Polysulfated xanthones: Multipathway development of a new generation of dual anticoagulant/antiplatelet agents

Article abstract of DOI:10.1021/jm2006589

A multipathway strategy was used to evaluate the in vitro and in vivo antithrombotic effects of a new synthetic family of sulfated small molecules. Polysulfated xanthonosides showed highly effective anticoagulation effects in vitro, both in plasma (clotting times) and in whole human blood (thromboelastography), as well as in vivo (ip administration, mice). Physicochemical properties were assessed for mangiferin heptasulfate (7), which showed high solubility and stability in water and in human plasma and no putative hepatotoxicity in vivo. Mangiferin heptasulfate (7) was found to be a direct inhibitor of FXa, while persulfated 3,6-(O-β-glucopyranosyl)xanthone (13) acted as a dual inhibitor of FXa (directly and by antithrombin III activation). By impedance aggregometry, compounds 7 and 13 exhibited the antiplatelet effect by inhibition of both arachidonic acid and ADP-induced platelet aggregation. Dual anticoagulant/antiplatelet agents, such as sulfated xanthonosides 7 and 13, are expected to lead to a new therapeutic approach for the treatment of both venous and arterial thrombosis.

Full text of DOI:10.1021/jm2006589

ARTICLE
pubs.acs.org/jmc
Polysulfated Xanthones: Multipathway Development of a New
Generation of Dual Anticoagulant/Antiplatelet Agents
Marta Correia-da-Silva,† Emília Sousa,† Bꢀarbara Duarte,‡ Franklim Marques,‡ Fꢀelix Carvalho,§
Luís M. Cunha-Ribeiro,|| and Madalena M. M. Pinto*,†
†Centro de Química Medicinal, Universidade do Porto (CEQUIMED-UP), Laboratꢀorio de Química Org^anica e Farmac^eutica,
Departamento de Química, Faculdade de Farmꢀacia, Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
‡Unidade de Anꢀalises Clínicas, Departamento de Ci^encias Biolꢀogicas, Faculdade de Farmꢀacia, Universidade do Porto, Rua Aníbal
Cunha 164, 4050-047 Porto, Portugal
§REQUIMTE, Laboratꢀorio de Toxicologia, Departamento de Ci^encias Biolꢀogicas, Faculdade de Farmꢀacia, Universidade do Porto,
Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
Servico de ImunoHemoterapia, Centro de Trombose, Hemostase e Biologia Vascular, Hospital de S. Jo~ao, 4200-319 Porto, Portugal
)
S Supporting Information
b
ABSTRACT: A multipathway strategy was used to evaluate
the in vitro and in vivo antithrombotic effects of a new
synthetic family of sulfated small molecules. Polysulfated
xanthonosides showed highly effective anticoagulation effects
in vitro, both in plasma (clotting times) and in whole human
blood (thromboelastography), as well as in vivo (ip adminis-
tration, mice). Physicochemical properties were assessed for
mangiferin heptasulfate (7), which showed high solubility and
stability in water and in human plasma and no putative
hepatotoxicity in vivo. Mangiferin heptasulfate (7) was found to be a direct inhibitor of FXa, while persulfated 3,6-(O-β-
glucopyranosyl)xanthone (13) acted as a dual inhibitor of FXa (directly and by antithrombin III activation). By impedance
aggregometry, compounds 7 and 13 exhibited the antiplatelet effect by inhibition of both arachidonic acid and ADP-induced
platelet aggregation. Dual anticoagulant/antiplatelet agents, such as sulfated xanthonosides 7 and 13, are expected to lead to a
new therapeutic approach for the treatment of both venous and arterial thrombosis.
’ INTRODUCTION
On the basis of these considerations, our research has been
focused on developing new small carbohydrate-based mol-
ecules that could preserve some molecular properties of the
polysaccharide anticoagulant agents but with reduced anionic
character, higher hydrophobic nature, and feasible synthesis. In
this direction, we have recently obtained a series of polysulfated
flavonosides (Figure 1) that combines both oligosulfated and
flavonic moieties.13 These compounds exhibited in vitro and
in vivo anticoagulant activity, and preliminary toxicological
studies suggested lack of acute side effects.13
Following these stimulating results, with the same purpose, we
decided to obtain a new group of synthetic small molecules. A
multipathway strategy of simultaneous investigation of param-
eters like stability, toxicity, and in vivo efficacy was followed to
associate appropriate physicochemical properties to the struc-
tures of the newly synthesized compounds as early as possible in
the discovery process.
Past research has highlighted the drawbacks of the thera-
peutic use of heparins.1ꢀ4 Their anionic nature can result in
significant interactions with multiple physiologically important
proteins, leading to many side effects.5,6 Heparin suffers from
low tissue permeability, short serum half-life, and poor
stability.1 Consequently, the pharmacokinetic properties of
heparin make it only adequate for intravenous application.7
Commercially available heparin is a natural product obtained
from either bovine or porcin sources, adding concerns about its
structure variability8 and the risk of contamination with animal
pathogens.9 The identification of a small sequence in the
molecule of heparin, which is responsible for the affinity of
heparin for antithrombin III (ATIII), has led to massive work
in the past 2 decades devoted to the synthesis of oligosacchar-
ides, especially DEFGH and its derivatives.10ꢀ12 While some
synthetic heparin-mimetic oligosaccharides show activity com-
parable to that of heparin, these molecules require a large
number of synthetic steps, making them costly and possibly
preventing their widespread clinical use.10ꢀ12 Other sulfated
saccharides, while simpler to synthesize, require a high level of
charge to show a significant anticoagulant effect.10ꢀ12
Because of their similarity, we reasoned that sulfated xantho-
nosides may exhibit similar activities to sulfated flavonosides
Received: February 22, 2011
Published: July 06, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Figure 1. Polysulfated flavonosides with anticoagulant activity.13
Because aryl C-glycosides are stable analogues of O-glycosides,36
resistant to metabolic processes, the C-glycoside mangiferin
emerged as an interesting derivative to be sulfated.
A xanthone O-glycosylated on both rings (12, Scheme 1) was
also planned to interact in an extended binding area of proteins
that are critical in the coagulation process, such as ATIII.37
According to this hypothesis, the sulfated diglycosylxanthone
(13, Scheme 1) would lead to a molecule with higher molecular
size and number of sulfate groups than the previous polysulfated
flavonosides and thus increase anticoagulant effects.
In this work, the development of new sulfated xanthones
having anticoagulant and antiplatelet properties is described.
Compounds were screened by in vitro clotting time assays in
human plasma and their effects in all components of human
whole blood evaluated with thromboelastography. In order to
understand the mechanism of action of the synthesized com-
pounds, enzyme inhibition assays were performed against factor
Xa (FXa), thrombin, prothrombin, activated protein C, and
ATIII. The platelet aggregation was also investigated in whole
blood with the platelet function analyzer (PFA-100) and with the
new impedance aggregometer Multiplate. In vivo studies in mice
were conducted for the direct FXa inhibitor to determine in vivo
anticoagulant activity and toxicity potential.
Figure 2. Schematic development of a new generation of cardiovascular
agents of xanthonic nature.
