Synthesis and pharmacological properties of a close analogue of an antithrombotic pentasaccharide (SR 90107A/ORG 31540)

Article abstract of DOI:10.1021/jm960726z

The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal models of venous thrombosis. We describe here the preparation and the ph

Full text of DOI:10.1021/jm960726z

1600
J . Med. Chem. 1997, 40, 1600-1607
Syn th esis a n d P h a r m a cologica l P r op er ties of a Close An a logu e of a n
An tith r om botic P en ta sa cch a r id e (SR 90107A/ORG 31540)†
Maurice Petitou,* Philippe Duchaussoy, Guy J aurand, Franc¸oise Gourvenec, Isidore Lederman,
J ean-Marc Strassel, Tereza Baˆrzu, Bertrand Cre´pon, J ean-Pascal He´rault, J ean-Claude Lormeau,
Andre´ Bernat, and J ean-Marc Herbert
Haemobiology Research Department, Sanofi Recherche, 195 route d’Espagne, 31036 Toulouse Cedex, France
Received October 15, 1996X
The synthetic pentasaccharide (1) corresponding to the heparin sequence which binds to, and
activates, antithrombin III (AT III) is a potent antithrombotic compound in several animal
models of venous thrombosis. We describe here the preparation and the pharmacological
properties of 34, an analogue of oligosaccharide 1 with the latter’s N-sulfates being replaced
by sulfate esters and hydroxyl groups being methylated. These structural modifications allow
a simpler and more efficient synthesis of such anionic oligosaccharides. Affinity for human
AT III, anti-factor Xa activity, ability to inhibit thrombin generation, antithrombotic activity
in a rat model of venous thrombosis, and elimination half-life in the rat have been determined
for 1 and 34. Surprisingly, introduction of O-sulfates in place of N-sulfates, and methylation
of hydroxyl groups, contributes to reinforce the binding to AT III, resulting in an improved
pharmacological profile.
In tr od u ction
of 1. This work allows us to precisely assess how
O-sulfation vs N-sulfation and alkylation of hydroxyl
groups influence the pharmacological properties of such
synthetic pentasaccharides.
The activity of heparin and low molecular weight
heparin, two widely used antithrombotic drugs, is
essentially due to their ability to interact with anti-
thrombin III (AT III), a serine protease inhibitor present
in plasma in a latent form. This interaction mainly
results in inhibition of the coagulation enzymes throm-
bin and factor Xa. Recent studies at the molecular level
have shown that, upon heparin binding, AT III under-
goes a conformational change which is sufficient to allow
factor Xa inhibition (allostery mechanism), whereas
thrombin inhibition further requires binding of both AT
III and thrombin to the same heparin molecule (tem-
plate mechanism).1 This explains why very short he-
parin fragments can promote selective factor Xa inhi-
bition without affecting thrombin. Indeed a unique
pentasaccharide sequence, which represents the binding
site of heparin to AT III, was shown to be a potent and
selective anti-factor Xa compound.2 We found that such
selective factor Xa inhibitors also displayed antithrom-
botic properties in animal models of venous thrombosis,3
and this prompted us to launch a research program
aimed at the preparation of pure synthetic antithrom-
botic compounds based on AT III mediated factor Xa
inhibition (for review, see ref 4). The lead compound
in this series (1, SR 90107A/ORG 31540) is the exact
copy of an AT III binding sequence present on heparin
chains. Despite its complex structure which requires
numerous synthesis steps,5 this compound is currently
undergoing clinical trials to prove the therapeutic
potential of this class of pure indirect factor Xa inhibi-
tors. To simplify the synthesis, various pentasaccha-
rides in which N-sulfate groups have been replaced by
O-sulfates and hydroxyl groups by O-alkyl groups have
been prepared.6-10 In this article, we report the syn-
thesis and the biological properties of 34, the exact copy
Ch em istr y
The N,O-sulfated pentasaccharide (1, Scheme 1) used
in this study was obtained as previously reported.4,5
This compound is an exact copy of the heparin sequence
required for AT III binding and therefore contains both
glucuronic and iduronic acid units, and N-sulfated
glucosamine. The strategy for the synthesis of such a
complex oligosaccharide consists in the preparation of
a fully protected molecule (2), the protective groups of
which are then sequentially removed to introduce the
desired substituents in the appropriate positions. Thus
in 2, O-acetyl substitutes for O-sulfate, O-benzyl for
hydroxyl, methoxycarbonyl for carboxylate, azido and
N-(benzyloxycarbonyl)amino for N-sulfate. For the
preparation of 2, glucuronic and iduronic acid units were
elaborated from glucose, while glucosamine units were
obtained either from glucose or from glucosamine itself.
Syn th esis of 34. Since 34 does not contain N-sulfate
groups but only sulfate esters, we used glucose as the
precursor of all units. It should be underlined as well
that the structures of 34 and 1 also differ by the
presence of methyl ethers in place of hydroxyl groups.
These apparently modest structural differences are of
considerable interest from the chemical synthesis stand-
point. On the one hand, the synthesis of 1 requires the
use of three levels of protecting groups: permanent
(removed at the very end of the synthesis to unmask
the hydroxy functions), semipermanent (removed before
introduction of the sulfate esters), and temporary (useful
to temporarily protect a hydroxyl group during the
elaboration of the pentasaccharidic backbone). So far
benzyl ethers and acetyl esters have been used as
permanent and semipermanent protecting groups, re-
spectively, while several other protecting groups were
temporarily employed.4 On the other hand, for the
†Dedicated to Pr. Dr. Hans Paulsen on the occasion of his 75th
anniversary.
* To whom correspondence should be addressed. Tel: +33 61 16 23
90. Fax: +33 61 16 22 86. E-mail: maurice.petitou@tls1.elfsanofi.fr.
X Abstract published in Advance ACS Abstracts, April 15, 1997.
S0022-2623(96)00726-1 CCC: $14.00 © 1997 American Chemical Society

Close Analogue of an Antithrombotic Pentasaccharide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1601
Sch em e 1. Structures of 1 and 34 and Their Fully Protected Precursor Pentasaccharides 2 and 33a
a
D, E, F, G, H are used throughout the text to designate the corresponding monosaccharide units.
synthesis of 34, hydroxyl groups do not need to be
unmasked at the end of the synthesis (the methyl that
protect them belong to the final structure), and further,
there is no need to differentiate between N- and O-
sulfates (only the latter are present in the final struc-
ture). As a consequence only two levels of protective
groups are required, the temporary ones during the
construction of the backbone and the semipermanent
ones that will be replaced by sulfates in 34. Thus the
benzyl group (ether or ester) can serve, together with
acetyl (or benzoyl) groups, to protect the positions to be
sulfated or to block the carboxyl functions. This allows
shorter synthetic routes and an easier deprotection-
functionalization sequence at the end of the synthesis.
