Flavonoids with an oligopolysulfated moiety: A new class of anticoagulant agents

Article abstract of DOI:10.1021/jm1013117

Polysulfated (oligo)flavonoids were synthesized and assayed for their in vitro and in vivo anticoagulant activities. The approach was based on molecular hybridization of two classes of anticoagulants, sulfated polysaccharides and sulfated flavonoids. The synthesis was optimized using microwave-assisted sulfation with triethylamine-sulfur trioxide. The obtained polysulfated flavonosides were highly effective in increasing clotting times and able to completely block the clotting process, in contrast to their corresponding aglycones. The thromboelastography proved that polysulfated flavonosides possess good whole blood anticoagulation activity. The following structure-activity relationships were found: 3-O-rutinosides (10, 13) were direct inhibitors of FXa, while 7-O-rutinosides (7, 8) showed inhibition of FXa by ATIII activation. Furthermore, compounds 7 and 13 were stable in plasma and active in vivo and preliminary toxicity studies would lead us to rule out acute side effects. From the overall results, the polysulfated flavonosides showed the potential as new effective and safe agents for anticoagulant therapy.

Full text of DOI:10.1021/jm1013117

J. Med. Chem. 2011, 54, 95–106 95
DOI: 10.1021/jm1013117
Flavonoids with an Oligopolysulfated Moiety: A New Class of Anticoagulant Agents
†
Marta Correia-da-Silva, Emı
Luıs M. Cunha-Ribeiro, and Madalena M. M. Pinto*
´
†
‡
‡
§
´
lia Sousa, B ꢀa rbara Duarte, Franklim Marques, F ꢀe lix Carvalho,
,†
†
Centro de Quı´mica Medicinal;Universidade do Porto (CEQUIMED-UP), Departamento de Ci e^ ncias Quı´micas, Laborat oꢀ rio de Quı´mica
‡
Org a^ nica e Farmac e^ utica, Faculdade de Farm aꢀ cia, Universidade do Porto, Rua Anı´bal Cunha 164, 4050-047 Porto, Portugal, Laborat oꢀ rio de
An aꢀ lises Clı´nicas, Faculdade de Farm aꢀ cia, Universidade do Porto, Rua Anı´bal Cunha 164, 4050-047 Porto, Portugal, REQUIMTE,
§
Departamento de Toxicologia, Faculdade de Farm aꢀ cia, Universidade do Porto, Rua Anı ´b al Cunha 164, 4050-047 Porto, Portugal, and Servi c- o de
ImunoHemoterapia, Centro de Trombose, Hemostase e Biologia Vascular, Hospital de S. Jo a~ o, 4200-319 Porto, Portugal
Received June 17, 2010
Polysulfated (oligo)flavonoids were synthesized and assayed for their in vitro and in vivo anticoagulant
activities. The approach was based on molecular hybridization of two classes of anticoagulants, sulfated
polysaccharides and sulfated flavonoids. The synthesis was optimized using microwave-assisted
sulfation with triethylamine-sulfur trioxide. The obtained polysulfated flavonosides were highly
effective in increasing clotting times and able to completely block the clotting process, in contrast to
their corresponding aglycones. The thromboelastography proved that polysulfated flavonosides possess
good whole blood anticoagulation activity. The following structure-activity relationships were found:
3
-O-rutinosides (10, 13) were direct inhibitors of FXa, while 7-O-rutinosides (7, 8) showed inhibition of
FXa by ATIII activation. Furthermore, compounds 7 and 13 were stable in plasma and active in vivo and
preliminary toxicity studies would lead us to rule out acute side effects. From the overall results, the
polysulfated flavonosides showed the potential as new effective and safe agents for anticoagulant therapy.
Introduction
molecular size, bioavailability was improved, allowing subcu-
13
1
,2
taneous administration. However, like heparin, these deriva-
14,15
tives are still heterogeneous and polydisperse
Heparin is a widespread anticoagulant agent, exerting its
therapeutic effect by binding the enzyme inhibitor antithrom-
and the risk of
inducing the immunological type of HIT has also been re-
a
bin III (ATIII ), which results in the inactivation of factor Xa
1
6
3
FXa) and thrombin. Heparin is obtained from either bovine
ported. The structural features associated with HIT have been
(
1
7
characterized and a heparin pentasaccharide, fondaparinux
(Figure 1), completely free of HIT activation, was developed
and approved.
or porcine sources, thus making heparin preparations struc-
turally diverse, varying in their molecular weight, monosac-
charide sequence, and sulfation pattern. The use of heparin
suffers froma number of limitations duetoitspolyanionic and
heterogeneous nature. Heparin therapy is limited to intrave-
1
8,19
4
,5
In contrast to unfractionated and LMW
20
heparins, fondaparinux has a defined composition. How-
ever, the synthesis of this pentasaccharide is accomplished
through a multistep, complex, and low-yielding procedure.
21
6
nous application and can be associated to cases of excessive
7
bleeding and liver toxicity. Furthermore, a significant num-
8
Therefore, the search for new alternative anticoagulant agents
with a well-defined composition, fewer secondary effects, and
feasible synthesis is a major challenge to medicinal chemists.
Our top priority was to develop new anticoagulant small
molecules based on a flavonoid scaffold. Several sulfated
flavonoids have already been described for their anticoagulant
ber of patients develop heparin-induced thrombocytopenia
9
HIT) after prolonged heparin exposure. Some problems
(
associated with heparin were minimized with the preparation
1
0,11
of low-molecular-weight heparins (LMWH), which in-
1
duce less bleeding complications. Because of the smaller
2
2
2
actions. Two sulfated flavonoids isolated from Flaveria biden-
0
0
0
0
tis, quercetin 3-acetyl-3 ,4 ,7-O-trisulfate and quercetin 3,3 ,4 ,7-
O-tetrasulfate (Figure 1), have already shown anticoagulant
actions in vitro by prolonging activated partial thromboplastin
(APTT) and prothrombin (PT) times through a heparin cofac-
*
To whom correspondence should be addressed. Phone: þ351
2
22078916. Fax: þ351 222003977. E-mail: madalena@ff.up.pt.
a
Abbreviations: ATIII, antithrombin III; FXa, factor Xa; HIT,
heparin-induced thrombocytopenia; LMWH, low-molecular-weight
heparins; HCII, heparin cofactor II; APTT, activated partial thrombo-
plastin time; PT, prothrombin time; EHBS, extended heparin binding
site; SOSA, selective optimization of side activities; DMA, dimethyla-
cetamide; MW, microwave; APTT
quired to double the time of clot formation in the APTT assay; PT
concentration of compound required to double the time of clot forma-
tion in the PT assay; TT, thrombin time; TT , concentration of com-
pound required to double the time of clot formation in the TT assay;
TEG, thromboelastography; PFA, platelet function analyzer; ip, intra-
peritoneal; ADP, adenosine 5 -diphosphate; EPI, epinephrine; GOT,
glutamic-oxaloacetic transaminase; GPT, glutamate-pyruvate-transa-
minase; SEM, standard error of the mean.
23
tor II (HCII)-dependent thrombin inhibition. Moreover,
0
0
quercetin 3,3 ,4 ,7-O-tetrasulfate was found to inhibit agonist-
24
induced human platelet aggregation. Also, the inhibitory
0
2
, concentration of compound re-
effects of quercetin 7-O-sulfate and quercetin 4 ,7-O-disulfate
sodium salts (Figure 1) on thrombin-induced platelet aggrega-
2
,
2þ
tion through a mechanism involving Ca influx inhibition were
Several sulfated flavonoids obtained by synthesis
were shown to act as activators of ATIII/FXa inhibition.
However, the activation effect achieved with these sulfated
2
25,26
reported.
27-30
0
2
7-30
small molecules on ATIII was weak.
As assessed by a
r
2
010 American Chemical Society
Published on Web 12/07/2010
pubs.acs.org/jmc

9
6
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Correia-da-Silva et al.
Figure 1. Pentasaccharide fondaparinux and flavonoids with anticoagulant activity.
Figure 2. Schematic representation of the strategies used herein for the development of new anticoagulant small molecules based on three
0
0
examples, namely fondaparinux, quercetin 3,3 ,4 ,7-O-tetrasulfate, and diosmin.
computational study based on the hydropathic interaction
HINT) analysis, the increase of the flavonoid molecular size
Compounds were screened by in vitro clotting time assays in
(
human plasma, and the effectofthe mostpromisingmolecules
was further evaluated in human whole blood using thromboe-
lastography and platelet function analyzer. To better under-
stand the mechanism of action of these new anticoagulant
molecules, in vitro enzyme inhibition assays were performed
against FXa, thrombin, and ATIII. Finally, stability and in
vivo studies were conducted for one representative direct and
indirect inhibitor of FXa to determine in vivo anticoagulant
activity and their toxicity potential.
was associated to an improvement of the binding affinity
toward ATIII. For instance, a dimeric compared to a mono-
meric molecule scored better because it could simultaneously
engage both the extended heparin (EHBS) and pentasacchar-
2
9
ide (PBS) binding sites of ATIII.