(Figure 2).14 Xanthones have a dibenzo-γ-pyrone nucleus and
include a large group of compounds, both of natural15 and
synthetic origin16 with a wide range of biological and pharma-
cological activities.17 These derivatives have been reviewed for
their cardiovascular effects18 and described as antiplatelet,19ꢀ21
antiarrhythmic, hypotensive,22 and antithrombotic agents.23,24
Nonetheless, few reports can be found concerning sulfated
derivatives of xanthones.25ꢀ27
’ CHEMISTRY
The synthesis of sulfated xanthones was first described in 2006
by a biotransformation of 1-hydroxy-2,3,5-trimethoxyxanthone
to 5-sulfate-1-hydroxy-2,3-dimethoxyxanthone and 5-sulfate-1-
hydroxy-2,3,7-trimethoxyxanthone in the Trichothecium roseum
fungi.26 A chemical procedure using pyridineꢀsulfur trioxide
adduct was first described by some of us in the synthesis of 3,6-
disulfate xanthone (3, Scheme 1).27 Herein, three sulfated
xanthones (6, 7, and 13) and one diglycosylxanthone (12) were
obtained for the first time, in moderate yields (Scheme 1).
Sulfation of commercially available mangiferin (5) with 4
equiv/OH of triethylamineꢀsulfur trioxide adduct in dimethy-
lacetamide at 65 °C (Scheme 1, step a) afforded mangiferin
20,30,40,60-tetrasulfate (6), and the use of higher quantities of
sulfating agent (Scheme 1, step b) furnished the 20,3,30,40,6,60,7-
heptasulfated derivative 7. In both products (6 and 7), sulfation
did not take place at the 5-hydroxyl group, as this is particularly
nonreactive because of the formation of a strong hydrogen bond
with the adjacent carbonyl at the 4-position.
Mangiferin (5, Scheme 1) was selected for sulfation as part of
our top priority of developing new molecules derived from
molecular modifications of known drugs,28 for several reasons
that follow. Modification of molecules that have alreadybeen used
in human therapy leads to compounds with more predictable and
less complex pharmacokinetics, lower incidence of side effects,
and less demanding clinical studies.29 Mangiferin is a natural
product and has been reported to be endowed with several
pharmacological activities including antioxidant,30 antiviral,
prevention of carcinogenesis,31 and inhibition of platetet
aggregation.21 Because of several interesting effects, mangiferin
has been widely used as a nutritional supplement in cosmetic
products and natural medicine.32 The wide use of mangiferin by
humans for many years suggests low toxicity not only for
mangiferin but also for their metabolites and analogues. Pro-
tective effects of mangiferin with regard to cardio,33,34 renal,33
liver, and brain35 toxicity are well documented. Furthermore,
mangiferin is a C-glycoside. Because of favorable pharmacological
profiles attributed primarily to the C-glycosyl moiety, aryl
C-glycosides have gained increasing popularity as drug candidates.36
To obtain the sulfated derivative 13, the building block 12 was
initially synthesized (Scheme 1, steps cꢀf). Despite the wide
occurrence and biological importance of flavonoids and other
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Scheme 1. Synthesis of Sulfated Glycosylxanthones 6, 7, and 13a
a Reagents and conditions: (a) SO3/Et3N (4 equiv/OH), DMA, 65 °C, 24 h; (b) SO3/Et3N (8 equiv/OH), DMA, 65 °C, 24 h; (c) furnace, 220 °C,
overnight; (d) 0.15 M aq K2CO3, TBHAS (2 equiv/OH), CHCl3, 50 °C, 36 h; (e) 0.08 M NaOMe, MeOH, room temp, overnight; (f) SO3/Et3N
(6 equiv/OH), MW, 30 min; (g) Py/SO3, DMA, 65 °C.27 The numbering used corresponds to the NMR assignments.
polyphenol glycosides, synthetic efforts toward efficient prepara-
tion of this group of natural products are rarely reported. 3,6-
Dihydroxyxanthone (9) was first obtained from 2,20,4,40-tetrahy-
droxybenzophenone (8) by a dehydrative process in the furnace
(220 °C) (Scheme 1, step c).38 Following, 3,6-(O-β-glucopyra-
nosyl)xanthone (12) was obtained by phase transfer catalyzed
(PTC) coupling reaction of 3,6-dihydroxyxanthone (9) and
acetobromo-β-D-glucose in aqueous K2CO3 and TBAHS at
50 °C (Scheme 1, step d).39 The same reaction was first conducted
in dry DMF with anhydrous K2CO3 at room temperature. How-
ever, the product was not isolated, probably because of the easy
hydrolysis of the acetyl groups from xanthone under these
coupling conditions. 3,6-(O-β-Glucopyranosyl)xanthone (12) was
obtained after deprotection of the glycosylated xanthone 3,6-(2,
3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)xanthone (11) with so-
dium methoxide in an overall 50% yield (Scheme 1, step e).
Sulfation of compound 12 using microwave (MW) irradiation
(Scheme 1, step f) allowed us to obtain the persulfated 3,6-(O-β-
glucopyranosyl)xanthone (13) in 30 min and with 60% yield.
The structure elucidation of compounds 6, 7, 12, and 13 was
established on the basis of IR, HRMS, and NMR techniques (see
Supporting Information for detailed discussion).
The potential of druglikeness of the synthesized polysulfated
glucosidic molecules was investigated by determining param-
eters, namely, solubility, chemical stability, and plasma stability
which are essential for preformulation. Stability of polysulfated
small, aromatic molecules has been rarely studied. The stability
of resveratrol 40-sulfate was investigated for 24 h at 37 °C in
RPMI 1640 cell culture medium which was used for MCF-7
cellular uptake studies. While resveratrol 40-sulfate was stable for
24 h, the nonsulfated resveratrol degraded ∼50% during 24 h
under these conditions.40 In another study, epicatechin persulfate
was found to be stable under neutral and basic conditions and
considerably unstable under acidic conditions.41 We have pre-
viously proved the human plasma stability for polysulfated fla-
vonoids O-rutinosides.13 In this work, we assessed not only the
stability in human plasma but also the chemical stability in aque-
ous solution at different storage temperatures in a range of con-
centrations and pH values for the polysulfated xanthone
C-glucoside 7 (Figures 3 and 4).
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Concerning stability in aqueous solutions, compound 7 was
found to be chemically stable for 15 days at several storage
conditions, room temperature, 4 °C, and ꢀ20 °C, at high, low,
and medium concentrations (Figure 3), i.e., the concentration
detected on each day was not significantly different from the
initial one. Furthermore, compound 7 was highly soluble in water
and did not produce crystals when stored at 4 °C or ꢀ20 °C.