The fully protected pentasaccharide 33 was chosen as
the key precursor of 34. Its elaboration represents the
biggest part of the synthesis work.
Hydrolysis of the isopropylidene group then delivered
the 4,6-diol 8 which had to be oxidized into the corre-
sponding uronic acid. This was achieved through
temporary protection of the primary alcohol as a tert-
butyldimethylsilyl ether, followed by levulinoylation of
the secondary hydroxyl group and direct J ones’ oxida-
tion of the silyl ether 10. Benzylation of the free acid
11 with benzyl bromide, in the presence of potassium
hydrogen carbonate, in DMF, and removal of the levuli-
nyl group, finally afforded the glycosyl acceptor 13 (64%
from 7).
The glucuronic acid containing synthon 25 was pre-
pared from 14 (Scheme 3).15 Since 25 must be engaged
in a glycosidation reaction with 13 to give an R-glyco-
sidic bond, we used the nonparticipating benzyl group
to protect the position 2 which later will be sulfated in
the final pentasaccharide. After methylation of the
secondary alcohols, trans-diaxial opening of the epoxide
by sodium benzylate, followed by acetylation, gave
O-(4,6-O-isopropylidene-2,3-di-O-methyl-â-D-glucopyra-
nosyl)-(1f4)-3-O-acetyl-1,6-anhydro-2-O-benzyl-â-D-glu-
copyranose, 17. The reaction sequence described above
for the preparation of 12 from 7 was then applied to
provide 22. No intermediate purification was required,
and pure 22 was isolated (46% from 14) after a single
column chromatography at the end of the sequence.
Acetolysis of the 1,6-anhydro ring, using the acetic
anhydride-trifluoroacetic acid system, followed by se-
lective removal of the anomeric acetate by benzylamine
in ether, gave 24 which was purified by column chro-
The glycosyl acceptor 5 was obtained from 311 after
methylation and reductive opening of the benzylidene
ring by the cyanoborohydride-tetrafluoroboric acid
system (Scheme 2).12 Condensation of 5 with the
known7 L-idose derivative 6 under henceforth classical
thioglycoside activation13,14 (N-iodosuccinimide, trifluo-
romethanesulfonic acid) led, as expected from the
participation of the benzoyl at position 2, to the ste-
reospecific formation of the desired disaccharide 7,
isolated in excellent yield (89%). The R-L configuration
of the new interglycosidic bond was confirmed by the
1
long-range coupling observed by 2D H NMR between
1
H-1 and H-3 of the idose ring in the C4 conformation.

1602 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Petitou et al.
Sch em e 2. Preparation of the L-iduronic Acid Containing Synthon 13a
a
Bn, benzyl; Bz, benzoyl; tBDMS, tert-butyldimethylsilyl. Reagents: (a) MeI, NaH, DMF; (b) HBF4, NaBH3CN, THF; (c) N-
iodosuccinimide, TsOH, PhCH3; (d) aqueous CF3COOH, CH2Cl2; (e) tBDMSCl, DMAP, Et3N, CH2Cl2; (f) Lev2O, DMAP, Et3N; (g) CrO3/
H2SO4, acetone; (h) BnBr, DMF, KHCO3; (i) NH2NH2, AcOH/pyridine.
Sch em e 3. Preparation of the D-Glucuronic Acid Containing Synthon 25a
a
Bn, benzyl; Bz, benzoyl; tBDMS, tert-butyldimethylsilyl. Reagents: (a) MeI, NaH, DMF; (b) BnONa, BnOH; (c) Ac2O, DMAP, Et3N,
CH2Cl2; (d) aqueous CF3COOH, CH2Cl2; (e) tBDMSCl, DMAP, Et3N, CH2Cl2; (f) Lev2O, DMAP, Et3N; (g) CrO3/H2SO4, acetone; (h) BnBr,
DMF, NaHCO3; (i) Ac2O, CF3COOH; (j) PhCH2NH2, Et2O; (k) CCl3CN, Cs2CO3, CH2Cl2.
Sch em e 4a
a
Reagents: (a) trimethylsilyl triflate, PhCH3; (b) PhCH2NH2, Et2O.
matography. Finally, reaction with trichloroacetonitrile
in the presence of cesium carbonate yielded a 3:2
mixture of R- and â-imidates 25 (52% from 22).
Reaction of equimolar amounts of 13 and 25 under
the classical conditions for imidate coupling16 delivered
the corresponding R-coupled tetrasaccharide 26 isolated
in 73% yield after column chromatography (Scheme 4).
The R-configuration was proved by the small coupling
constant (3.7 Hz) observed by 1H NMR between H-1 and
H-2 of the F unit. Removal of the levulinyl protection
then gave the tetrasaccharide 27 (96%) ready to be
glycosylated by 32.
to obtain an R-linkage after coupling with 27, and later
to introduce a sulfate at this position. Opening of the
epoxide ring of 2817 by sodium benzylate, followed by
methylation, acetolysis, and treatment with ethanethiol
in the presence of boron trifluoride, gave the thioglyco-
side donor 32, as a mixture of R- and â-anomers (78%
from 29, Scheme 5).
Condensation of 32 and 27 using triflic acid and
N-iodosuccinimide in a mixture of ether and dichloro-
ethane finally afforded the pentasaccharide 33 (62%).
The R-configuration was confirmed by 1H NMR analysis
(coupling constant between H-1 and H-2 of the D unit:
3.3 Hz). Final conversion of 33 into the target pen-
tasaccharide 34 was carried out in a very efficient way
The choice of a benzyl ether at position 2 of the
glycosyl donor 32 was, as for 25, dictated by the need

Close Analogue of an Antithrombotic Pentasaccharide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1603
Sch em e 5. Preparation of the Nonreducing End Unita
An tith r om botic Activity. Compounds 1 and 34 are
aimed at the prevention of venous thrombosis. The
Wessler model is the reference to assess the antithrom-
botic potency of the compounds in animal in vivo.21 The
well-known antithrombotic drugs heparin and low mo-
lecular weight heparin are highly efficient in this model.
Here again we found (Table 1) a direct correlation
between the affinity for AT III and the antithrombotic
activity of each compound.
a
Bn, benzyl. Reagents: (a) BnONa, BnOH; (b) MeI, NaH,
DMF; (c) Ac2O, CF3COOH; (d) EtSH, BF3‚Et2O, PhCH3.