The oligo-O-sulfated moiety is present in potent antico-
agulant polysaccharides, suggesting the incorporation of an
oligo-O-sulfated moiety into a flavonoid scaffold in order to
improve the anticoagulant potency by increasing both molec-
ular size and number of sulfate groups (Figure 2, A and B).
Modification of molecules that have already been safely used
in humans conducts to compounds with more predictable and
lesscomplex pharmacokinetics, lower incidence of side effects,
Chemistry
Diosmin (1), hesperidin (2), rutin (3), and trihydroxyethyl-
rutin (4) were selected as the building blocks for the synthesis
of polysulfated flavonosides (Scheme 1). Additionally, in order
to study structure-activity relationship, the respective aglycones
diosmetin (5) and hesperetin (6) were submitted to sulfation
(Scheme 1).
3
1
and less demanding clinical studies. On the basis of these
considerations, we selected for sulfation several flavonoids
glycosides already used in human therapy as venotropic
3
2
agents (Figure 2C). This strategy follows the principles of
1
SOSA (selective optimization of side activities) approach.
3
Flavonoids 5 and 6 were initially obtained by hydrolysis of
1 and 2, respectively, in a sulfuric acid-acetic acid-water
In this work, the synthesis, the in vitro and in vivo anti-
coagulant profile, and preliminary toxicological investigation
of several polysulfated flavonoids/flavonosides are described.
3
3
medium (Scheme 1, a). Compounds 1-6 were then sub-
mitted to sulfation with triethylamine-sulfur trioxide adduct in

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 97
a
Scheme 1. Reagents and Conditions for the Synthesis of Compounds 5-13
a
Reagents and conditions: (a) sulfuric acid-acetic acid-water (1:30:19); (b) triethylamine-sulfur trioxide adduct (4 equiv/OH for compounds 1-4, 6;
equiv/OH for compound 5), DMA, 65 °C; (c) triethylamine-sulfur trioxide adduct (10 equiv/OH), DMA, MW.
8
0
0
dimethylacetamide (DMA) (Scheme 1, b). Sulfation was first
attempted with tetrabuthylammonium hydrogen sulfate and N,
metin 3 ,7-O-disulfate (11), and hesperetin 3 ,7-O-disulfate (12)
were obtained in moderate yields (Scheme 1). Compounds 7,
0
0
N -dicyclohexylcarbodiimide, a method extensively used in sul-
8, 10, and 12 are described for the first time. Diosmetin 3 ,7-
O-disulfate (11) was previously isolated from the leaves of
Lachenalia unifolia, and rutin nonasulfate (9) was previously
3
obtained by synthesis.
The sulfation of the hydroxyl at 5-position did not occur in
flavonoids 7-12, which can be explained by the hydroxyl
binding with the adjacent carbonyl at the 4-position. Surpris-
ingly, for compounds 7 and 8, sulfation did not take place in
3
4
fation of flavonoids; nonetheless, a complex mixture of sulfated
derivatives was obtained (data not shown). The triethylami-
ne-sulfur trioxide adduct in DMA (4-8 equiv/OH) allowed us
35
6
2
8,30
to achieve a highly degree of sulfation.
Following conven-
0
0
000 00 000 00 000
tional heating (Scheme 1, b), diosmin 2 ,2 ,3 ,3 ,4 ,4 -O-
0
0
000 00 000 00 000
hexasulfate (7), hesperidin 2 ,2 ,3 ,3 ,4 ,4 -O-hexasulfate
0
0
000
0
00 000
0
00 000
00 00
(8), rutin 2 ,2 ,3 ,3 ,3 ,4 ,4 ,4 ,7-O-nonasulfate (9), 3 ,4 -
bis(2-O-sulfate ethoxy)-7-(2-O-sulfate ethoxy)-rutin (10), dios-
0
the 3 hydroxyl, in contrast to compounds 11 and 12. This fact

9
8
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Correia-da-Silva et al.
Figure 3. Effects of polysulfated flavonoids (7, 8, 10-13) on APTT, PT, and TT clotting assays using human pooled plasma, expressed as ratio
of clotting time in the presence/absence of compound. Data points represent the average of three experiments performed in duplicate with a
standard deviation <10%. NC, no coagulation.
a
Table 1. Effects of Polysulfated Flavonosides 7, 8, 10, and 13 in the Clotting Assays
activated partial thromboplastin time (APTT)
prothrombin time (PT)
thrombin time (TT)
b
b
b
compd
total inhibition
APTT
2
total inhibition
5
PT
2
total inhibition
TT
>5
na
2
7
8
1.25
1.25
0.435
0.413
0.145
0.066
2.234
3.615
0.835
0.649
1
1
0
3
0.625
0.312
2.5
1.25
5
5
0.867
0.877
a
-3
Values in concentrations expressed in 10 M represent the average of three independent experiments with a standard deviation <10%; positive
= 1.9 U/mL, and TT
b
control, heparin: APTT
M (P > 0.05).
2
= 0.4 U/mL, PT
2
2
= 0.2 U/mL. The concentration required to double clotting time. na: not active at 5 ꢀ
-
3
10
can be explained because the sulfation becomes progressively
difficult, due to anion crowding, considering a large number
of hydroxyl groups.
APTT, PT, and thrombin time (TT). The three clotting assays
recorded interactions at different stages of the coagulation
process, providing key information about the mode of action.
The APTT identifies the time interval required for clot for-
mation in response to a nonphysiological stimulus that leads
to primary activation of the intrinsic pathway; the PT assay
measures the activity of coagulation factors of the extrinsic
pathway, and the TT screens the formation of fibrin from
fibrinogen after the addition of known amounts of thrombin
to the plasma sample. The anticoagulant effect of the de-
sulfated starting materials (2-4) was also evaluated in order
to explore their potential influence in the anticoagulant effect
of the sulfated molecules. Because of solubility issues, only
diosmin (1) was not considered because the final concentra-
tion of DMSO interfered with the clotting assays.
3
7
The major bottleneck in the synthetic procedure was the
isolation of the polysulfated compounds from the crude prod-
uct. Because of the presence of an oligo-O-sulfated moiety, the
flavonoids derivatives synthesized are highly soluble in water.
To remove water-soluble impurities, dialysis with cellulose
membrane (cutoff 1000) was necessary for compounds 8-10
4
1
(see Supporting Information).
To optimize the reaction, namely the time needed for sul-
fation of flavonoids glycosides with a high number of hydro-
xyl groups (1-4), new conditions were investigated by using
microwave (MW) irradiation (Scheme 1, c). Recently, sulfa-
tion with MW was described using trimethyamine-sulfur
3
7
trioxide in CH CN. In the case of compounds 1-4, DMA
3
The results for the polysulfated flavonoids (7, 8, 10-13), on
APTT, PT, and TT for the range of investigated concentra-
tions are summarized in Figure 3. For the most potent com-
pounds (7, 8, 10, and 13), the concentration needed to double
the coagulation times (APTT , PT , TT ) was calculated
was found a more suitable solvent than CH CN. To avoid the
presence of partially sulfated products, a 10 equiv/OH of the
3
3
7
sulfating complex was used.
When compared to conventional heating, the MW-assisted
sulfation allowed the synthesis of highly sulfated derivatives in
shorter reaction times (30 min). Additionally, rutin persulfate 13
was obtained (Scheme 1, c) in quantitative yield (99%) and with
2
2
2
(
Table 1). Compound 11 was not tested at a final concentra-
-
3
tion of 5 ꢀ 10 M (not soluble), and compound 9 was not
obtained with the desirable purity to be tested (<95%).
The investigated sulfated compounds (7, 8, 10, and 13)
showed anticoagulant properties in a dose-dependent manner
3
8
high purity (> 95%) in contrast to the previously described as
well as compound 10 with an increased yield (from 20 to 89%).
The structure elucidation of compounds 5-13 was estab-
lished on the basis of IR, HRMS, and NMR techniques (see
Supporting Information for detailed discussion). For agly-
(
Figure3). Thenonsulfated flavonoids2-4 were inactiveinall
-3
clotting time assays (at the concentration tested, 5 ꢀ 10 M,
no significant difference was observed between the compound
and the control; data not shown).