Compound 7 did not degrade at the range of pH values selected
to mimic the in vivo oral dosing (Figure 4), i.e., the % of
compound 7 remaining at 120 min was higher than 85%.42
Finally, compound 7 was found stable after 3 h at 37 °C in human
plasma, i.e., the concentration after 3 h was not significantly
different from the initial one. This was consistent with the plasma
stability previously reported for diosmin hexasulfate (1)13,43 and
rutin decasulfate (4).13
’ RESULTS AND DISCUSSION
The anticoagulant activity of the sulfated molecules 6, 7, 10,
and 13 was screened in vitro in human plasma by activated partial
thromboplastin (APTT), prothrombin (PT), and thrombin time
(TT) assays. The nonsulfated parent compounds 5 and 12 and
sodium sulfate were also considered in order to explore the
influence of the sulfate group on the anticoagulant activity.
The results for the sulfated derivatives tested (6, 7, 10, and 13)
on APTT, PT, and TT are summarized in Figure 5. For the most
potent compounds the concentration needed to double the
coagulation times (APTT2, PT2, TT2) was calculated (Table 1).
The nonsulfated compounds 5 and 12 and sodium sulfate solution
were inactive in all clotting time assays (at the concentration
tested, 5 ꢁ 10ꢀ3 M, no significant difference was observed
between the compound and the control; data not shown).
The investigated polysulfated xanthonosides 6, 7, and 13
showed anticoagulant properties in a dose dependent manner
in all clotting times (Figure 5). The nonglycosylated xanthone
disulfate 10 was only active, in PT and TT, at the highest
concentration tested (5.00 ꢁ 10ꢀ3 M).
From the overall results, it can be concluded that compound 7,
highly soluble in water, is stable either in water or in plasma.
Prolongation of APTT and PT was observed for all the sulfated
xanthones (6, 7, 10, and 13). Polysulfated xanthones 6, 7, and 13
were able to completely block these clot formation pathways at
high concentrations (Figure 5). Xanthone 13 was the most potent
compound (APTT2 = 6.4 ꢁ 10ꢀ5 M, PT2 = 7.1 ꢁ 10ꢀ5 M).
Sulfated compound 10 only increased the APTT and PT approxi-
mately 1.3 and 1.1 times, respectively, at 5.00 ꢁ 10ꢀ3 M.
The TT was sensitive to the presence of compounds 6, 7, and
13, which were able to completely prevent thrombin activation,
at the highest concentration tested (Figure 5).
Figure 3. Stability of compound 7 in aqueous solutions at storage
conditions at room temperature, 4 °C, and ꢀ20 °C.
Table 1. Concentrations Needed To Double the Clotting
Timesa for Sulfated Xanthones 6, 7, 10, and 13
compd
APTT2
PT2
TT2
6
0.39
0.21
>5
nd
nd
7
1.60
>5
1.96
na
10
13
0.06
0.71
0.61
aValues in concentrations expressed in 10ꢀ3 M represent the average of
three independent experiments with a standard deviation of <10%. Positive
control, heparin: APTT2 =0.4U/mL,PT2 =1.9U/mL,andTT2 =0.2U/mL.
nd: not determined. na: not active at 5.00 ꢁ 10ꢀ3 M (P > 0.05).
Figure 4. StabilityꢀpH profile for compound 7 (1.00 ꢁ 10ꢀ5 M).
Figure 5. Effects of sulfated derivatives (6, 7, 10, and 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 of <10%. NC, no coagulation: clotting time values greater than 165 s (PT and TT) and 110 s (APTT). /, P < 0.05.
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Table 2. In Vitro Inhibitory Activitya of Polysulfated
Xanthones 7 and 13 against Human FXa and ATIII Inhibition
of FXa in the Presence of CBS31.39 (1.25 ꢁ 10ꢀ4 M)
glucopyranosyl)xanthone (13) a dual direct and indirect FXa
inhibitor. At 1.00 ꢁ 10ꢀ3 M compound 13 (FXa inhibition of
66%) and even compound 7 (FXa inhibition of 52%) were more
potent on FXa than the earlier obtained direct FXa inhibitors
ethoxyrutin nonasulfate (3, FXa inhibition of 39%) and rutin
persulfate (4, FXa inhibition of 32%).13 By comparison of the
indirect FXa inhibition results, compound 13 (ATIII/FXa in-
hibition of 89%) was also more active than the earlier obtained
ATIII/FXa inhibitors diosmin hexasulfate (1, ATIII/FXa inhibi-
tion of 39%) and hesperidin hexasulfate (2, ATIII/FXa inhibition
of 46%).13 The MichaelisꢀMenten fit for both xanthones 7 and
13 suggests a noncompetitive inhibition against FXa (data not
shown), which is in accordance with previously reported data for
some sulfated benzofuran small molecules.44 Dual direct and
indirect FXa inhibitors, like compound 13, or direct FXa
inhibitors, like compound 7, have advantages regarding heparins
or fondaparinux primarily because they can inhibit both circulat-
ing and clot-bound enzymes. Indirect FXa inhibitors require
ATIII as a cofactor and do not inhibit FXa bound to the
prothrombinase complex. In contrast, direct FXa inhibitors
directly engage the active center of the FXa molecule and thus
inhibit the free and the attached FXa to the prothrombinase
complex.45
None of the compounds 7 and 13 showed any influence on
thrombin activity (% of PPACK thrombin inhibition at 1.9ꢁ 10ꢀ9
M was equal to 37 ( 5%) even in the presence of ATIII (data not
shown), which highlights their FXa selectivity. FXa activates
clotting over a wider concentration range compared to thrombin,
in both in vitro and in vivo systems.46 Consequently, FXa inhibitors
compared with thrombin inhibitors are expected to have a wider
therapeutic window and thus to reduce the need for coagulation
monitoring. Because of the absence of effect in the amidolytic
compared to the coagulation assay, the action in the TT assay
for compounds 7 and 13 does not seem not to be the result of an
inhibition of thrombin but instead mediated by an interaction
with the thrombin action on fibrinogen or fibrinogen itself, which
cannot be measured in the chromogenic substrate assay.
To assess if the anticoagulant effect of sulfated xanthones
7 and 13 could also interfere with the protein C pathway, the
determination of activated protein C (APC) in citrated human
plasma was evaluated by a functional test. Protein C was activated
by a specific activator derived from the venom of Agkistrodon c.
contortrix, and the quantity of enzyme thus formed was measured
by its amidasic activity on the synthetic substrate CBS 42.46.
At 2.50 ꢁ 10ꢀ4 M, compounds 7 and 13 had no significant
influence in the protein C activity (Figure 6). Only at high
concentrations (2.00 ꢁ 10ꢀ3 M), compounds 7 and 13 decreased
the APC to pathological levels (less than 50%, Figure 6). Never-
theless, compounds 7 and 13, at the concentration that led to
approximately 40% of APC inhibition, also showed deeply
decreased prothrombin levels (Figure 6). Since low levels of
prothrombin were observed, the risk of thrombosis due to the
APC inhibition for these compounds is not expected, as it
happens for heparin and oral anticoagulants.47ꢀ50
human FXa
ATIII/FXa
compd
% inhibb
IC50 (ꢁ10ꢀ4 M)
% inhibb
IC50 (ꢁ10ꢀ4 M)
7
52.10 ( 2.67
66.26 ( 2.13
9.70 ( 0.02
7.40 ( 0.05
41.0 ( 6.00
nd
13
88.82 ( 5.30
1.90 ( 0.03
a Each value represents the average ( SEM of three independent
experiments done in duplicate. b Results expressed as percentage of
inhibition at 1.00 ꢁ 10ꢀ3 M. The % of rivaroxaban FXa inhibition at
5.00 ꢁ 10ꢀ9 M was equal to 52.00 ( 3.62 (IC50 = (4.59 ( 1.63) ꢁ
10ꢀ9 M). The % of LMWH ATIII/FXa inhibition at 0.20 U/mL was
equal to 97.96 ( 0.58.