P h a r m a cok in etics. Renal excretion is the main
route of elimination of the pentasaccharides.22 As the
rate of elimination is proportional to the concentration
of free pentasaccharide in plasma, then one may expect
this rate to be negatively correlated to the affinity for
AT III.22 Such a correlation was indeed confirmed, with
34 having a half-life considerably longer than that of 1
(Table 1).
Ta ble 1. In Vitro and in Vivo Properties of the
Pentasaccharides 1 and 34a
1
34
KD for AT-III (nM)
58 ( 3
850 ( 27
0.13
(0.10-0.17)
81
(55-103)
0.7 ( 0.1
7 ( 2
1040 ( 80
0.11
anti-Xa activity (units/mg)
thrombin generation inhibition
(IC50, µM)
antithrombotic activity
(ED50, µg/kg)
elimination half-life (t1/2, h)
(0.09-0.17)
20
(12-28)
3.8 ( 1.5
Con clu sion
a
Values are means ( SD (n ) 3) or 95% confidence interval.
Compared to the complex synthesis of 1, the prepara-
tion of 34 is much simpler. Replacing N-sulfates by
O-sulfates avoids the final N-sulfation step as well as
the use of the hazardous azido group. Furthermore,
methylation of the hydroxyl groups at the very begin-
ning of the synthesis assures their protection and allows
benzyl ethers and benzyl esters to serve, together with
acetates, for the protection both of the hydroxyls to be
sulfated and the carboxylates. This is of considerable
interest since it offers many different possibilities for
the preparation of compounds of this family. Regarding
the biological properties of 34, they are very similar to
those of the reference compound 1. Interestingly 34
displays a somewhat better affinity for AT III, which
may explain its slightly higher potency in the pharma-
cological tests reported here and its slightly higher half-
life. From a more fundamental point of view, since the
interaction of AT III and the oligosaccharide is not
altered by the present structural modifications, one may
conclude that NH and OH groups are not hydrogen bond
donors in the interaction with AT III. Further studies
are necessary to assess the contribution to the binding
of O- vs N-sulfates and methyl vs hydroxyl groups.
as follows: hydrogenolysis of benzyl ethers and benzyl
esters, saponification by sodium hydroxide in methanol,
and sulfation using triethylamine-sulfur trioxide com-
plex in dimethylformamide. Pure 34 was obtained
directly (88% yield over the three steps) after gel
permeation chromatography first in 0.2 M sodium
chloride and then in water.
Biologica l P r op er ties in Vitr o a n d in Vivo
Affin ity for AT III. Plasma AT III is the intended
target of the synthetic pentasaccharides 1 and 34. It
was therefore of prime importance to assess the influ-
ence of the structural modifications described above on
binding affinity. Interaction of the pentasaccharides
with AT III induces a change in the intrinsic protein
fluorescence which allows titration of the complex
formed and determination of the affinity of the different
ligands.18 We used this method to determine the KD
values reported in Table 1. The substitution of O-sul-
fates for N-sulfates and the introduction of methyl
groups resulted in an increase (8 fold) of the affinity for
AT III.
In h ibition of F a ctor Xa . Binding of the pentasac-
charides to AT III induces a conformational change
which results in an accelerated inhibition of blood
coagulation factor Xa by AT III.2 The anti-factor Xa
activity was determined, in buffer, by an amidolytic
method adapted from Teien and Lie.19 Activity of 1 and
34 paralleled their AT III affinity, and a good correlation
was found between their anti-factor Xa activity and the
natural log value of their Kd.
In h ibition of Th r om bin Gen er a tion . An efficient
venous antithrombotic agent must either inhibit throm-
bin or impair thrombin generation. The pentasaccha-
rides are not thrombin inhibitors; it is therefore of prime
importance to assess their ability to inhibit thrombin
generation. As compared to factor Xa inhibition assay
which is performed in buffer, thrombin generation
inhibition was carried out in plasma. Thrombin is
generated through proteolysis of prothrombin by factor
Xa in the prothrombinase complex, the rate of thrombin
generation is therefore largely dependent on available
factor Xa,20 and the same ranking as for factor Xa
inhibition was therefore expected and indeed observed
for 1 and 34 (Table 1).
Exp er im en ta l Section
All compounds were homogeneous by TLC analysis and had
spectral properties consistent with their assigned structures.
Melting points were determined in capillary tubes in a Mettler
apparatus and are uncorrected. Optical rotations were mea-
sured with a Perkin-Elmer Model 241 polarimeter at 22 ( 3
°C. Compound purity was checked by TLC on silica gel 60
F254 (E. Merck) with detection by charring with sulfuric acid.
Unless otherwise stated, column chromatography were per-
formed on silica gel 60, 40-63 or 63-200 µm (E. Merck). 1H
NMR spectra were recorded with Bruker AC 200, AM 250, AC
300, or AM500 instruments, for solution in CDCl3 or D2O.
Before analysis in D2O, samples were passed through a Chelex
(Bio-Rad) ion exchange column and lyophilized three times
from D2O. Chemical shifts are relative to external TMS when
the spectra are recorded in CDCl3 and to external TSP when
the spectra are recorded in D2O. MS analyses were performed
on a ZAB-2E instrument (Fisons). Elemental analyses were
performed on a Fisons elemental analyzer.
Factor Xa (71 nkat per vial) and S-2222 substrate (Bz-Ile-
Glu-Gly-Arg-pNA) were from Chromogenix (Mo¨lndal, Sweden).
Rabbit brain thromboplastin was from Sigma (Saint Quentin
Fallavier, France). Platelet poor plasma (PPP) was obtained
after centrifugation (15 min, 1500g) from blood collected from
the ante-cubital vein of normal healthy volunteers using 3.8%

1604 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Petitou et al.
sodium citrate as anticoagulant (blood/citrate, 9:1, v/v). Hu-
man tissue factor (Innovin) was from Baxter-Dade (Miami,
FL).
A solution of the above crude 11 in DMF (100 mL) was
treated, at room temperature, with BnBr (16 mL, 130.5 mmol)
and KHCO3 (6.7 g, 67 mmol) overnight. MeOH (35 mL) was
added, and the product was extracted with Et2O, washed with
H2O, dried, and concentrated. Column chromatography (1:1
cyclohexane/EtOAc) then gave methyl O-(benzyl 2-O-benzoyl-
4-O-levulinyl-3-O-methyl-R-L-idopyranosyluronate)-(1f4)-2,6-
di-O-benzyl-3-O-methyl-R-D-glucopyranoside (12, 11.6 g): TLC
Rf 0.54, 1:1 cyclohexane/EtOAc.