3
9,40
cones 5 and 6, data are in accordance to literature.
35
For
diosmetin 3 ,7-O-disulfate (11), rutin nonasulfate (9), and
0
36
Prolongation of APTT (Figure 3) was observed for all the
compounds (7, 8, 10-13). Flavonosides 7, 8, 10, and13 doubled
3
rutin decasulfate (13) their structural data are reported for
6
the first time in this work (see Experimental Section).
APTT (APTT ) at concentrations in the micromolar range and
were able to completely block this clot formation pathway
2
Results and Discussion
(Figure 3 and Table 1). The flavonoside 13 was the most potent
The anticoagulant activity of the sulfated molecules 7, 8,
and 10-13 was initially screened in vitro in human plasma by
in prolonging APTT (APTT = 66 μM). The sulfated aglycone
11 prolonged no more than 1.65-fold the APTT at the highest
2

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 99
a
-4
-4
b
Table 2. Effects of Polysulfated Flavonosides 7, 8, 10 (6.25 ꢀ 10 M), and 13 (1.25 ꢀ 10 M) in Whole Human Blood on the Parameters Obtained
from Thromboelastography (TEG)
InTEM
ExTEM
FibTEM
c
d
e
f
c
d
e
f
f
compd
reference range
R (s)
100-240
K (s)
30-110
µ (deg)
70-83
MA (mm)
50-72
R (s)
38-79
K (s)
34-156
µ (deg)
63-83
MA (mm)
50-72
MA (mm)
9-25
control
188 ( 21g
267 ( 7*
82 ( 3 g
229 ( 9*
214 ( 32*
122 ( 2*
176 ( 43*
77 ( 3 g
57 ( 5*g
57 ( 6*
67 ( 1*g
61 ( 6*
57 ( 2
45 ( 2*
40 ( 2*
64 ( 1*
67 ( 3
49 ( 3
59 ( 1
105 ( 7*
120 ( 26*
117 ( 34
67 ( 1
68 ( 5
70 ( 2
69 ( 2
54 ( 2
54 ( 1
59 ( 3*
61 ( 1*
46 ( 2*
10 ( 1
8 ( 1
8 ( 1
8 ( 1
7 ( 1
g
g
7
8
150 ( 2*
116 ( 23
111 ( 9
g
g
g
g
g
300 ( 12*g
g
g
g
g
g
1
1
0
3
560 ( 82*
g
g
g
g
g
g
261 ( 7*
48 ( 2*
257 ( 85*
51 ( 11*
a
b
Data points represent the average ( standard error of the mean (SEM) of three independent experiments done in duplicate. At higher
concentrations, no clot formation was observed. R: clotting time expressed in seconds (s). K: clotting formation time expressed in seconds (s).
µ angle expressed in degrees (°). MA: maximum amplitude expressed in mm. * P < 0.05.
c
d
e
f
g
-
3
concentration tested (0.5 ꢀ 10 M), while the sulfated aglycone
-
3
1
2 prolonged 1.1 fold at 5 ꢀ 10 M.
Prolongation of PT(Figure3) was observedfor compounds
, 8, 10, and 13 in a dose-dependent manner. Nonetheless, this
7
effect was observed for higher concentrations than in APTT.
Compounds 7, 8, 10, and 13 were also able to double the PT
(Figure 3 and Table 1) and, with one exception (compound 8),
were also able to completely inhibit this clotting pathway. The
sulfated aglycones 11 and 12 only increased the PT 1.1 and 1.2
times, respectively (Figure 3), at the highest concentration
-
3
-3
tested (0.5 ꢀ 10 and 5 ꢀ 10 M, respectively).
The TT was sensitive to the presence of compounds 10 and
3 that were able to completely block this clotting process at
1
5
1
-
3
ꢀ 10 M (Figure 3 and Table1). Compound 7 increased TT
-3
.6-fold at the highest concentration tested (5 ꢀ 10 M). The
-3
clotting times for flavonoside 8 (5 ꢀ 10 M), sulfated agly-
-
3
-3
cones 11 (0.5 ꢀ 10 M), and 12 (5 ꢀ 10 M) were not signi-
ficantly different (P > 0.05) from control (Figure 3).
In summary, compounds 7, 8, and 10-13 prolonged APTT
and PT, but only compounds 10 and 13 influenced the TT
pathway.
A structure-activity relationship can be established con-
cerning the anticoagulant action and the number of sulfate
groups, i.e., the anticoagulant effects increased with the in-
crease of the number of sulfates. Decasulfated compound 13
was more potent than nonasulfated compound 10, and com-
pound 10 more than hexasulfated compounds 7 and 8; the
disulfated compounds 11 and 12 were only slightly active in
prolonging the clotting times. The weak anticoagulant effect
showed by sulfated aglycones 11 and 12 when compared to the
related sulfated flavonosides 7 and 8 also highlights the
importance of the oligo-O-sulfated moiety in the anticoagulant
activity. Flavonosides 7, 8, 10, and 13 were more potent than
Figure 4. Thromboelastography (TEG) representative tracings of
polysulfated flavonoside 13 (1.25 ꢀ 10 M).
-4
While clotting tests evaluate one isolated segment of the
coagulation system (secondary hemostasis), TEG provides
data about the coagulation process as a whole, from the
beginning of clot formation, through clot stabilization, and
4
3
ending with fibrinolysis. In contrast to plasma clotting tests,
TEG screens the thrombodynamic properties of the entire
blood. The clot is induced under a low shear environment
4
3
resembling an in vivo sluggish venous flow. Thus, TEG
allows acquisition of quantitative information about the
kinetics of clot formation and growth as well as the maximal
2
3,42
the previously described sulfated flavonoids.
Additionally,
when comparing different classes of flavonoids, namely fla-
vones (compounds 7 and 11) with flavanones (compounds 8
and 12, respectively), comprising the same number of sulfates,
it can be noted a slightly preference for flavone scaffolds, in the
prolongation of the clotting times, namely APTT.
Among the three clotting times, the APTT was the most
sensitive test to the presence of sulfated compounds. On the
basis of these results, flavonosides 10 and 13 may be impli-
cated in the conversion of fibrin to fibrinogen by thrombin
while flavonosides 7 and 8 may be acting similarly to heparin
43
strength and stability attained by clots (the quality of clots).
The parameters measured by TEG are: R, the period of time
from initiation of the test to the initial fibrin formation
(clotting time) and is due only to plasma factors; K, the time
from beginning of clot formation until the amplitude of
thromboelastogram reaches 20 mm (clotting formation time);
µ angle, the acceleration/kinetics of fibrin build up and cross-
linking (clot formation and growth); and MA, the strength
and stability of the formed clot (maximum clot firmness/
maximum amplitude strength of clot). Modified thromb-
oelastogram was obtained by adding to the whole blood an
intrinsic activator (InTEM) or an extrinsic activator (ExTEM).
InTEM and ExTEM are global tests for plasmatic coagulation
factors, fibrin polymerization, and platelet contribution. It was
also possible to eliminate platelet function from the TEG
(prolonging APTT more than PT).
To better characterize the anticoagulant profile, the effect
on the whole blood of the most promising sulfated molecules
(7, 8, 10, and 13) was studied by thromboelastography (TEG)
(Table 2). In Figure 4, the TEG representative tracings for
polysulfated flavonosides (e.g., compound 13) are illustrated.

1
00 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Correia-da-Silva et al.
sample and study the fibrin status by adding cytochalasin D
and Ca (FibTEM).
PFA-100 system, the closure of a defined aperture is caused by
platelets which are activated by a collagen-coated membrane
and an additional agonist, adenosine 5 -diphosphate (ADP)
2þ
0
In the presence of compounds 7, 8, 10, and 13, the thromb-
oelastogram traces showed a hypocoagulable blood and there
was no evidence of fibrinolytic activity (Figure 4). Com-
pared to control values, compounds 7, 8, 10, and 13 increase
not only the clotting time (R) but also increase the clotting
formation time (K) and decrease µ (deceleration progress of
the clot) and MA values (Table 2). Because K and µ are a
combination of both plasma and platelet factors and MA is
largely dependent on platelet function and integrity of the
fibrin clot, it is evident that an action on platelets may also
be implicated in the antithrombotic effect of compounds 7, 8,
or epinephrine (EPI). At the aperture exists a shear stress of
about 5000/s, which is about twice the level inside a moder-
ately stenosedartery. Plateletsadhereatthe apertureand form
aggregates consisting of platelets bound to each other mainly
by von Willebrand factor. When a clot is formed, further
blood flow is prevented and this time is registered as the
closure time. Hence, the closure time refers to the process of
platelet plug formation, including adhesion and aggregation
at high shear stress forces. The PFA-100 closure times in the
presence of polysulfated flavonosides 7, 8, 10, and 13 are col-
lected in Table 3.