In summary, all the sulfated compounds (6, 7, 10, and 13)
prolonged APTT and PT, and compounds 6, 7, and 13 also
prolonged TT pathway. As the three clotting assays recorded
interactions at different stages of the coagulation process, pre-
liminary information about the mode of action can be obtained.
As the sulfated compounds 6, 7, and 13 prolonged APTT, PT
(APTT more than PT), and TT, their mechanism of action may
be implicated in the common pathway of the coagulation cascade.
A structureꢀactivity relationship can be recognized concern-
ing the anticoagulant action and the number of sulfate groups,
i.e., the anticoagulant effects increased with the increase of the
number of sulfates. Xanthone 3,6-β-D-glucopyranoside persulfate
(13), with eight sulfate groups, was more potent than heptasulfated
mangiferin 7, and compound 7 was more potent than tetrasulfated
compound 6. The disulfated xanthone 10 was only slightly active in
prolonging the clotting times. Nevertheless, by comparison of these
results with the earlier obtained for polysulfated flavonosides,13
xanthone 13 with eight sulfate groups (APTT2 = 6.4 ꢁ 10ꢀ5 M)
was not significantly different from decasulfated flavonoid rutin (4,
APTT2 = 6.6 ꢁ 10ꢀ5 M) and xanthone 6 (APTT2 = 3.9 ꢁ 10ꢀ4 M)
with only four sulfate groups had the same order of potency as
hexasulfated diosmin (1, APTT2 = 4.1 ꢁ 10ꢀ4 M) and
hesperidin (2, APTT2 = 4.4 ꢁ 10ꢀ4 M).
Compounds 7 and 13 were further evaluated for selectivity
against the critical enzymes targeted by current anticoagulant
therapy: thrombin, FXa, and ATIII. Inhibition of thrombin and
FXa, in the presence and absence of ATIII, was followed by
spectrophotometric determination of the product formed after
amide bond cleavage of the chromogenic substrates chromozym
TH and CBS 31.39, respectively, and the initial rate was
compared with that obtained in the absence of the compound.
Reference inhibitors rivaroxaban (direct FXa inhibitor), LMWH
(ATIII/FXa activator), and PPACK (direct thrombin inhibitor)
were also tested in the same conditions. Kinetic studies of the
inhibitory activity of sulfated xanthones 7 and 13 against FXa
were fitted by the MichaelisꢀMenten equation (nonlinear
regression analysis, data not shown).
Both xanthones showed a direct inhibitory effect on FXa
(Table 2), with compound 13 exhibiting higher potency than
compound 7. For compound 13 this effect was significantly (P <
0.05) enhanced by the presence of ATIII, suggesting also an
indirect action on FXa by activation of ATIII. For compound 7
the percentage of FXa inhibition in the presence of ATIII was not
significantly different from that obtained in its absence (P >
0.05). Therefore, mangiferin 20,3,30,40,6,60,7-heptasulfate (7) was
found to be a direct FXa inhibitor and persulfated 3,6-(O-β-
To have a global picture of the anticoagulant effect of sulfated
molecules 7 and 13 and to warrant anticoagulation activity in
whole blood, thromboelastography (TEG) analyses were carried
out. Classical clotting tests are performed in citrated plasma and
provide information about only one isolated segment of the
coagulation system (secondary hemostasis). The clot quality and
the impact of platelets on coagulation are not assessed by these
assays. In contrast, TEG is performed in whole blood and the clot
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Figure 6. Dose-dependent effect of compounds 7 and 13 on activated protein C (APC) and prothrombin levels. Data points represent the average (
SD of three independent experiments done in duplicate. /, P < 0.05.
Table 3. Effectsa of Polysulfated Xanthones 7 and 13 (3.12 ꢁ
10ꢀ4 M) in Whole Human Blood on the Parameters Obtained
from InTEM-Thromboelastography
InTEM
compd
R (s)b
K (s)c
µ (deg)d
A10 (mm)e
blood (saline)
188
82
77
57
7
247*f
469*f
86
114*f
73
68*f
50
51*f
13
aEach value represents the average of two independent experiments. bR,
clotting time expressed in seconds (s). c K, clotting formation time
expressed in seconds (s). d µ, angle expressed in degrees (deg). A10,
e
amplitude expressed in mm at 10 min. f /, P < 0.05.
In the presence of compounds 7 and 13, abnormal thromboe-
lastograms were obtained when an intrinsic activator was added
in InTEM test (Table 3), an effect similar to that observed in the
presence of a factor deficiency (anticoagulants or hemophilia).52
Compared to control values, compound 7 increased only the
clotting time (R), while compound 13 increased also the clotting
formation time (K) and decreases µ and A10 (Table 3). This
indicates that the kinetics of fibrin polymerization and network-
ing and the strength of the formed clot, which is highly
dependent on platelets, are significantly retarded by the presence
of sulfated compound 13, while compound 7 is only affecting the
clotting factors. Nevertheless, normal thromboelastograms were
obtained in the presence of compounds 7 and 13 when the
platelet-specific effect on A10 of TEG tracings is eliminated
(FibTEM test, Figure 7). These results allowed the conclusion
that an action on platelets would also be implicated in the mode
of action of compounds 7 and 13.
The overall TEG results showed where the imbalance of the
hemostasis system was located. In the presence of compounds 7
and 13, there were no signals of fibrinolysis. The blood became
hypocoagulable in the presence of both compounds. The enzy-
matic reactions were decreased, as shown by the R parameter.
There was a low fibrinogen level as depicted by the µ parameter,
and the platelet function was decreased, as represented by the A10
parameter.
Figure 7. Thromboelastography (TEG) representative tracings of
compound 13 (3.12 ꢁ 10ꢀ4 M) and control (saline).
is induced under a low shear environment resembling an in vivo
sluggish venous flow. TEG displays qualitatively two distinct parts
of hemostasis (the part that produces the clot and the part that
causes the breakdown of the clot) and allows the acquisition of
quantitative information about the kinetics of clot formation and
growth as well as the clot strength and stability attained by clots.51
The anticoagulanteffects of sulfatedcompounds7 and 13 inwhole
human blood were evaluated at 3.12 ꢁ 10ꢀ4 M. Modified
thromboelastograms were obtained (Figure 7, e.g., compound
13) by adding to the whole blood an intrinsic activator (InTEM)
or an extrinsic activator (ExTEM). It was also possible to eliminate
platelet contribution from the TEG sample and study the fibrin
partof the clot by adding cytochalasinD and Ca2+ (FibTEM). The
parameters obtained from the InTEM thromboelastogram were R,
the period of time from initiation of the test to the initial fibrin
formation (clotting time); 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 buildup and cross-linking (clot formation and growth);
and A10, the amplitude of the clot at 10 min (Table 3).