A solution of hydrazine hydrate (1 M in 3:2 pyridine/AcOH,
67 mL) was added to a cooled (0 °C) solution of the above 12
in pyridine (67 mL). After 30 min at 0 °C, concentration
followed by column chromatography (14:1 CH2Cl2/EtOAc)
yielded 13 (6.58 g, 64% from 7): 1H NMR (CDCl3) δ 8.00-
7.15 (m, 20H, 4Ph), 5.15 (d, 1H, H-1′), 4.57 (d, 1H, H-1), 3.48,
3.47, 3.32 (3s, 3 OCH3). Anal. (C43H48O13) C, H.
Meth yl 2,6-Di-O-ben zyl-3-O-m eth yl-r-D-glu cop yr a n o-
sid e (5). MeI (1.6 mL, 25 mmol) was added, at 0 °C, to a
solution of methyl 2-O-benzyl-4,6-O-benzylidene-R-D-glucopy-
ranoside11 (3, 6.3 g, 16.9 mmol) and NaH (0.6 g, 20 mmol) in
DMF (60 mL). After 1 h, MeOH (5 mL) was introduced
dropwise, and after 15 min the product was extracted with
EtOAc. The solution was washed with aqueous 10% Na2S2O3
and H2O, dried (Na2SO4), and concentrated. Column chroma-
tography gave methyl 2-O-benzyl-4,6-O-benzylidene-3-O-meth-
yl-R-D-glucopyranoside (4, 8.44 g, 79%): 1H NMR (CDCl3) δ
7.24-7.51 (m, 10H, 2Ph), 5.51 (s, 1H, :CHPh), 3.66 (s, 3H,
OCH3), 4.58 (d, 1H, J ) 3.7 Hz, H-1), 3.65, 3.38 (2s, 6H,
2OCH3); TLC, Rf 0.60, 15:1 CH2Cl2/acetone.
HBF4 (54% solution in Et2O, 26 mL, 188 mmol) was added
dropwise, with stirring, to a cooled (0 °C) solution of 4 (8 g, 21
mmol) and NaBH3CN (11.8 g, 188 mmol) in THF (110 mL).
After 75 min the solution was filtered (Celite) and diluted with
CH2Cl2, and NaOH was added until basic pH. The solution
was then washed with brine and H2O, dried (Na2SO4), and
concentrated. Column chromatography (20:1 CH2Cl2/acetone)
afforded 5 (5.7 g, 70%): 1H NMR (CDCl3) δ 7.36-7.24 (m, 10H,
2Ph), 3.66, 3.36 (2s, 6H, 2OCH3); TLC Rf 0.30, 18:1 CH2Cl2/
acetone.
Meth yl O-(2-O-Ben zoyl-4,6-O-isopr opyliden e-3-O-m eth -
yl-r-L-id op yr a n osyl)-(1f4)-2,6-d i-O-ben zyl-3-O-m eth yl-r-
D-glu cop yr a n osid e (7). Triflic acid in toluene (0.15 M, 1.5
mL) was added under argon to a stirred, cold (-20 °C) solution
of ethyl 2-O-benzoyl-4,6-O-isopropylidene-3-O-methyl-1-thio-
R,â-L-idopyranoside7 (6, 5.94 g, 15.5 mmol), 5 (6.03 g, 15.5
mmol), and N-iodosuccinimide (8.8 g, 39 mmol) in toluene (200
mL) containing finely grounded 4 Å molecular sieves. After 1
h solid NaHCO3 (125 mg) was introduced, and 15 min later
the solution was filtered, diluted with CH2Cl2, washed with
H2O, dried (Na2SO4), and evaporated. Column chromatogra-
phy (30:1, then 28:1, then 26:1 CH2Cl2/acetone) gave 7 (11.18
g, 89%): 1H NMR (CDCl3) δ 8.10-7.21 (m, 15H, 3Ph), 5.06 (d,
1H, J ) 3.1 Hz, H-1′), 4.55 (d, 1H, J ) 3.7 Hz, H-1), 3.58, 3.44,
3.29 (3s, 3 OCH3), 1.46, 1.47 (2s, 6H, :C(CH3)2). Anal.
(C18H24O8) C, H.
Meth yl O-(Ben zyl 2-O-ben zoyl-3-O-m eth yl-r-L-id op y-
r a n osylu r on a t e)-(1f4)-2,6-d i-O-b en zyl-3-O-m et h yl-r-D-
glu cop yr a n osid e (13). Aqueous CF3COOH (70%, 22 mL)
was added to a solution of 7 (9.9 g, 14 mmol) in CH2Cl2 (500
mL). After 40 min at room temperature the solution was
diluted with CH2Cl2, washed with cold saturated aqueous
NaHCO3 and H2O, and dried (Na2SO4). Concentration yielded
crude methyl O-(2-O-benzoyl-3-O-methyl-R-L-idopyranosyl)-
(1f4)-2,6-di-O-benzyl-3-O-methyl-R-D-glucopyranoside (8, 9.39
g): 1H NMR (CDCl3) δ 8.0-7.15 (m, 15H, 3Ph), 5.04 (1H, J )
2 Hz, H-1′), 4.57 (d, 1H, J ) 3.5 Hz, H-1), 3.67, 3.49, 3.34 (3s,
3 OCH3), 2.60 (2H, OH).
The above crude 8 (9 g) dissolved in CH2Cl2 (36 mL) was
heated at 50 °C for 4 h with Et3N (3 mL, 22 mmol), 4-(dimeth-
ylamino)pyridine (210 mg, 1.74 mmol), and tert-butyldimeth-
ylsilyl chloride (1.09 g, 20.3 mmol). Levulinic anhydride (5g,
22.5 mmol), Et3N (3.2 mL, 22.5 mmol), and 4-(dimethylamino)-
pyridine (400 mg, 3.26 mmol) were then added. After 4 h the
mixture was diluted with CH2Cl2, successively washed with
5% aqueous KHSO4, H2O, saturated aqueous NaHCO3, and
H2O, dried (Na2SO4), and concentrated to yield methyl O-(2-
O-benzoyl-4-O-levulinyl-3-O-methyl-6-O-(tert-butyldimethylsi-
lyl)-R-L-idopyranosyl)-(1f4)-2,6-di-O-benzyl-3-O-methyl-R-D-
glucopyranoside (10, 14.3 g): TLC Rf 0.76, 1:1 cyclohexane/
EtOAc.
O-(Ben zyl 4-O-levu lin yl-2,3-d i-O-m eth yl-â-D-glu cop y-
r a n osylu r on a te)-(1f4)-3-O-a cetyl-1,6-a n h yd r o-2-O-ben -
zyl-â-D-glu cop yr a n ose (22). O-(4,6-O-Isopropylidene-â-D-
glu copyr a n osyl)-(1f4)-1,6:2,3-dia n h ydr o-â-D-m a n n opy-
ranose15 (14, 22 g, 63.5 mmol) was methylated as described
for the preparation of 4 to give O-(4,6-O-isopropylidene-2,3-
di-O-methyl-â-D-glucopyranosyl)-(1f4)-1,6:2,3-dianhydro-â-D-
mannopyranose (15, 21.7 g): TLC Rf 0.67, 1:1 toluene/acetone.