1
0, and 13. Moreover, normal thromboelastograms were ob-
tained in the presence of these compounds when the platelet-
specific effect on MA of TEG tracings is eliminated (FibTEM
test).
Compounds 7 and 8 prolonged the collagen-ADP closure
times. However, it is unlikely that a platelet dysfunction exists
in the presence of compounds 7 and 8 because the collagen-
EPI times were not significantly different (P > 0.05) from
control. PFA-100 closure times of both collagen-ADP and
collagen-EPI cartridges were markedlylonger in the presence
of compounds 10 and 13 (Table 3). Because results of both
tests were abnormal, it is likely that compounds 10 and 13
caused a platelet dysfunction. Further studies should be
performed to support the antiplatelet action of compounds
From these data, it can be inferred that polysulfated flavo-
nosides 7, 8, 10, and 13 may interfere with coagulation cascade
factors and/or platelet function. Therefore, their influence on
platelets, thrombin, FXa, and ATIII were studied.
To measure platelet adhesion and aggregation in whole
4
4,45
blood, a platelet function analyzer (PFA) was used.
In the
1
0 and 13. The possibility of a dual anticoagulant and platelet
antiaggregatory activities for compounds 10 and 13 could
a
-4
Table 3. Effect of Polysulfated Flavonosides 7, 8, 10 (6.25 ꢀ 10 M),
-4
b
and 13 (1.25 ꢀ 10 M) on Collagen-ADP or EPI Induced Platelet
46
enhance their potential as antithrombotic agents.
Aggregation in Human Whole Blood
Compounds 7, 8, 10, and 13 were further evaluated for
selectivity against the critical enzymes targeted by current
anticoagulant therapy, thrombin, FXa, and ATIII (Table 4).
Inhibition of thrombin and FXa, in the presence and in ab-
sence of ATIII, was followed by spectrophotometric determi-
nation of the product formed after amide bond cleavage of the
chromogenic substrates Chromozym TH and CBS 31.39, re-
spectively and the initial rate compared with that obtained in
the absence of the compound. Reference inhibitors, EDTA
(direct FXa inhibitor), LMWH (ATIII/FXa activator), and
compd
reference range
collagen-ADP
71-118
collagen-EPI
94-193
control
119 ( 8
162 ( 16*
139 ( 7*
204 ( 44*c
168 ( 5
171 ( 3
167 ( 17
>300
c
7
8
c
c
1
0
3
c
1
189 ( 13*
244 ( 45*
a
Results expressed in closure time expressed in seconds (s) and
b
represent the average ( SEM of three independent experiments. At
higher concentrations, no clot formation was observed. *P < 0.05.
c
a
Table 4. In Vitro Inhibitory Activity of Polysulfated Flavonosides 7, 8, 10, and 13 against Human FXa and ATIII Inhibition of FXa in the Presence of
CBS 31.39
human FXa
ATIII/FXa
-6
-6
-6
-6
substratef
25 ꢀ 10
M
125 ꢀ 10
M
25 ꢀ 10
M
125 ꢀ 10
M
7
8
1
1
-
3
3
2
1
0
ꢀ 10
ꢀ 10
M
M
nd
na
na
23.35 ( 2.91
na
na
43.69 ( 2.63
22.58 ( 1.76
na
50.66 ( 2.64
38.70 ( 5.48
28.91 ( 2.77
-
-
3
.5 ꢀ 10
M
-
3
2
1
0
ꢀ 10
ꢀ 10
M
M
na
na
na
na
na
na
49.56 ( 8.59
40.18 ( 3.55
30.50 ( 1.76
58.12 ( 2.91
46.21 ( 5.68
39.72 ( 3.38
-3
-
3
.5 ꢀ 10
M
0
3
-
3
2
1
0
ꢀ 10
ꢀ 10
M
M
nd
41.94 ( 20.81
25.66 ( 4.84
53.27 ( 6.57
38.83 ( 7.81
na
56.01
47.65 ( 3.08
28.15 ( 1.06
33.57 ( 4.87
24.80 ( 1.67
20.30 ( 2.07
-3
-
3
.5 ꢀ 10
M
-
3
2
1
0
ꢀ 10
ꢀ 10
M
M
40.18 ( 1.79
30.94 ( 3.96
na
42.70 ( 3.49
31.98 ( 0.94
22.4 ( 3.04
31.38 ( 1.83
30.50 ( 4.16
29.33 ( 1.17
33.11 ( 1.22
20.70 ( 0.92
na
-3
-
3
.5 ꢀ 10
M
EDTA
-
3
1
0 ꢀ 10
M
45.74 ( 0.13
na
58.96 ( 6.98
na
73.51 ( 0.44
nd
72.14 ( 1.38
nd
-3
1
ꢀ 10
M
LMWH
.20 U/mL
0
na
na
96.31 ( 0.51
97.96 ( 0.58
a
Results expressed as percentage of inhibition; each value represents the mean ( SEM of three independent experiments. nd: not determined. na: not
active at the concentration tested (P > 0.05).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 101
Figure 5. APTT, PT, and TT prolonging activities of compound 7 and 13 in mice at 150 μmol/kg, after 30, 60, and 120 min of ip administration,
expressed as ratio of the clotting time of compound-treated mice to clotting time of saline-treated mice (control group). Data points represent
the average ( SEM of three independent experiments.
Figure 6. Plasma levels of glutamic-oxaloacetic transaminase (GOT) and glutamate-pyruvate-transaminase (GPT) in mice 30, 60, and 120 min
after ip administration of compound 7 and 13 at 150 μmol/kg, expressed as U/L. Data points represent the average ( SEM of four independent
experiments.
5
2
PPACK (direct thrombin inhibitor) were also tested in the
same conditions. Various concentrations of the sulfated in-
thrombin inhibitors are able to inhibit clot-bound thrombin,
they are unable to block the continuing production of thrombin
from prothrombin.
-
3
53,54
hibitors (0.5, 1, and 2 ꢀ 10 M) and CBS 31.39 (25 and 125 ꢀ
In contrast, FXa inhibitors prevent the
-
6
1
0
M) were used (Table 4). Kinetic studies of the inhibitory
activity of sulfated flavonosides 7, 8, 10, and 13 against FXa
conversion of prothrombin to thrombin and have no influence
in the previously formed thrombin; they allow a basal level of
thrombin activity necessary for primary hemostasis mainte-
and ATIII/FXa were fitted by the Michaelis-Menten equation
53,54
(nonlinear regression analysis, see Supporting Information).
nance, resulting in less bleeding complications.
itors have advantages over thrombin inhibitors.
FXa inhib-
3,54
On the
5
None of the compounds 7, 8, 10, and 13 showed any in-
fluence on thrombin activity (% of PPACK thrombin inhibi-
basis of these considerations, direct FXa inhibitors 10 and 13
are potentially safer alternatives to the existing anticoagulant
agents.
Previously to in vivo assays in animals, stability studies in
plasma were performed for one representative direct (13) and
indirect (7) inhibitor of FXa. Three hours time point was
selected tocover the time period usedinthepresent in vivoand
in vitro experiments. Compound 7 and 13 were stable after 3 h
in human plasma (concentrations of at least 95% of the initial
one were detected). This is consistent with the previously
-
9
tion at 1.9 ꢀ 10 M was equal to 37 ( 5%) even in the
presence of ATIII (datanot shown). Compounds7, 10, and 13
showed a direct inhibitory effect on FXa (Table 4). For
compound 7, this effect was significantly (P < 0.05) enhanced
bythe presence of ATIII, suggesting an indirect actionon FXa
by activation of ATIII. Compound 8 showed an inhibitory
effect on FXa onlyinthe presence of ATIII. Forcompound 10
and 13, the percentage of FXa inhibition in the presence of
ATIII was not significantly different from blank (P>0.05).
Therefore, compounds 10 and 13 are direct FXa inhibitors.
As previously discussed, compounds 7 and 8 may act similarly
to heparin. The position of the oligo-O-sulfated moiety on the
molecular scaffold seems to be correlated with this effect:
while3-O-rutinosides (compounds10and 13) weredirect FXa
inhibitors, 7-O-rutinosides (compounds 7 and 8) were ATIII
activators of FXa inhibition. In the presence of compounds 10
and 13, the Vmax values did not change significantly, while the
effective Km increased, suggesting a competitive inhibition
5
5
reported stability for rutin decasulfate (13).