In vivo studies in mice were also performed for compound 7 to
preliminarily assess its anticoagulant and toxicity effects. To
estimate the in vivo anticoagulant activity, APTT, PT, and TT
were determined after 150 and 300 μmol/kg intraperitoneal (ip)
administration or at 120 min after 300 μmol/kg of oral admin-
istration by gavage. These values were compared with the APTT,
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Figure 8. Dose-dependent APTT, PT, and TT prolonging activities of compound 7 after ip administration in mice, 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. /, P < 0.05.
Table 4. Antiaggregant Effectsa of Nonsulfated (5 and 12) and Sulfated (7 and 13) Xanthones (6.25 ꢁ 10ꢀ4 M) on Whole Human
Blood Evaluated with PFA-100 and Multiplate Analyzers
PFA-100 closure time (s)
multiplate AUC (U)b
Col test
whole blood
Col/ADP
Col/EPI
TRAP test
ASPI test
ADP test
ADP-HS test
ref values
71ꢀ118
119
94ꢀ193
168
nd
84ꢀ128
106
71ꢀ115
72
72ꢀ125
60
57ꢀ113
72
43ꢀ100
46
blood (saline)
Na2SO4
nd
131
87
84
78
47
ASA
5
nd
nd
104
19*c
49*c
77
54
nd
nd
115
72
77
65
44
12
7
nd
nd
110
84
66
68
43
146*c
174*c
165
268*c
112
44*c
29*c
52*c
44*c
31*c
25*c
30*c
22*c
13
91
aEach value represents the average of two independent experiments done in duplicate. nd: not determined. bArea under the aggregation curve (AUC)
expressed in U (1 U = 10 AU ꢁ min). c /, P < 0.05.
PT, and TT values obtained with the saline-treated mice group
and expressed as a ratio (Figure 8). Plasma levels of glutamicꢀ
oxaloacetic transaminase/aspartate aminotransferase (GOT/AST)
and glutamateꢀpyruvateꢀtransaminase/alanine aminotrans-
ferase (GPT/ALT) were determined after the ip administration
of these molecules.
After 30 min of ip administration, compound 7 was highly
active, increasing the APTT to the desirable therapeutic range
(1.5ꢀ2.3 times the mean of the normal APTT range,53 Figure 8).
The rapid onset of action revealed by compound 7 is a desirable
feature compared to oral anticoagulants drugs acenocoumarol
and warfarin sodium. Moreover, at both concentrations com-
pound 7 was still active after 120 min; it is reasonable to speculate
that a desirable duration of action will be sustained.53 After oral
administration, compound 7 was not active (results not shown).
These results are in accordance with the results previously
obtained for polysulfated flavonosides.13
After ip administration of compound 7 at 300 μmol/kg, GOT
and GPT plasma levels were not significantly different (125 and
46 U/L, respectively) from those of saline treated mice (108 and
40 U/L, respectively) (P > 0.05). These preliminary results,
obtained after a single acute dose, suggest that compound 7 has
low hepatotoxic potential, in contrast to unfractionated and
LMW heparins.3,54,55
After obtaining the in vivo results, we decided to further
investigate the effects of sulfated derivatives (7 and 13) on
platelets. Thus, the platelet aggregation in whole blood in the
presence of sulfated xanthones 7 and 13 (Table 4, Figure 9) was
evaluated by measuring the collagen/adenosine 50-diphosphate
(Col/ADP) and collagen/epinephrine (Col/EPI) closure times
and impedance platelet aggregometry induced by thrombin
receptor-activating peptide (TRAP test), arachidonic acid
(ASPI test), collagen (Col test), ADP (ADP test), and ADP
and prostaglandin E1 (ADP-HS test). The parent nonsulfated
compounds 5 and 12, a highly ionic salt (sodium sulfate), and
acetylsalicylic acid (ASA, cyclooxygenase-1 inhibitor) were also
tested in the same conditions.
The Col/EPI test cartridge is the primary cartridge used to
detect platelet dysfunction induced by intrinsic platelet defects,
von Willebrand disease, or exposure to platelet inhibiting agents.
The Col/ADP test cartridge is used to indicate if abnormal results
obtained with Col/EPI test cartridge may have been caused by the
effect of ASA. Usually, a pattern of abnormal Col/EPI and normal
Col/ADP is observed with ASA ingestion cases. While ASA has
been shown to increase the Col/EPI closure time,56 clopidogrel
has been shown to increase the Col/ADP closure time.57 Closure
times do not vary with heparin administration.57 Considering
compounds 7 and 13, PFA-100 closure times of both Col/ADP
and Col/EPI cartridges were markedly longer with xanthone 13,
and only the Col/ADP closure time was increased with xanthone
7 at 6.25 ꢁ 10ꢀ4 M (Table 4).
None of the investigated compounds affected the TRAP test,
which is affected by GpIIb/IIIa antagonists and thrombin inhibi-
tors The ASPI test specifically measures the activation by arachi-
donic acid, the substrate of cyclooxygenase 1. Cyclooxygenase
forms thromboxane A2, which is a potent platelet activator. When
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Figure 9. Multiplate traces in ASPI test. During a measurement period of 6 min the change in electrical impedance is calculated from the mean values of the
two curves: control, saline; ASA, acetylsalicylic acid (final concentration, 6.25 ꢁ 10ꢀ4 M); 7 and 13, sulfated compounds (final concentration, 6.25 ꢁ 10ꢀ4 M).
the platelet cyclooxygenase is blocked, the formation of throm-
boxane A2 is inhibited and no alteration or minor platelet
activation is recorded. In the Col test, collagen activates the
collagen receptor, which leads to a release of endogenous arachi-
donic acid. The addition of xanthones 7 and 13 into the blood led
to a reduced aggregation response in both the ASPI test (Table 4,
Figure 9) and the Col test (Table 4).
of cardiovascular agents, with a defined composition and feasible
synthesis. Additionally, the less charge density allows a systemic
anticoagulant action after intraperitoneal administration, with
these compounds not likely to induce hemorrhagic complica-
tions or hepatic toxicity.
’ EXPERIMENTAL SECTION
The results obtained in the ASPI and Col tests in the presence of
xanthones 7 and 13 (Table 4), when compared with blank and
ASA, revealed their ability to disrupt the arachidonic acid pathway.