Freshly prepared sodium benzylate (1 M in benzyl alcohol,
315 mL) was slowly added to the above crude 15 (21.7 g). The
mixture was heated during 4 h at 80 °C and neutralized with
Dowex 50 H+, and O-(4,6-O-isopropylidene-2,3-di-O-methyl-
â-D-glucopyranosyl)-(1f4)-1,6-anhydro-2-O-benzyl-â-D-glucopy-
ranose was obtained (16, 23 g) after filtration on a silica gel
pad (5:1 toluene/acetone): TLC Rf 0.21, 4:1 toluene/acetone.
Et3N (44 mL, 313 mmol), 4-(dimethylamino)pyridine (1.28
g, 10.5 mmol), and Ac2O (25 mL, 261 mmol) were added to a
solution of 16 (23 g, 52 mmol) in CH2Cl2 (390 mL). After 30
min the mixture was successively washed with 5% aqueous
KHSO4, H2O, saturated aqueous NaHCO3, and H2O and dried
(Na2SO4). Concentration gave O-(4,6-O-isopropylidene-2,3-di-
O-methyl-â-D-glucopyranosyl)-(1f4)-3-O-acetyl-1,6-anhydro-
2-O-benzyl-â-D-glucopyranose (17, 23.4 g) used as such in the
next step: TLC Rf 0.37, 4:1 toluene/acetone.
The isopropylidene group of 17 was removed as described
for the preparation of 8 to give O-(2,3-di-O-methyl-â-D-glu-
copyranosyl)-(1f4)-3-O-acetyl-1,6-anhydro-2-O-benzyl-â-D-glu-
copyranose (18, 21.6 g): TLC Rf 0.51, 2:3 toluene/acetone.
This compound was then silylated and levulinylated as
described for the preparation of 10 to give crude O-(4-O-
levulinyl-2,3-di-O-methyl-6-O-(tert-butyldimethylsilyl)-â-D-glu-
copyranosyl)-(1f4)-3-O-acetyl-1,6-anhydro-2-O-benzyl-â-D-glu-
copyranose (20, 54.2 g): TLC Rf 0.52, 2:1 toluene/acetone.
Oxidation of the crude residue obtained in the preceding
step, as described for the preparation of 11, gave the crude
acid O-(4-O-levulinyl-2,3-di-O-methyl-â-D-glucopyranosyluron-
ic acid)-(1f4)-3-O-acetyl-1,6-anhydro-2-O-benzyl-â-D-glucopy-
ranose (21, 37.9 g): TLC Rf 0.32, 12:2:0.6:1 EtOAc/pyridine/
AcOH/H2O.
Esterification of 21, as described for the synthesis of 12,
followed by column chromatography (5:1 toluene/acetone),
afforded pure O-(benzyl 4-O-levulinyl-2,3-di-O-methyl-â-D-
glucopyranosyluronate)-(1f4)-3-O-acetyl-1,6-anhydro-2-O-ben-
zyl- â-D-glucopyranose (22, 20 g, 46% from 14): 1H NMR
(CDCl3) δ 7.29-7.25 (m, 10H, 2Ph), 5.40 (d, 1H, J ) 1 Hz,
H-1), 4.69 (d, 1H, J ) 7 Hz, H-1′), 3.57, 3.50 (2s, 2 OCH3),
2.3-2.6 (m, 4H, levulinyl CH2CH2), 2.12, 2.03 (2s, 6H, 1Ac and
levulinyl CH3); TLC Rf 0.45, 2:1 toluene/acetone.
O-(Ben zyl 4-O-levu lin yl-2,3-d i-O-m eth yl-â-D-glu cop y-
r a n osylu r on a te)-(1f4)-3,6-d i-O-a cetyl-2-O-ben zyl-r,â-D-
glu cop yr a n osyl Tr ich lor oa cetim id a te (25). CF3COOH (10
mL, 130 mmol) was added to a solution of the above 22 (8 g,
11.7 mmol) in Ac2O (112 mL, 1.17 mol). After 1.5 h the
solution was concentrated. Toluene (10 mL) was evaporated
twice from the residue to eliminate traces of Ac2O. Crude
O-(benzyl 4-O-levulinyl-2,3-di-O-methyl-â-D-glucopyranosyl-
uronate)-(1f4)-1,3,6-tri-O-acetyl-2-O-benzyl-R,â-D-glucopyra-
nose (23, 9.55 g) was obtained: TLC Rf 0.36, 3:1 toluene/
acetone.
A solution of CrO3 (3.55 g, 35.5 mmol) in aqueous H2SO4
(3.5 M, 14.5 mL) was slowly added to a cooled (0 °C) solution
of the above crude 10 in acetone (700 mL). After 3 h CH2Cl2
was introduced, the mixture was poured into iced H2O, stirred
vigorously, washed with H2O until neutral, and dried. Con-
centration gave the acid methyl O-(2-O-benzoyl-4-O-levulinyl-
3-O-methyl-R-L-idopyranosyluronic acid)-(1f4)-2,6-di-O-benzyl-
3-O-methyl-R-D-glucopyranoside (11, 12.46 g): TLC Rf 0.34,
15:1 CH2Cl2/MeOH.

Close Analogue of an Antithrombotic Pentasaccharide
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11 1605
Benzylamine (49 mL, 453 mmol) was added to a solution of
the above crude 23 (9.55 g) in Et2O (400 mL). After 6 h at
room temperature the solution was washed with 1 M aqueous
HCl and H2O, dried, and concentrated. Column chromatog-
raphy (2:1 toluene/acetone) afforded pure O-(benzyl 4-O-
levulinyl-2,3-di-O-methyl-â-D-glucopyranosyluronate)-(1f4)-
3,6-di-O-acetyl-2-O-benzyl-R,â-D-glucopyranose (24, 6.35 g,
73% from 22): TLC Rf 0.43, 2:1 toluene/acetone. Anal.
(C37H46O16) C, H.