Subsequently, to assess the putative in vivo efficacy, we
performed in vivo studies in mice to measure anticoagulant
activity and putative hepatic toxicity for compounds 7 and
13. To estimate the in vivo anticoagulant activity, APTT,
PT, and TT were determined at 30, 60, and 120 min after
150 μmol/kg intraperitoneal (ip) administration or at 120 min
after 300 μmol/kg oral administration by gavage. These
values were compared with the APTT, PT, and TT values
obtained with saline-treated mice groups and expressed as a
ratio (Figure 5). Considering the previous reported hepato-
(see Supporting Information).
A high-priority goal in the search for new antithrombotic
9
,56,57
agents is the discovery of small molecules that selectively inhibit
FXa or thrombin. Direct inhibitions of these targets have been
suggested to be a better strategy because they do not require an
toxic effects of heparin and LMWH,
it was of interest
to estimate any possible drug-induced hepatocellular injury
of these compounds. Plasma levels of glutamic-oxaloacetic
transaminase/aspartate aminotransferase (GOT/AST) and
glutamate-pyruvate-transaminase/alanine aminotransferase
(GPT/ALT) were evaluated following the ip administration
4
7
intermediary protein to mediate the anticoagulant effect.
48
49
Three new oral drugs, rivaroxaban and apixaban as FXa
direct inhibitors, and dabigatran etexilate, as a thrombin
50
51
58
direct inhibitor, were successfully developed. Although direct
(Figure 6).

1
02 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Correia-da-Silva et al.
After ip administration, both compounds 7 and 13 were
highlyactive, increasing the APTTtothe desirable therapeutic
Inc. (California, US). MW reactions were performed using a
MicroSYNTH 1600 from Millestone (ThermoUnicam, Portugal)
synthesizer in sealed reaction vessels (30 mL). TLC was performed
using Polygram cel 400 UV254 0.1 mm (Macherey-Nagel,
Germany) (BuOH-CH COOH-H O 4:2:6 and 5:2:3) and Merck
5
9
range (1.5-2.3 times the mean of the normal APTT range,
Figure 5). In contrast, heparin does not produce systemic
6
0
3
2
anticoagulant action after ip administration. Moreover,
both compounds were still active after 120 min, particularly
compound 13, being reasonable to speculate that they will
3
silica gel 60 (GF254) plates (CHCl :MeOH 9:1). Compounds were
visually detected by absorbance at 254 and/or 365 nm. Melting
points were obtained in a K o€ fler microscope and are uncorrected.
IR spectra were measured on an ATI Mattson Genesis series FTIR
6
1,62
have a desirable sustained duration of action.
Because
compounds 7 and 13 showed stability in plasma, the anti-
coagulant activity at 120 min cannot be attributed to decom-
position products such as desulfonated byproduct or parent
compounds. Furthermore, rutin decasulfate (13) has pre-
(
(
software: WinFirst v.2.10) spectrophotometer in KBr microplates
cm ). Hand C NMR spectra were taken in DMSO-d at room
6
-
1
1
13
temperature, on Bruker Avance 300 and 500 instruments (300.13
or 500.13 MHz for H and 75.47 or 125.77 MHz for C). Chemical
shifts are expressed in δ (ppm) values relative to tetramethylsilane
1
13
5
5
viously shown favorable pharmacokinetic profile. After oral
administration, none of the compounds tested (7 and 13) were
active(resultsnot shown). These resultsmightbe relatedtothe
previously reported behavior of rutin decasulfate (13) in
(TMS) as an internal reference. Coupling constants are reported
13
in hertz (Hz). C NMR assignments were made by 2D HSQC and
HMBC experiments (long-range C, H coupling constants were
optimized to 7 and 1 Hz). MS spectra were recorded as FAB (Fast
Atom Bombardment) or EI (Electronic Impact) mode on a VG
Autospec Q spectrometer (m/z), and HRMS mass spectra were
measured on a APEX III mass spectrometer, recorded as ESI
6
3
Caco-2 cells. If required, formulations could be developed
to achieve oral administration because delivery agents have
already demonstrated to improve oral absorption of macro-
6
4
molecules like heparin. The rapid onset of action revealed by
compounds 7 and 13 is a desirable feature compared to oral
anticoagulants drugs, acenocoumarol and warfarin sodium.
Considering the plasma levels of transaminases, no signifi-
cant alteration (P > 0.05) of GOT and GPT plasma levels was
observed after administration of both compounds 7 and 13
(
ꢀ
Electrospray) mode in Centro de Apoio Cientı
´
fico e Tecnol oꢀ xico
each compound was determined by HPLC-DAD analysis. Except
for compound 9, all tested compounds, whether synthesized or
purchased, possessed a purity of at least 95%.
HPLC Chromatographic Conditions. The HPLC analyses
were carried out on a System SMI pump series II (Gloucester,
UK) equipped with a Rheodyne 7125 injector fitted with a 20 μL
loop, a TSP-UV6000LP detector, and a Chromquest for Windows
NT integrator and using a C-18 Nucleosil column (5 μm, 250 mm
(
1
Figure 6). In general, after severe damage, GOT levels rise
0 to 20 times or higher than normal whereas GPT can reach
5
8
levels up to 50 times greater than normal. Thus, these pre-
liminary results suggested that these compounds will not in-
duce hepatic toxicity, in contrast to unfractionated and LMW
ꢀ
4.6 mm I.D.), from Macherey-Nagel (D u€ ren, Germany). Acet-
onitrile was of HPLC grade from Merck. HPLC ultrapure water
was generated by a Milli-Q system (Millipore, Bedford, MA,
USA). The mobile phases were degassed for 15 min in an ultrasonic
bath before use. The mobile phase used was 10 mM of phosphate
buffer with 25 mM of TBA (adjusted to pH 8 with TEA) and
acetonitrile (55:45) at a constant flow rate of 1.0 mL/min.
9
,56,57
heparins.
From the overall results, these derivatives are expected to
possess minimal toxicity and appear to have the potential to
lead to effective and safe therapeutics.
Conclusion
General Procedure for the Hydrolysis of Flavonoids Glycosides
Procedure a). Flavonoid glycosides 1 (3.29 mmol) and 2 (4.91
(
The incorporation of an oligo-O-sulfated moiety into a
flavonoid scaffold was a successful strategy to improve the
anticoagulant potency of sulfated flavonoids. Polysulfated
flavonosides proved to be active in vitro both in plasma and in
whole human blood as well as in vivo, surpassing existing
anticoagulant flavonoids. This study provided interesting
SAR by revealing sulfated 7-O-rutinosides as indirect FXa
inhibitors and sulfated 3-O-rutinosides as direct FXa inhibi-
tors. In contrast to unfractionated and LMW heparins, the
polysulfated oligoflavonoids correspond to compounds with
a defined composition and with less charge density that
allowed a systemic anticoagulant action after ip administra-
tion and are not expected to induce hemorrhagic complica-
tions and hepatic toxicity, as it was preliminarily proved with
the in vivo studies in mice.
mmol) were kept under reflux in 200 mL of a mixture of sulfuric
acid-acetic acid-water (1:30:19) for 12 h (see Supporting In-
formation for experimental details of each purification process).
General Procedure for Sulfation with Conventional Heating
(Procedure b). To a solution of a flavonoid (1-6, 0.8-3 mmol) in
DMA (15 mL), triethylamine-sulfur trioxide adduct (4-8 equiv/
OH) was added and the suspension was heated at 65 °C for 24 h.
The mixture was poured into acetone (150 mL) under basic
conditions (a few mL of triethylamine) and left at 4 °C for 24 h.
The crude oil formed was washed with acetone and ether and then
dissolved in aqueous solution of 30% sodium acetate (5 mL).
Generally, the suspension was added dropwise in ethanol to
precipitate the sodium salt of the sulfated derivative (see Support-
ing Information for experimental details of each syntheses and
purification process).
General Procedure for Microwave-Assisted Sulfation(Procedure
c). To a solution of a flavonoid (1-4, 0.8 mmol) in DMA (10 mL),
triethylamine-sulfur trioxide adduct (10 equiv/OH) was added.
The reaction vessel was sealed, and the mixture was kept stirring
and heated for 30 min at 100 °C under MW irradiation. The reac-
tion mixture was ramped to 100 °C using the following power
The derivatives resulting from chemical molecular modifi-
cations of known therapeutic agents have high chances to
produce safer anticoagulant agents and this study provided
interesting data to be explored by rational design in the future.