The ADP test is sensitive to ADP receptor inhibitors, and because
of the addition of prostaglandin E1, the ADP-HS test has even an
increased sensitivity toward these inhibitors. Both sulfated
xanthones (7 and 13) also affected the ADP and ADP-HS tests
(Table 4), in contrast to ASA. Dual antiplatelet therapy with ASA
anda thienopyridine is often recommended for high-risk patients.58
ASA has only a modest effect, preventing cardiovascular events
in approximately 25% of patients with atherosclerosis.58 The
thienopyridines (clopidogrel, ticlopidine) are only slightly more
effective.58 The modest efficacy of these drugs may be related to
the fact that there are multiple alternative pathways for platelet
activation. The different antiaggregant behavior of these small
sulfated molecules affords an opportunity to discover different
molecules from the known antiplatelet agents with possibly
different pharmacological profile. Furthermore, molecules such as
xanthones 7 and 13, which simultaneously target the antiplatelet
and anticoagulant pathways, could be useful to prevent and treat
both venous and arterial thrombosis. In the past 5 years, dual
anticoagulant/antiplatelet agents have already been described,59ꢀ61
although such dual agents have not yet achieved clinical develop-
ment. From the overall results and putative in vivo efficacy, with no
suggestion of acute side effects, xanthones 7 and 13 show potential
to lead a new class of therapeutic agents in the prevention and
treatment of thromboembolic events.62
Chemistry. Triethylamineꢀsulfur trioxide adduct (S 5139) was
purchased from Fluka (Spain), and mangiferin (5, M 3547), 2,20,4,40-
tetrahydroxybenzophenone (8, T16403), 2,3,4,6-tetra-O-acetyl-β-D-glu-
copyranosyl bromide (A1750), and sodium methoxide 0.5 M in
methanol (71751) were purchased from Sigma-Aldrich (Spain). The
solvents used were products pro analysis or HPLC grade of the firms
Sigma-Aldrich and Fluka. Spectra/Por dialysis membrane, MWCO
1000, was purchased from Spectrum Laboratories, Inc. (CA, U.S.).
MW reactions were performed in sealed reaction vessels (30 mL) using a
MicroSYNTH 1600 synthesizer from Millestone (ThermoUnicam,
Portugal). TLC was performed using Polygram cel 400 UV254 0.1
mm (Macherey-Nagel, Germany) (BuOHꢀCH3COOHꢀH2O 4:2:6
and 5:2:3) and Merck silica gel 60 (GF254) plates (CHCl3/MeOH 9:1).
Compounds were visually detected by absorbance at 254 and/or
365 nm. Melting points were obtained in a K€ofler microscope and are
uncorrected. IR spectra were measured on an ATI Mattson Genesis
series FTIR spectrophotometer (software WinFirst, version 2.10), in
KBr microplates (cmꢀ1). 1H and 13C NMR spectra were taken in
DMSO-d6 at room temperature on Bruker Avance 300 and 500
1
instruments (300.13 or 500.13 MHz for H and 75.47 MHz for 13C).
Chemical shifts are expressed as δ (ppm) values relative to tetramethyl-
silane (TMS) as an internal reference. Coupling constants are reported
in hertz (Hz). 13C NMR assignments were made by 2D HSQC and
HMBC experiments (long-range C, H coupling constants were opti-
mized to 7 and 1 Hz). HRMS mass spectra were measured on a APEX III
mass spectrometer, recorded as ESI (electrospray ionization) mode in
Centro de Apoio Científico e Tecnolꢀoxico ꢀa Investigation (CACTI,
University of Vigo, Spain). The HPLC analyses were carried out on a
system SMI pump series II (Gloucester, U.K.) 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 ꢁ 4.6 mm i.d.), from Macherey-
Nagel (D€uren, Germany). Acetonitrile was of HPLC grade from Merck.
HPLC ultrapure water was generated by a Milli-Q system (Millipore,
Bedford, MA, U.S.). The mobile phases were degassed for 15 min in an
ultrasonic bath before use. The purity of each compound was deter-
mined by HPLC-DAD analysis.13 All tested compounds, whether
synthesized or purchased, possessed a purity of at least 95%.
’ CONCLUSION
The sulfation as a molecular modification of the xanthonoid
scaffold was a successful strategy to obtain analogues of polysulfated
flavonosides with higher anticoagulant potency. Two polysulfated
xanthonosides proved to be dual acting inhibitors of thrombosis,
combining anticoagulant and antiplatelet effects into a single
molecule. Both showed antiplatelet activity by inhibition of arachi-
donic acid and ADP-induced platelet aggregation, while concerning
the anticoagulant mechanism of action, heptasulfated mangiferin
(7) was found to be a direct FXa inhibitor and persulfated
diglycosidic xanthone (13) a dual direct and indirect FXa inhibitor.
We believe that in contrast to unfractionated and LMW
heparins, polysulfated xanthonosides represent a new generation
General Procedure for Sulfation of Mangiferin (5). To a
solution of mangiferin (5, 1 mmol) in DMA (15 mL), triethylamineꢀ
sulfur trioxide adduct (4 equiv/OH led to compound 6; 8 equiv/OH led
to compound 7) was added, and the suspension was heated at 65 °C for
24 h. The mixture was poured into acetone (150 mL) under basic
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conditions (a few milliliters 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 dropwise in ethanol to precipitate the sodium salt
of the sulfated derivative. The solid obtained was further purified from
other salts (monitored by IR) using a Spectra/Por 6 regenerated
cellulose MWCO 1000.
HRMS (ESI+) m/z calcd for C25H28O14Na 575.137 13, found
575.137 00.
Synthesis of 3,6-(O-β-Glucopyranosyl)xanthone Persulfate
(13). To a solution of the xanthone glycoside 12 (0.53 g, 0.97 mmol)
in DMA (10 mL), triethylamineꢀsulfur trioxide adduct (7.9 g, 6 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 reaction mixture was ramped to 100 °C using the following
powerꢀtemperature steps: (1) 200 W, room temperature to 80 °C,
during 1 min; (2) 200 W, 80ꢀ100 °C, for 29 min. After cooling, the
mixture was poured into acetone (150 mL) under basic conditions
(a few milliliters 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 dropwise in ethanol to precipitate the sodium salt of the sulfated
derivative. The solid obtained was further purified from other salts
(monitored by IR) using a Spectra/Por 6 regenerated cellulose MWCO
1000 (13, 0.8 g, 60%). IR (KBr) υmax: 3600ꢀ3400, 1614, 1251, 1104,
1027, 799, 612. 1H NMR (DMSO-d6, 300.13 MHz) δ: 8.11 (2H, d, J =
8.9 Hz, H-1, H-8), 7.14 (2H, d, J = 2.1 Hz, H-4, H-5), 7.10 (2H, dd, J =
8.9 and 2.1 Hz, H-2, H-7), 5.64 (1H, d, J = 3.7 Hz, H-10), 4.65 (1H, dd,
J = 8.3 and 2.9 Hz, H-60a), 4.44 (1H, t, J = 2.9 Hz, H-60b), 4.36ꢀ4.31
(1H, m, H-50), 4.05 (1H, brt, H-30), 4.01 (1H, brt, H-20), 3.90 (1H, t,
J = 9.5 Hz, H-40). 13C NMR (DMSO-d6, 75.47 MHz) δ: 174.3 (C-9),
162.5 (C-3, C-6), 157.2 (C-4a, C-10a), 127.7 (C-1, C-8), 115.9 (C-8a,
C-9a), 113.9 (C-2, C-7), 103.8 (C-4, C-5), 98.1 (C-10), 75.4 (C-50), 74.2
(C-30), 71.0 (C-20), 67.1 (C-40), 62.0 (C-60). HRMS (ESI+) m/z calcd for
C25H20O38S8Na9 1390.647 20, found 1390.654 06.