Trichloroacetonitrile (4.5 mL, 85 mmol) and cesium carbon-
ate (5 g, 15.3 mmol) were added under argon to a solution of
24 (6.33 g, 8.48 mmol) in CH2Cl2 (105 mL). After 1.5 h the
solution was filtered and concentrated. Column chromatog-
raphy of the residue (6:1 toluene/acetone) afforded a mixture
(R/â 62:38) of the O-(benzyl 4-O-levulinyl-2,3-di-O-methyl-â-
D-glucopyranosyluronate)-(1f4)-3,6-di-O-acetyl-2-O-benzyl-
R,â-D-glucopyranosyl trichloroacetimidates (25, 5.4 g, 71%):
TLC Rf 0.34 and 0.43, 3:1 toluene/acetone; 1H NMR (CDCl3) δ
8.70-8.60 (2s, 1H, dNH of R- and â-anomers), 7.17-7.35 (m,
10H, 2Ph), 6.44 (d, J ) 3.5 Hz, 0.62H, H-1R), 5.83 (d, J ) 7.5
Hz, 0.38H, H-1â), 3.48, 3.50 (2s, 6H, 2OMe), 2.20-2.60 (m,
4H, levulinyl CH2CH2), 2.11, 2.06, 2.05, 1.93, 1.85, 1.57 (6s,
Ac and levulinyl CH3).
Meth yl O-(Ben zyl 2,3-d i-O-m eth yl-â-D-glu cop yr a n osyl-
u r on ate)-(1f4)-O-(3,6-di-O-acetyl-2-O-ben zyl-r-D-glu copy-
r a n osyl)-(1f4)-O-(b en zyl 2-O-b en zoyl-3-O-m et h yl-r-L-
id op yr a n osylu r on a te)-(1f4)-2,6-d i-O-ben zyl-3-O-m eth yl-
r-D-glu cop yr a n osid e (27). Trimethylsilyl triflate (40 µL)
was added under argon to a stirred, cooled (-20 °C) solution
of the above imidates 25 (1.81 g, 2 mmol) and 13 (1.57 g, 2
mmol) in toluene (50 mL) containing 4 Å molecular sieves (3.3
g). After 30 min, solid NaHCO3 (0.1 g) was introduced, and
stirring was prolonged overnight. The solution was filtered,
washed with H2O, dried, and concentrated. Column chroma-
tography (3:2 cyclohexane/EtOAc) provided pure methyl O-
(benzyl 2,3-di-O-methyl-4-O-levulinyl-â-D-glucopyranosylur-
onate)-(1f4)-O-(3,6-di-O-acetyl-2-O-benzyl-R-D-glucopyranosyl)-
(1f4)-O-(benzyl 2-O-benzoyl-3-O-methyl-R-L-idopyranosyl-
uronate)-(1f4)-2,6-di-O-benzyl-3-O-methyl-R-D-glucopyrano-
side (26, 2.24 g, 73%): 1H NMR (CDCl3) δ 7.10-8.04 (m, 30H,
6Ph), 5.26 (d, 1H, J ) 2.6 Hz, H-1 G unit), 4.95 (d, 1H, J ) 3.7
Hz, H-1 F unit), 4.51 (d, 1H, J ) 3.5 Hz, H-1 H unit), 4.20 (d,
1H, J ) 7.9 Hz, H-1 E unit), 3.47, 3.42, 3.32, 3.28, 3.25 (5s,
15H, 5OMe), 2.26-2.58 (m, 4H, levulinyl CH2CH2), 2.12, 2.07,
1.92 (3s, 9H, 2Ac and levulinyl CH3). Anal. (C80H22O28) C, H.
The levulinyl group of 26 (2.21 g, 1.47 mmol) was selectively
removed as described for the preparation of 13 to give pure
(TLC) methyl O-(benzyl 2,3-di-O-methyl-â-D-glucopyranosy-
luronate)-(1f4)-O-(3,6-di-O-acetyl-2-O-benzyl-R-D-glucopyra-
nosyl)-(1f4)-O-(benzyl 2-O-benzoyl-3-O-methyl-R-L-idopyra-
nosyluronate)-(1f4)-2,6-di-O-benzyl-3-O-methyl-R-D-gluco-
pyranoside (27, 1.98 g, 96%) after column chromatography (1:1
cyclohexane/EtOAc): TLC Rf 0.37, 1:1 cyclohexane/EtOAc; 1H
NMR (CDCl3) δ 7.18-8.04 (m, 30H, 6Ph), 5.27 (d, 1H, J ) 3.1
Hz, H-1 G unit), 4.98 (d, 1H, J ) 3.7 Hz, H-1 F unit), 4.53 (d,
1H, J ) 3.5 Hz, H-1 H unit), 4.21 (d, 1H, J ) 7.7 Hz, H-1 E
unit), 3.58, 3.42, 3.32, 3.29, 3.28 (5s, 5OMe), 2.06, 1.89 (2s,
2Ac).
J ) 2.9 Hz, H-1R), 5.55 (d, J ) 7 Hz, H-1â), 3.67, 3.54 (2s, 6H,
2OMe) 2.14, 2.08, 2.05 (3s, 6H, OAc).
Thioethanol (0.2 mL, 2.6 mmol) was added to a solution of
31 (0.5 g, 1.3 mmol) in toluene (25 mL) followed by BF3‚Et2O
complex (1.3 mL, 1.3 mmol, dropwise addition). After 1.5 h
aqueous saturated NaHCO3 (15 mL) was introduced, and the
solution was diluted with CH2Cl2, washed with H2O and brine,
dried (Na2SO4), and concentrated. Column chromatography
of the residue (5:1, then 4:1, 3:1 hexane/EtOAc) afforded 32
(0.46 g, 91% from 30) as a colorless syrup: 1H NMR (CDCl3)
δ 7.30-7.42 (m, 5H, Ph), 5.30 (d, J ) 5.1 Hz, H-1R), 4.79 (d, J
) 8.1 Hz, H-1â), 3.66, 3.64, 3.53, 3.52 (4s, 6H, OMe), 2.42-
2.69 (m, 2H, SCH2CH3), 2.08 (s, OAc), 1.15-1.38 (m, 3H,
SCH2CH3); TLC Rf 0.37 1:1 hexane/EtOAc. Anal. (C19H28O6S)
C, H, S.