-
temperature steps: (1) 200 W, room temperature-80 °C, during
Experimental Section
1
min; (2) 200 W, 80-100 °C, for 29 min. After cooling, the mix-
Chemistry. Triethylamine-sulfur trioxide adduct (S 5139)
was purchased from Fluka (Spain), and diosmin (1, D 3525),
hesperidin (2, S 5139), rutin (3, R 2303), and trihydroxyethylru-
tin (4, 91950) were purchased from Sigma-Aldrich (Spain). The
solvents used were products pro analysis or HPLC grade from
Sigma-Aldrich and Fluka. Spectra/Por Dialysis membrane,
MWCO 1000, was purchased from Spectrum Laboratories,
ture was poured into acetone (150 mL) under basic conditions
(a few mL of triethylamine) and left at 4 °C for 24 h. The crude oil
formed was washed with acetone and ether and then dissolved in
aqueous solution of 30% sodium acetate (5 mL). The suspension
was added dropwisein ethanol to precipitate the sodium salt of the
sulfated derivative 7 and 13. In the case of compounds 8 and 10,
thesolidobtainedwasfurtherpurifiedfromothersalts(monitored

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 103
0
by IR) by dialysis using a Spectra/Por 6 regenerated cellulose
MWCO 1000.
Diosmin 2 ,2 ,3 ,3 ,4 ,4 -O-Hexasulfate (7). Yellow solid,
yield 91% (procedure b) and 86% (procedure c); mp 184-
Diosmetin 3 ,7-O-Disulfate (11). Yellow solid, yield 14%
(procedure b); mp >340 °C (ethanol). IR (KBr) υmax: 3600-
3400, 2963, 2875, 1655, 1606, 1510, 1489, 1261, 1043. H NMR
0
0
000 00 000 00 000
1
6
(DMSO-d , 300.13 MHz) δ: 12.81 (1H, s, 5-OH), 8.09 (1H, d,
0
0
1
1
1
85 °C (ethanol). IR (KBr) υmax: 3600-3400, 1653, 1616,
J = 2.0 Hz, H-2 ), 7.82 (1H, dd, J = 8.7 and 2.0 Hz, H-6 ), 7.18
0
1
508, 1261, 1047, 806. H NMR (DMSO-d , 500.13 MHz) δ:
(1H, d, J = 8.7 Hz, H-5 ), 7.01 (1H, d, J = 1.8 Hz, H-8), 6.75
0
6
0
2.83 (1H, s, 5-OH), 8.79 (1H, s, 3 -OH), 7.99 (1H, d, J = 2.2 Hz,
0
(1H, s, H-3), 6.57 (1H, d, J = 1.8 Hz, H-6), 3.86 (3H, s, 4 -
0
13
). C NMR (DMSO-d
H-2 ), 7.89 (1H, dd, J = 8.8 and 2.2 Hz, H-6 ), 7.24 (1H, d, J =
OCH
(C-7), 160.5 (C-2), 159.7 (C-5), 156.4 (C-9), 154.1 (C-4 ), 142.8
3
6
, 75.47 MHz) δ: 182.0 (C-4), 164.0
0
0
8
6
5
.8 Hz, H-5 ), 6.77 (1H, d, J = 1.9 Hz, H-8), 6.56 (1H, s, H-3),
00
0
0
0
0
0
0
.39 (1H, d, J = 1.9 Hz, H-6), 5.71 (1H, d, J = 2.8 Hz, H-1 ),
0
(C-3 ), 122.7 (C-6 ), 122.1 (C-1 ), 118.7 (C-2 ), 112.7 (C-5 ), 105.7
(C-10), 103.7 (C-3), 102.3 (C-6), 97.7 (C-8), 55.8 (OCH ). MS
]). HRMS (ESI ) m/z calcd for
C H O S 457.98123, found 457.98071.
0
00
00
.02 (1H, d, J = 11.0 Hz, H-1 ), 4.59-4.55 (3H, m, H-4 , H-2
3
0
00
000
þ
-
and H-4 ), 4.43 (1H, t, J = 3.5 Hz, H-3 ), 4.35 (1H, d br, J =
0
(FAB ): 426 ([M - OCH
3
00
00
7
H-3 ), 3.84 (3H, s, 4 -OCH
.9 Hz, H-2 ), 4.27-4.25 (1H, m, H-5 ), 4.03 (2H, t, J = 9.8 Hz,
1
6
10 12 2
0
0
0
000
0
3
), 3.80-3.50 (1H, m, H-5 ),
Hesperetin 3 ,7-O-Disulfate (12). Brown solid, yield 5%
(procedure b); mp >340 °C (ethanol). IR (KBr) υmax: 3600-
0
0
3
H-6 ). C NMR (DMSO-d
.52-3.40 (2H, under H O, H-6 ), 1.23 (3H, d, J = 6.2 Hz,
2
0
00 13
1
3400, 1622, 1637, 1516, 1043, 1265, 798. H NMR (DMSO-d
6
, 125.77 MHz) δ: 181.9 (C-4), 164.1
C-2), 162.6 (C-7), 161.2 (C-5), 157.1 (C-9), 154.2 (C-4 ), 142.7
6
,
0
(
(
(
(
(
(
500.13 MHz) δ: 12.51 (1H, s, 5-OH), 7.55 (1H, d, J = 2.0 Hz,
H-2 ), 7.17 (1H, dd, J = 8.6 and 2.0 Hz, H-6 ), 7.0 (1H, d, J = 8.6
0
0
0
0
0
0
0
C-3 ), 123.2 (C-6 ), 122.0 (C-1 ), 118.7 (C-2 ), 113.0 (C-5 ), 105.5
0
00
00
0
C-10), 103.5 (C-3), 99.7 (C-6), 97.9 (C-1 ), 97.5 (C-1 ), 95.1
0
Hz, H-5 ), 6.89 (1H, d, J = 2.2 Hz, H-8), 6.71 (1H, d, J = 2.2 Hz,
0
0
000
00
00
000
C-8), 75.8 (C-3 ), 75.3 (C-5 ), 74.3 (C-2 ), 74.0 (C-4 ), 73.4
0
H-6), 5.4 (1H, d, J = 12 Hz, H-2), 3.78 (3H, s, 4 -OCH
3
), 2.90
(2H, m, under H O, H-3). MS (EI ): 430 ([M-OCH ]). HRMS
0
000
000
00
þ
C-4 ), 73.1 (C-2 ), 71.1 (C-3 ), 68.8 (C-5 ), 67.4 (C-6 ), 55.9
0
2
3
000
þ
-
4 -OCH
3
), 18.0 (C-6 ). MS (FAB ): m/z 1245 ([M þ Na þ
(ESI ): m/z calcd for C16
00 000
H
12
O
12
S
2
462.97617, found 462.77777.
þ
þ
0
00 000
0
00 000
H] ). HRMS (ESI ): m/z calcd for C28
9645, found 1242.80127.
H
26
O
33
S
6
Na
7
1242.
Rutin 2 ,2 ,3 ,3 ,3 ,4 ,4 ,4 ,5, 7-O-Decasulfate (13). White
solid, yield 99% (procedure c); mp 222-225 °C (ethanol). IR
7
0
0
000 00 000 00 000
1
Hesperidin 2 ,2 ,3 ,3 ,4 ,4 -O-Hexasulfate (8). Orange
crystals, yield 70% (procedure b) and 58% (procedure c); mp
(KBr) υmax: 1643, 1515, 1430, 1265, 1024, 820. H NMR (DMSO-
0
d
6
, 300.13 MHz) δ: 8.28 (1H, dd, J = 2.2 and 8.9 Hz, H-6 ), 8.18
(1H, d, J = 2.2 Hz, H-2 ), 7.65 (1H, d, J = 8.9 Hz, H-5 ), 7.26 (1H,
d, J = 2.2 Hz, H-8), 7.11 (1H, d, J = 2.2 Hz, H-6), 5.88 (1H, d, J =
2.0 Hz, H-1 ), 5.15 (1H, s, H-1 ), 4.85-3.88 (10H, rutinose), 1.23
(3H, d, J = 6.2 Hz, H-6 ). C NMR (DMSO-d , 75.47 MHz) δ:
0
0
1
1
55-157 °C (water). IR (KBr) υmax: 3600-3400, 1641, 1518,
1
254, 1050, 805. H NMR (DMSO-d
6
, 300.13 MHz) δ: 11.99
0
0
00
000
(
(
1H, s, 5-OH), 8.90 (1H, s, 3 -OH), 7.96 (1H, d, H-2 ), 7.04-6.89
0
0
000 13
2H, H-6 and H-5 ), 6.07 (2H, H-8 and H-6), 5.67-5.52 (1H, m,
6
0
00
0
H-2), 4.93 (1H, brs, H-1 ), 4.43 (3H, s, 4 -OCH ), 4.20 (1H, d,
3
172.9 (C-4), 157.5 (C-7), 156.2 (C-5), 152.6 (C-9), 152.4 (C-2),
0
0
0
0
0
0
J = 9.0 Hz, H-1 ), 4.05-3.71 (10H, rutinose), 3.10 (2H, dd, J =
1
146.3 (C-4 ), 143.6 (C-3 ), 137.6 (C-3), 125.5 (C-6 ), 123.8 (C-1 ),
120.2 (C-5 ), 119.1 (C-2 ), 110.8 (C-10), 107.9 (C-1 ), 101.8 (C-1 )
0
00 13
0 0 00 00
4.5 and 7.3 Hz, H-3), 1.23 (3H, d, J = 6.1 Hz, H-6 ). C NMR
0
00
(
1
1
(
(
DMSO-d , 75.47 MHz) δ: 197.1 (C-4), 164.7 (C-7), 163.1 (C-5),
99.7 (C-6), 97.6 (C-8), 76.9-66.9 (9C, rutinose), 18.0 (C-6 ).