Stability Studies. Human Plasma. The plasma stability of com-
pound 7, at 2.50 ꢁ 10ꢀ5 M final concentration, was measured 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). Incubations were performed in
Eppendorf tubes on a bath shaker at 37 °C for 180 min. The reaction was
quenched with 4ꢁ cold acetonitrile (HPLC grade), followed immediately
by mixing and centrifugation during 15 min at 14 000 rpm. The time zero
samples were quenched immediately after the sample was added to
plasma. After filtration (Millipore), the supernatant was directly trans-
ferred to HPLC vials and analyzedby HPLC. Five pH values (1, 5, 6.7, 7.4,
9.1) were selected. The following buffers HCl (pH 1.0), sodium acetate
(0.05 M, pH 5.0), potassium phosphate (0.1 M, pH 6.8), PBS (pH 7.4),
and sodium boric acid (0.05 M, pH 9.1) were prepared. Then 20 μL of
water stock compound solution was added to 1980 μL of each buffer
solution to obtain a final test compound concentration of 1 ꢁ 10ꢀ5 M.
The samples were incubated in a temperature-controlled autosampler at
37 °C. The HPLC autosampler was programmed to inject samples
periodically at 0, 60, 120, and 180 min. The experiments were run at
three different concentrations of water solutions (1.00 ꢁ 10ꢀ2, 1.00 ꢁ
10ꢀ4, 1.00 ꢁ 10ꢀ6 M). The stability challenged samples were allowed to
sit 1, 7, and 15 days at laboratory temperature, 4 °C, and ꢀ20 °C. For
HPLC chromatographic conditions, the mobile phase used for chemical
and plasma stability studies of compound 7 was 50 mM sodium acetate
buffer (adjusted to pH 4.5 with acetic acid, HPLC grade) and acetonitrile
(90:10) at a constant flow rate of 1.0 mL/min.
Mangiferin 200,300,400,600-O-Tetrasulfate (6). Brown solid (0.183 g,
0.22 mmol, 22%), mp >340 °C (water). IR (KBr) υmax: 3600ꢀ3400,
1622, 1258, 1127, 1067, 804, 616. 1H NMR (DMSO-d6, 300.13 MHz)
δ: 13.67 (1H, s, 1-OH), 10.70 (1H, brs, 6-OH), 10.40 (1H, brs, 7-OH),
9.86 (1H, brs, 3-OH), 7.39 (1H, s, H-8), 6.87 (1H, s, H-5), 6.32 (1H, s,
H-4), 5.30ꢀ5.20 (1H, m, H-30), 4.85 (1H, d, J = 5.5 Hz, H-10), 4.77
(1H, brt, H-20), 4.49 (1H, m, H-40), 4.12 (1H, m, H-50), under water
(2H, H-60). 13C NMR (DMSO-d6, 75.47 MHz) δ: 179.1 (C-9), 169.7
(C-3), 161.5 (C-1), 156.2 (C-4a), 154.0 (C-6), 150.5 (C-10a), 143.6
(C-7), 111.7 (C-8a), 108.1 (C-8), 107.5 (C-2), 102.6 (C-5), 101.2 (C-
9a), 93.3 (C-4), 79.7 (C-50), 76.4 (C-20), 74.4 (C-30), 71.5 (C-40), 70.2
(C-60), 68.3 (C-10).
Mangiferin 20,3,30,40,6,60,7-O-Heptasulfate (7). Orange solid
(1.04 g, 0.93 mmol, 93%), mp >340 °C (water). IR (KBr) υmax
:
1
3600ꢀ3400, 1614, 1251, 1117, 1028, 804, 600. H NMR (DMSO-d6,
300.13 MHz) δ: 13.43 (1H, s, 1-OH), 8.26 (1H, s, H-8), 7.71 (1H, s,
H-5), 7.23 (1H, s, H-4), 5.39 (1H, t, J = 7.4 Hz, H-30), 4.77 (1H, brt,
H-20), 4.76 (1H, brd, H-10), 4.73 (1H, m, H-40), 4.09 (1H, d, J = 9.9 Hz,
H-60a), 3.90 (1H, m, H-50), 3.76 (1H, t, J = 9.6 Hz, H-60b). 13C NMR
(DMSO-d6, 75.47 MHz) δ: 180.0 (C-9), 162.0 (C-1), 159.0 (C-3),
155.8 (C-4a), 151.5 (C-6), 150.9 (C-10a), 140.9 (C-7), 113.9 (C-8),
113.4 (C-8a), 109.7 (C-2), 106.3 (C-5), 103.3 (C-9a), 96.4 (C-4), 79.4
(C-50), 77.6 (C-10), 73.3 (C-30), 72.3 (C-20), 69.5 (C-40), 67.6 (C-60).
HRMS (ESI+) m/z calcd for C19H11O32S7Na8 1158.645 45, found
1158.644 46.
Synthesis of 3,6-Dihydroxyxanthone (9). 3,6-Dihydroxyx-
anthone (9, 1.8 g, 7.9 mmol, 98%) was obtained from 2,20,4,40-tetrahydroxy-
benzophenone (8, 2 g, 8.1 mmol) after 24 h in the furnace (220 °C).38
Synthesis of 3,6-(O-β-Glucopyranosyl)xanthone (12).
TBAHS (3 g, 8.8 mmol, 2 equiv/OH) and 3,6-dihydroxyxanthone (9,
0.5 g, 2.2 mmol) were mixed in CHCl3 (20 mL) at 50 °C. 2,3,4,6-Tetra-
O-acetyl-R-D-glucopyranosyl bromide (1.8 g, 4.4 mmol, 1 equiv/OH)
was added to the suspension followed by 0.15 M aqueous K2CO3
(100 mL, 15 mmol) and left to react during 36 h. After cooling, the
reaction mixture was filtered and the solid washed with dichloro-
methane. The filtrate was concentrated, alkalinized by adding aqueous
sodium bicarbonate solution, and extracted with dichloromethane (3 ꢁ
30 mL). The organic phases were collected, dried over MgSO4 and the
solvent was removed under reduced pressure to afford compound 11
(1.2 g). Compound 11 was treated with 0.5 M sodium methoxide
(35 mL, 17.5 mmol, 2 mol/glucose) in 220 mL of methanol (extensively
dried over activated 4 Å molecular sieves) and left stirring at room
temperature overnight. The reaction solution was further purified by
solid phase extraction (silica flash cartidges GraceResolv). The acet-
oneꢀmethanol (80:20) fractions were collected and the solvent was
evaporated under reduced pressure to afford a brown solid correspond-
ing to compound 12 (0.61 g, 1.1 mmol, 50%). IR (KBr) υmax
:
1
Biological Activity. Clotting Assays. Human blood was ob-
tained from 10 healthy donors 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. Compounds 6, 7, 10, 12,
and 13 were dissolved in water, and compound 5 was dissolved in water
with 10% DMSO. The final concentration of sulfated compounds (6, 7,
3600ꢀ3400, 1625, 1440, 1263, 1065, 595, 489. H NMR (DMSO-d6,
500.13 MHz) δ: 8.10 (2H, d, J = 8.8 Hz, H-1, H-8), 7.22 (2H, d, J = 2.2
Hz, H-4, H-5), 7.11 (2H, dd, J = 8.8 and 2.2 Hz, H-2, H-7), 5.51 (1H, brs,
30-OH), 5.26 (1H, brs, 40-OH), 5.18 (1H, d, J = 8.8 Hz, H-10), 4.71 (1H,
brs, 60-OH), 3.69 (1H, m, H-60a), 3.50 (1H, under water, H-60b), 3.46
(1H, under water, H-50), 3.35 (1H, t, J = 8.8 Hz, H-30), 3.30 (1H, t, J =
8.3 Hz, H-20), 3.20 (1H, t, J = 8.8 Hz, H-40). 13C NMR (DMSO-d6, 75.47
MHz) δ: 174.3 (C-9), 162.5 (C-3, C-6), 157.4 (C-4a, C-10a), 127.6 (C-
1, C-8), 115.8 (C-8a, C-9a), 114.6 (C-2, C-7), 103.1 (C-4, C-5), 99.8 (C-
10), 77.2 (C-50), 76.4 (C-30), 73.2 (C-20), 69.6 (C-40), 60.7 (C-60).