Meth yl O-(6-O-Acetyl-2-O-ben zyl-3,4-d i-O-m eth yl-r-D-
glu cop yr a n osyl)-(1f4)-O-(b en zyl 2,3-d i-O-m et h yl-â-D-
glu copyr an osylu r on ate)-(1f4)-O-(3,6-di-O-acetyl-2-O-ben -
zyl-r-D-glu cop yr a n osyl)-(1f4)-O-(ben zyl 2-O-ben zoyl-3-
O-m eth yl-r-L-idopyr an osylu r on ate)-(1f4)-2,6-di-O-ben zyl-
3-O-m eth yl-r-D-glu cop yr a n osid e (33). Triflic acid (3.4 µL,
0.038 mmol) was added under argon to a stirred, cold (-20
°C) solution of 32 (0.15 g, 0.384 mmol), 27 (0.45 g, 0.32 mmol),
and N-iodosuccinimide (0.087 g, 0.384 mmol) in 2:1 1,2-dichlo-
roethane/Et2O (21 mL) containing 4 Å molecular sieves (0.6
g). After 30 min solid NaHCO3 (10 mg) was added, and 15
min later the solution was filtered, diluted with CH2Cl2,
washed with 10% aqueous Na2S2O3 and H2O, dried (Na2SO4),
and evaporated. Column chromatography (16:1 CH2Cl2/ace-
tone) gave pure (TLC, Rf 0.5, 1:1 cyclohexane EtOAc) 33 (0.38
g, 62%): 1H NMR (CDCl3) δ 7.18-8.04 (m, 35H, 7Ph), 5.44 (d,
1H, J ) 3.3 Hz, H-1 D unit), 5.25 (d, 1H, J ) 2.2 Hz, H-1 G
unit), 4.95 (d, 1H, J ) 3.5 Hz, H-1 F unit), 4.52 (d, 1H, J ) 3.3
Hz, H-1 H unit), 4.15 (d, 1H, J ) 8.1 Hz, H-1 E unit), 3.56,
3.49, 3.43, 3.41, 3.32, 3.28 (7s, 7OMe), 2.09, 2.06, 1.87 (3s, 9H,
3Ac).
Meth yl O-(3,4-Di-O-m eth yl-2,6-d i-O-su lfo-r-D-glu cop y-
r a n osyl)-(1f4)-O-(2,3-d i-O-m et h yl-â-D-glu cop yr a n osyl-
u r on ic a cid )-(1f4)-O-(2,3,6-tr i-O-su lfo-r-D-glu cop yr a n o-
syl)-(1f4)-O-(3-O-m e t h yl-2-O-su lfo-r-L-id op yr a n osyl-
u r on ic acid)-(1f4)-3-O-m eth yl-2,6-di-O-su lfo-r-D-glu copy-
r a n osid e, Deca sod iu m Sa lt (34). A solution of 33 (0.25 g,
0.144 mmol) in DMF (11 mL) was stirred during 36 h under a
weak stream of H2 in the presence of 10% Pd/C catalyst (0.25
g). After filtration the solution was concentrated to give
methyl O-(6-O-acetyl-3,4-di-O-methyl-R-D-glucopyranosyl)-
(1f4)-O-(2,3-di-O-methyl-â-D-glucopyranosyluronic acid)-(1f4)-
O-(3,6-di-O-acetyl-R-D-glucopyranosyl)-(1f4)-O-(2-O-benzoyl-
3-O-methyl-R-L-idopyranosyluronic acid)-(1f4)-3-O-methyl-R-
D-glucopyranoside (0.170 g): TLC Rf 0.87, 5:5:1:3 EtOAc/
pyridine/AcOH/H2O.
Aqueous NaOH (5 M, 2.9 mL) was added to a solution of
the above crude compound in MeOH (26 mL). After 40 min
Dowex 50 H+ was introduced until neutral pH. The solution
was concentrated, and the residue was layered on top of a
Sephadex G 25 column (1.6 × 100 cm) eluted with H2O.
Concentration of the pooled fractions gave methyl O-(3,4-di-
O-methyl-R-D-glucopyranosyl)-(1f4)-O-(2,3-di-O-methyl-â-D-
glucopyranosyluronic acid)-(1f4)-O-(R-D-glucopyranosyl)-(1f4)-
O-(3-O-methyl-R-L-idopyranosyluronic acid)-(1f4)-3-O-methyl-
R-D-glucopyranoside (0.132 g): TLC Rf 0.14, 5:5:1:3 EtOAc/
pyridine/AcOH/H2O.
Eth yl 6-O-Acetyl-2-O-ben zyl-3,4-d i-O-m eth yl-1-th io-r,â-
D-glu cop yr a n osid e (32). The epoxide ring of 1,6:2,3-dian-
hydro-4-O-methyl-â-D-mannopyranose17 (28, 6.39 g, 40.4 mmol)
was opened as descibed for the preparation of 16 to give 1,6-
anhydro-2-O-benzyl-4-O-methyl-â-D-glucopyranose (28, 10.51
g, 97%) after column chromatography (5:1, then 4:1, 3:1, 1:2,
1:3 hexane/EtOAc): 1H NMR (CDCl3) δ 7.26-7.34 (m, 5H, Ph),
5.43 (m, 1H, H-1), 3.47 (1s, 3H, OMe).
Compound 28 (3.57 g, 13.4 mmol) was methylated as
described for the preparation of 4 to give 1,6-anhydro-2-O-
benzyl-3,4-di-O-methyl-â-D-glucopyranose (29, 3.45 g, 89%)
after column chromatography (2:1 hexane/EtOAc): 1H NMR
(CDCl3) δ 7.26-7.38 (m, 5H, Ph), 5.42 (m, 1H, H-1), 3.47, 3.31
(2s, 6H, 2OMe).
Et3N/SO3 complex (1.05 g, 5.82 mmol) was added to a
solution of the above compound (0.132 g) in DMF (6 mL), and
the solution was heated at 50 °C for 20 h. NaHCO3 (1.8 g
dissolved in H2O) was then introduced, and the solution was
layered on top of a Sephadex G-25 column (1.6 × 100 cm)
equilibrated in 0.2 M NaCl. The fractions were pooled,
concentrated, and dessalted on the same gel filtration column,
equilibrated in H2O. Lyophilization then gave 34 (0.23 g, 88%
from 33): LSIMS (liquid secondary ion mass spectrometry),
negative mode, m/ z 1791 (M - Na)-, 1769 (M - 2Na + H)-;
1H NMR (D2O) δ 5.53 (d, 1H, J ) 3.7 Hz, H-1 D unit), 5.45 (d,
1H, J ) 3.7 Hz, H-1 F unit), 5.12 (d, 1H, J ) 2.6 Hz, H-1 G
unit), 5.05 (d, 1H, J ) 3.5 Hz, H-1 H unit), 4.65 (d, 1H, J )
7.7 Hz, H-1 E unit), 3.60, 3.59, 3.55, 3.54, 3.49, 3.42 (s, OMe).
Acetolysis of the anhydro ring of 30 (3.2 g, 11.44 mmol) was
performed as described for the preparation of 23 to give crude
1,6-di-O-acetyl-2-O-benzyl-3,4-di-O-methyl-â-D-glucopyra-
nose (31, 4.4 g): 1H NMR (CDCl3) δ 7.33 (m, 5H, Ph), 6.23 (d,

1606 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 11
Petitou et al.
34 was >95% pure (chiral detection) by HPLC on a Carbopac
PA100 column eluted with a linear 1.1-2.0 M NaCl gradi-
ent.