HRMS (ESI ): m/z calcd for C H O S Na 1653.9576, found
1652.53247.
6
þ
0
0
0
0
62.8 (C-9), 148.0 (C-4 ), 146.5 (C-3 ), 131.2 (C-1 ), 130.2 (C-6 ),
000
27 20 46 10
11
0
0
17.9 (C-2 ), 114.1 (C-5 ), 112.2 (C-10), 103.5 (C-1 ), 96.4
0
0
0
0
Biological Activity. Clotting Assays. Human blood was col-
lected from 10 healthy donors aged between 25 and 45 years old
without history of bleeding or thrombosis and who had not taken
any medication known to affect blood coagulation and platelet
function for 2 weeks. Venous blood was obtained and transferred
to a plastic tube. Nine volumes of blood were decalcified with one
volume of 3.8% sodium citrate solution. Blood was centrifuged
for 20 min at 2400g, and the pooled plasma was stored at -20 °C
until use. Compound 9 was not obtained with the desirable purity
to be tested in biological assays. Sulfated compounds 7, 8, and
10-13 were dissolved in water. The final concentration of sul-
C-1 ), 95.4 (C-6), 91.5 (C-8), 78.4 (C-2), 75.8 (C-3 ), 74.2
00 00 000 00 000
C-5 ), 73.5 (C-2 ), 72.3 (C-4 ), 71.5 (C-4 ), 70.9 (C-2 ),
000 000 00 0
8.9 (C-3 ), 67.5 (C-5 ), 61.5 (C-6 ), 55.7 (4 -OCH ), 45.9
3
6
0
00
þ
þ
(
HRMS (ESI ): m/z calcd for C H O S Na 1244.81155,
C-3), 15.2 (C-6 ). MS (FAB ): m/z 1244 ([M þ Na þ H] ).
þ
2
8
28 33
6
7
found 1244.81504.
0
0
000
0
00 000
0
00 000
Rutin 2 ,2 ,3 ,3 ,3 ,4 ,4 ,4 ,7-O-Nonasulfate (9). Yellow
solid, yield 24%(procedure b, purity <95%); mp >340 °C
1
(
water). IR (KBr) υmax: 1652, 1615, 1500, 1258, 1055, 808. H
NMR (DMSO-d , 500.13 MHz) δ: 12.60 (1H, s, 5-OH), 8.14
6
0
0
(1H, d, J = 8.9, H-2 ), 8.06 (1H, m, H-6 ) 7.65 (1H, d, J = 8.9,
0
H-5 ), 6.89 (1H, s, H-8), 6.53 (1H, s, H-6), 4.94 (1H, d, J = 10.2,
-3
fated compounds in these assays ranged from 5 ꢀ 10 M to
-
3
-
3
0
0
000
0.05 ꢀ 10 M. Sulfated derivative 11 was not tested at 5 ꢀ 10
H-1 ), 4.91 (1H, s, H-1 ), 4.76-4.03 (10H, rutinose), 1.24 (3H,
-
2
0
00 13
M because it was not soluble in water at 1 ꢀ 10 M. Compounds
d, J = 5.2, H-6 ). C NMR (DMSO-d , 125.77 MHz) δ: 172.0
6
-
3
2
-4 were tested at a single concentration (5 ꢀ 10 M, 10%
(
(
(
(
C-4), 160.2 (C-7), 159.1 (C-5), 156.9 (C9), 156.4 (C-2), 146.7
0
0
0
0
0
DMSO). Heparin (0.05-5 UI/mL) was used as positive control.
In the control group, water (control for sulfated compounds 7, 8,
and 10-13) or 10% DMSO (control for desulfated compounds
2-4) was used. APTT, PT, and TT tests were performed using an
ACL100 coagulometer (IZASA, Portugal). The following com-
mercial kits were used: 49735320 (IZASA, Portugal) for the
APTT, 20002900 (IZASA, Portugal) for the PT, and 9758515
(IZASA, Portugal) for the TT. The assays were carried out ac-
cording to the respective instructions of the manufacturers. For
APTT assay, citrated normal human plasma (25 μL) was mixed
with 25 μL of sample solution at various concentrations and
incubated for 2 min at 37 °C. Then APTT assay reagent (56 μL)
was added and the mixture was incubated for 6 min at 37 °C.
C-4 ), 143.3 (C-3 ), 133.7 (C-3), 125.3 (C-6 ), 122.6 (C-1 ), 119.8
00
0
00
C-5 ), 118.1 (C-2 ), 104.2 (C-10), 101.9 (C-1 ), 100.3 (C-1 ) 99.1
0
00
C-6), 97.0 (C-8), 81.1-66.9 (9C-rutinose), 17.9 (C-6 ).
0
0
00
3
,4 -Bis(2-O-sulfate ethoxy)-7-(2-O-sulfate ethoxy)-rutin (10).
Orange solid, yield 20% (procedure b) and 89% (procedure c); mp
1
93-195 °C (ethanol). IR (KBr) υmax: 3600-3400, 1649, 1610,
1
6
1258, 1059, 800. H NMR (DMSO-d , 300.13 MHz) δ: 12.62 (1H,
0
0
s, 5-OH), 7.96 (1H, m, H-6 ), 7.65 (1H, d, J = 2.0 Hz, H-2 ), 7.24
0
(
d, J = 2.0 Hz, H-6), 5.68 (1H, d, J = 3.3 Hz, H-1 ), 4.41 (1H, s,
1H, d, J = 8.8 Hz, H-5 ), 6.93 (1H, d, J = 2.0 Hz, H-8), 6.36 (1H,
0
0
0
00
-
H-1 ), 4.27 (6H, m, OCH
2
CH
), 3.99-3.07 (10H, rutinose), 0.98 (3H, d, H-6 ).
NMR (DMSO-d , 75.47 MHz) δ: 177.6 (C-4), 164.2 (C-7), 160.7
2 3 2
OSO ), 4.11 (6H, m, OCH -
-
3
000 13
C
CH
2
OSO
6
0
0
0
0
(
(
(
C-5), 156.6 (C-2), 156.4 (C-9), 150.5 (C-4 ), 147.6 (C-3 ), 134.4
0
2
CaCl (50 μL, 25 mmol/L) was added, and clotting times were
0
C-3), 124.2 (C-2 ), 122.3 (C-6 ), 120.0 (C-1 ), 113.6 (C-2 ), 113.0
0
recorded during 110 s. The PT assay was carried out as follows:
citrated normal human plasma (25 μL) was mixed with 25 μL of
a solution of sample at various concentrations and incubated for
2 min at 37 °C. Then PT assay reagent (100 μL), preincubated for
2 min at 37°C, was added and clottingtimes wererecorded during
0
000
00
C-6 ), 112.8 (C-5 ), 105.3 (C-10), 100.7 (C-1 ), 100.5 (C-1 ),
8.9 (C-6), 98.5 (C-8), 73.92-64.10 (9C, rutinose), 56.07 (6C-
9
OCH CH OH), 45.80 (6C-OCH CH OH), 17.78 (C-6 ). HRMS
0
00
2
þ
2
2
2
(
ESI ): m/z calcd for C33
33
H O
46
S
9
Na10 1682.67, found 1682.67.

1
04 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
65 s. For TT assay, citrated normal human plasma (25 μL) was
Correia-da-Silva et al.
