 U/mL) to the mixture and the clotting time was measured. The
After the blood samples were centrifuged, the platelet poor plasma samples
were used for the measurement of their anti-fXa activities or anticoagulant
concentration of inhibitor required to double the clotting time (CT2) was
estimated from the concentration-response curve by a regression analysis.
11. 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 the 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.
activities. Anti-Xa activity: Plasma (5
NaCl–0.2% BSA buffer (pH 7.4; 40 L), H2O (5
(10 L). A reaction was started by the addition of 0.75 M S-2222 (40
l
L) was mixed with 0.1 M Tris–0.2 M
L) and 0.1 U/mL human fXa
L). After
l
l
l
l
the mixture was stirred for 10 s at rt, the increase of optical density (OD/min)
was measured at 405 nm. The anti-fXa activity (inhibition %) was calculated as
follows: Anti-fXa activity = 1 À [(OD/min) of sample/(OD/min) of control].