P h a r m a cok in etics. Pharmacokinetics were performed in
rats after iv bolus injection of the compounds. The jugular
vein was canulated under general anesthesia (sodium pento-
barbitone 30 mg/kg, iv), and the animals were allowed to
recover from the surgery overnight. The pentasaccharides
were injected via the catheter at a dose of 100 nmol/kg. Blood
was collected in a 0.129 M citrate solution (1/10; v/v). Con-
centrations of pentasaccharides in plasma were determined
by measuring the anti-factor Xa activity as described above.
Each pentasaccharide was used as its own reference. Elimi-
nation half-lives were calculated by the slope of the terminal
phase using a computational method (Siphar/PC, version 4.0).
The results are mean ( SEM, n ) 4.
Bin d in g Con sta n ts. Fluorescence measurements were
performed as descrided by Atha et al.18 using a Perkin-Elmer
LS-50 type spectrofluorimeter; excitation λ ) 280 nm, emission
λ ) 338 nm; equipped with a thermostated sample compart-
ment, at 37 °C, under continuous stirring. Oligosaccharides
were added into the cuvette containing 2 mL of Tris-HCl
buffer, 0.01 M, pH 7.0, 0.15 M NaCl, and 5-60 nM human
AT-III (purified according to Mc Kay23). The ratio and the
concentrations of AT III-oligosaccharide complexes were
calculated considering a 1:1 reaction stoichiometry, and dis-
sociation constants (KD) were determined by Scatchard analy-
sis using a specific application of RS/1 computer program (BBN
Software Product Corp., Cambridge, MA). The results are
mean ( SEM, n ) 3.
An ti-fa ctor Xa Activity. Anti-factor Xa activity was
determined by an amidolytic method (modification of the
procedure of Teien and Lie19): factor Xa (7.5 nkat/mL in 20
mM Tris/maleate buffer, pH 7.4, NaCl 150 mM; 100 µL) was
incubated during 2 min with AT III (0.5 unit/mL in 20 mM
Tris/maleate buffer, pH 7.4, NaCl 150 mM; 100 µL) at 37 °C
in the presence of the oligosaccharides (at various concentra-
tions in 20 mM Tris/maleate buffer, pH 7.4, NaCl 150 mM;
100 µL). To measure residual factor Xa, S-2222 substrate (Bz-
Ile-Glu-Gly-Arg-pNA; 1 mM in 50 mM Tris/HCl buffer, pH 8.4,
NaCl 175 mM, EDTA 2 7.5 mM; 100 µL) was added. The
reaction was stopped 2 min later by addition of 50% aqueous
acetic acid (100 µL), and the absorbance at 405 nm was read.
The percentage of inhibition was then calculated [inhibition
% ) 100 × (A405 of buffer control - A405 of sample)/A405 of buffer
control], and the activity of the compounds was determined
by comparison with a calibrated standard, using Excel 4.0
Software (Microsoft, Redmond USA). The results are mean
( SEM, n ) 3.
Th r om bin Gen er a tion Test. The thrombin generation
methodology was adapted from Hemker et al.20 who used a
poor substrate of thrombin (S-2222, Bz-Ile-Glu-Gly-Arg-pNA)
to allow easier monitoring of the reaction. Under the condi-
tions of the assay, the very low concentration of factor Xa, as
compared to thrombin, could not be detected and does not
interfere with the determination of thrombin. Briefly, in a
plastic disposable semimicrocuvette were successively added,
at 37 °C, rabbit brain thromboplastins (50 µL, i.e. the amount
necessary to obtain a coagulation time of 90 s in the Quick
assay, see ref 20), S-2222 substrate (1.6 mM, in 50 mM Tris-
HCl buffer, pH 7.35, NaCl 0.15 M; 50 µL), the compound to be
tested (in 50 mM Tris-HCl buffer, pH 7.35, NaCl 0.15 M; 50
µL), and CaCl2 (0.25 M in H2O; 50 µL). Thrombin generation
was initiated by the addition of defibrinated platelet poor
plasma (100 µL). The absorbance at 405 nm was plotted vs
time during 20 min. The area under the curve and the IC50
were determined, using the four-parameter logistic model, with
a confidence interval of 95%. The adjustment was obtained
by nonlinear regression using the Levenberg-Marquard al-
gorithm in RS/1 software (BBN Software Product Corp.,
Cambridge, MA). The results are mean, and 95% confidence
interval, n ) 3.
An tith r om botic Activity. Thrombus formation by a
combination of stasis and hypercoagulability was induced as
described by Vogel et al.21 Male Sprague-Dawley rats (250-
300 g; Iffa Credo, France) were anesthetized with sodium
pentobarbitone (30 mg/kg, ip). The vena cava was exposed
and dissected free from surrounding tissue. Two loose sutures
were prepared 0.7 cm apart on the inferior vena cava, and all
collateral veins were ligated. The various pentasaccharides
were administered iv in the dorsal penile vein 5 min before
thrombosis induction. Human tissue factor (1 ng/kg) was then
injected iv, in the dorsal penile vein, during 30 s, and 10 s
after the end of the injection, stasis was established by
tightening the two sutures. Stasis was maintained for 20 min,
and the thrombus formed was removed, rinsed, dried overnight
at 60 °C, and weighed. The results are expressed as ED50 (50%
reduction in thrombus weight) mean and 95% confidence
interval, n ) 6.
Ack n ow led gm en t. This work is part of a collabora-
tion between N. V. Organon (Oss, The Netherlands) and
Sanofi Recherche. We thank the Sanofi Recherche
“Service d’Analyse de Recherche” staff (C. Picard, Head)
for elemental and HPLC analyses (M. Maftouh, S.
Albugues); NMR analyses (C. Ponthus, D. Albene, M.
Rival), and MS analyses (F. Uzabiaga, C. Dhers, C.
Monteiro). Our thanks also go to P. Sizun for recording
high-field NMR spectra.
Refer en ces
(1) Olson, S. T.; Bjo¨rk, I. Regulation of Thrombin Activity by
Antithrombin and Heparin. Semin. Thromb. Hemostasis 1994,
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(2) Choay, J .; Petitou, M.; Lormeau, J . C.; Sinay¨, P.; Casu, B.; Gatti,
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a Synthetic
Pentasaccharide with High Affinity for Antithrombin III and
Eliciting High Anti-Factor Xa Activity. Biochem. Biophys. Res.
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(3) Walenga, J . M.; Petitou, M.; Lormeau, J . C.; Samama, M.;
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Heparin Pentasaccharide in a Rabbit Stasis Thrombosis Model
Using Different Thrombogenic Challenges. Thromb. Res. 1987,
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