1
reader (BioTeck Instruments, Vermont, US). Test compounds
were diluted in water. Reference inhibitors PPACK (direct throm-
bin inhibitor, D-Phe-L-Pro-L-Arg-CMK), LMWH (ATIII/FXa
activator), and EDTA (direct FXa inhibitor) were assayed in
the same conditions. A 40 μL of test compound at 1 mM, final was
diluted with 70 μL of buffer and warmed at 37 °C for 2 min before
the addition of 20 μL of ATIII solution. For the direct inhibition
of test compounds on thrombin or FXa, buffer was incorporated
in place of ATIII solution. After the mixture was incubated at
37 °C for 10 min, 20 μL of thrombin solution or FXa solution was
added. After 10 min of incubation at 37 °C, the enzyme reactions
were initiated by the addition of chromogenic substrate, immedi-
ately placed in the microplate reader and the color developed from
the release of p-nitroaniline from each substrate was monitored
every 12 s for 20 min at 405 nm on a microplate reader. Absorbance
was calculated by subtracting the absorbance at each point with the
absorbance measured without the enzyme. The control was per-
formed using water in place of the test compound. The initial slope
of the absorbance curve in the presence of the test compound was
compared to that of the control to obtain the percentage of
inhibition. Data were fitted by the Michaelis-Menten equation.
Measurements were carried out in duplicate and repeated at least
three times on different days (n = 6).
Stability Studies. The plasma stability of tested compounds,
at 25 μM final concentration, was performed in human plasma
previously diluted 1:1 with phosphate buffer (PBS, pH 7.4).
Three independent samples, plus respective blank and controls,
were analyzed for each time (time zero and 180 min). Incuba-
tions were performed in eppendorfs, on a bath shaker, at 37 °C
for 180 min. After the addition of tetrabutylammonium (TBA)
buffer (10 mM potassium dihydrogen phosphate and 25 mM
TBA, adjusted to pH 8 with TEA), the reaction was quenched
with 4ꢀ cold acetonitrile (HPLC grade), followed immediately
by mixing and centrifugation during 15 min at 14000 rpm. The
time zero samples were quenched immediately after the sample
was added to plasma. After filtration (Millipore), the super-
natant was evaporated and the residue dissolved with mobile-
phase (50 μM final concentration), and transferred to HPLC
vials.
In Vivo Studies. Animals were kept in polyethylene cages with
wood shavings as bedding and maintained in a temperature-
controlled room at 20 ( 1 °C, with a 12/12 h lighting schedule
(lights on at 08:00 h, off at 20:00 h), and relative humidity of
50% for at least 2 weeks prior to use. Water was available ad
libitum at all times, but food was not available at the day of the
experiments. Housing and experimental treatment of the mice
were conducted under the European Community guidelines for
the use of experimental animals (European Convention for the
Protection of Vertebrate Animals Used for Experimental and
Other Scientific Purposes, 1986, and Protocol of Amendment
to the European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes,
1998). All experiments were performed using adult male CD1
mice (30-35 g body wt; Charles River Laboratories, Criffa,
Barcelona, Spain). Animals were grouped and housed, four per
cage/group. The tested drugs were dissolved in saline (0.9%
NaCl) and administered to animals ip at 150 μmol/kg or orally
at 300 μmol/kg, by gavage, in a volume of 0.1 mL/40 g body
weight. Citrated blood was collected from the portal vein into
syringes containing citrate, 30, 60, and 120 min after ip admin-
istration, or 120 min after oral administration. Platelet-poor
plasma was prepared by centrifugation for measurement of
APTT, PT, and TT and transaminases levels (GOT, GPT).
Levels of GOT and GPT were measured on the Roche Cobas
Mira Plus automated chemistry analyzer. All data were ex-
pressed as relative fold values, compared with the values ob-
tained with control group.
mixed with 25 μL of sample solution at various concentrations
and incubated for 2 min at 37 °C. Then 100 μL of thrombin solu-
tion (100 U/mL), preincubated for 5 min at 37 °C, was added and
clotting times were recorded during 165 s. The clotting times were
recorded in seconds. Each measurement was performed in dupli-
cate and repeated three times on different days (n = 6). Coagula-
tion time prolonging ratio was calculated comparing the clotting
time in the presence of each tested compound with that obtained
when water was used instead of test compound. The concentration
required to double the clotting time was calculated from linear
regression analysis of each individual concentration-response
curve.
Thromboelastography. Blood samples were collected from
healthy donors into siliconized Vacutainer tubes containing
3
.8% trisodium citrate such that a ratio of citrate/whole blood
of 1:9 (v/v) was maintained and TEG was performed within 5 h
of blood collection. The TEG assay was performed according to
the manufacturer’s guidelines using a thromboelastography coa-
gulation analyzer Rotem delta (Pentapharm GmbH, Germany).
All disposables supplies were purchased from Pentapharm GmbH.
Sulfated compounds were dissolved in saline and diluted in the
-
4
whole blood (1:16) to a final concentration of 6.25 ꢀ 10
M
-
4
(
compounds 7, 8, and 10) or 1.25 ꢀ 10 M (compound 13). In the
control group, saline was added to the whole blood (1:16). As soon
as each compound was added to the blood, the program was acti-
vated and allowed to run until 60 min. Clot formation is monitored
at 37 °C in an oscillating plastic cylindrical cuvette (cup) and a
coaxially suspended stationary piston (pin). During clot forma-
tion, fibrin fibrils physically link the cup to the pin and the pin starts
to move. The movement of the pin is recorded as amplitude (mm).
The stronger the clot, the more the pin moves with the cup and the
higher MA is. InTEM and ExTEM were performed by adding a
contact activator or tissue factor, respectively, and FibTEM, by
2þ
adding cytochalasin D and Ca
.
Platelet Function. In vitro platelet function was evaluated
using a PFA-100 device (Dade Behring, Frankfurt, Germany)
according to the manufacturer’s instructions. Compound solu-
tion (125 μL) was added to whole blood (1875 μL) to a final con-
-4
centration of 6.25 ꢀ 10 M (compounds 7, 8, and 10) or 1.25 ꢀ
-
4
1
0
M (compound 13). This mixture was divided and applied
into two cartridges. Both cartridges hold a membrane with a
central aperture and are coated with collagen; one cartridge con-
tains epinephrine and the other ADP. The time required for plate-
lets to occlude the central aperture in the membrane is reported as
the closure time.
Chromogenic Substrate Hydrolysis Assays. Human FXa
27nkat/mL) and human thrombin (137 NIH/mL) were obtained
(
from Diagnostica Stago (Roche, Portugal). Bovine ATIII was
obtained from Sigma-Aldrich (Germany) (50 UI/mL). The chro-
mogenic substrates used were Chromozym TH (Tosyl-Gly-Pro-
Arg-p-nitroaniline(pNA), Roche Applied Science) for thrombin
and CBS 31.39 (CH SO -D-Leu-Gly-Arg-pNA, AcOH; Diagnos-
3
2
tica Stago) for FXa. Stocksolutions of the enzymeswereobtained
by restitution according to the manufacture instructions and
assay solutions were prepared by fresh dilution with the assay
buffer. The assay buffer was 50 mM Tris/HCl at pH 8.3, contain-
ing 227 mM NaCl and 0.1% (w/v) bovine serum albumin (BSA,
Sigma-Aldrich) instead of PEG-8000. The final enzyme concen-
trations were 0.5 U/mL, 0.06 U/mL, and 1 U/mL for FXa,
thrombin, and ATIII, respectively. The final substrate concentra-
-6
-6
tions in the reactions were 178 ꢀ 10 M and 125 ꢀ 10 M (in
water) for Chromozym TH and CBS31.39, respectively. For the
kinetic studies of CBS 31.39 hydrolysis by FXa in the presence of
compounds 7, 8, 10, and 13, various concentrations of the sulfated
-
3
inhibitors (0.5, 1, and 2 ꢀ 10 M) and CBS 31.39 (25 and 125 ꢀ
-6
10
M) were investigated. Under the experimental conditions,
less than 10% of the substrate was consumed in all assays [ε
Data were tested for statistical significance by nonparametric
two-tailed Mann-Whitney test using GraphPad Prism 5 soft-
ware. A value of P < 0.05 was considered significant.
4
05 nm
-
1
p-nitroaniline) = 10.4 mmol L cm ]. The chromogenic as-
-1
(
3
3
says were carried out at 37 °C in 96-well plates on a microplate

Article
Acknowledgment. We thank Funda c- ~a o para a Ci ^e ncia e a
Tecnologia (FCT), I&D 4040/2007, FEDER, and POCI for
financial support and for the Ph.D. grant to Marta Correia da
Silva (SFRH/BD/22962/2005). We thank Dr. Luciana Ricca
Gon c- alves andDr. Gra c- a Oliveirafor criticaldiscussionofthe
TEG and PFA results and technicians of the Coagulation
Laboratory from the Servi c- o de ImunoHemoterapia, Hospital
de S. Jo ~a o for technical support.
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