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10, and 13) in these assays ranged from 5.00 ꢁ 10ꢀ3 to 5.00 ꢁ 10ꢀ5 M.
Only a single concentration was tested for nonsulfated compounds 5and 12
and sodium sulfate solution (5.00 ꢁ 10ꢀ3 M). Heparin (0.05ꢀ5 UI/mL)
was used as positive control. In the control group, water (control
for compounds 6, 7, 10, 12, and 13) or 10% DMSO (control for
compound 5) was used. APTT, PT, and TT tests were performed using
an ACL100 coagulometer (IZASA, Portugal). The following commer-
cial kits were used: no. 0020006800 (HemosIL, SynthASil, IZASA,
Portugal) for the APTT, no. 0008469810 (HemosIL, PT-fibrinogen,
IZASA, Portugal) for the PT, and no. 0009758515 (HemosIL, throm-
bin time, IZASA, Portugal) for the TT. The assays were carried out as
previously reported.13 Each measurement was performed in duplicate
and repeated three times on different days (n = 6). Coagulation 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 concentrationꢀresponse curve. For clotting time values
greater than 165 s (PT and TT) and 110 s (APTT), values of 165 and
110 s, respectively, were arbitrarily assigned for statistical analysis.
Chromogenic Substrate Hydrolysis Assays. Human FXa
(27 nkat/mL) and human thrombin (137 NIH/mL) were obtained from
Diagnostica Stago (Roche, Portugal). Bovine ATIII was obtained from
Sigma-Aldrich (Germany) (50 U/mL). The chromogenic substrates
used were Chromozym TH (tosyl-Gly-Pro-Arg-p-nitroaniline (pNA),
Roche Applied Science) for thrombin and CBS 31.39 (CH3SO2-D-Leu-
Gly-Arg-pNA, AcOH; Diagnostica Stago) for FXa. Stock solutions of the
enzymes were obtained according to the manufacturers' 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, containing 227 mM NaCl
and 0.1% (w/v) bovine serum albumin (BSA, Sigma-Aldrich) instead of
PEG-8000. The final enzyme concentrations were 0.5, 0.06, and 1 U/mL
for FXa, thrombin, and ATIII, respectively. The final substrate concen-
trations inthe reactions were 178 and125 μM (in water) forChromozym
TH and CBS31.39, respectively. For the IC50 determination of CBS
31.39 hydrolysis by FXa, variousconcentrations of the inhibitors 7 and 13
clotting time (PT) was determined after mixing the plasma with factor II
deficient plasma. High and low calibration curves were prepared with
calibrated plasma using dilutions automatically performed by the instru-
ment (ACL100 coagulometer, IZASA, Portugal). Linearity range was
1.56ꢀ6.25% (r2 = 0.999) for low calibration curve, and linearity range was
25ꢀ100% (r2 = 0.999) for high calibration curve. Normal plasma (STA
PreciClot Plus I, Diagnostica Stago, Roche, Portugal) and pathological
plasma (STA PreciClot Plus II, Diagnostica Stago, Roche, Portugal) were
used for checking the accuracy and reproducibility of results.
Thromboelastography. Blood samples from healthy donors were
collected into siliconized Vacutainer tubes containing 3.8% trisodium
citrate such that a ratio of citrate/whole blood of 1:9 (v/v) was maintained.
Sulfated compounds were dissolved in saline and diluted in the whole
blood (1:16) to a final concentration of (3.12ꢀ6.25) ꢁ 10ꢀ4 M. In the
control group saline was added to the whole blood (1:16). The TEG assay
was performed as previously described13 using a thromboelastograph
coagulation analyzer Rotem delta (Pentapharm GmbH, Germany). All
disposable supplies were purchased from Pentapharm GmbH.
In Vivo Studies. 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 Pur-
poses, 1986, and Protocol of Amendment to the European Convention
for the Protection of Vertebrate Animals Used for Experimental and
Other Scientific Purposes, 1998) and as previously described.13
Platelet Function Closure Times. Blood samples from healthy
donors were collected into tubes containing hirudin at a final concen-
tration of 25 μg/mL and analyzed within 0.5ꢀ1 h after collection. In
vitro platelet function was evaluated using a PFA-100 device (Dade
Behring, Frankfurt, Germany) as previously reported.13
Multiplate Electrical Impedance Aggregometry. Blood sam-
ples from healthy donors were collected into tubes containing hirudin at
a final concentration of 25 μg/mL and analyzed within 0.5ꢀ1 h after
collection. Stock solutions of compounds 7, 12, 13, ASA, and sodium
sulfate were prepared in saline and nonsulfated parent compound 5 in
DMSO (the final concentration of DMSO was 0.5% to eliminate the
effect of the solvent on the aggregation). Tested solutions (125 μL,
saline) were added to whole blood (1875 μL) to a final concentration of
6.25 ꢁ 10ꢀ4 M. The assays were carried out according to the respective
instructions of the manufacturer (multiplate, Dynabyte, Munich,
Germany): An amount of 300 μL of whole blood at room temperature
was added to 300 μL of preheated saline at 37 °C. After an incubation
time of 180 s, 20 μL of the selected agonist solution (Dynabyte, Munich,
Germany) was added, giving final concentrations of thrombin receptor-
activating peptide (TRAP-6) at 32 μM (TRAP test), arachidonic acid at
0.5 mM (ASPI test), collagen at 3.2 μg/mL (Col test), ADP at 6.5 μM
(ADP test), or prostaglandin E1 at 9.4 nM (ADP-HS test). The
instrument continuously measures the change in resistance during 6
min, which is proportional to the amount of platelets adhering to the
electrodes and transforms it to arbitrary “aggregation units” (AU). These
are plotted against time (min) and give the area under the curve (AUC)
in U (1 U = 10 AU ꢁ min), calculated from the mean values of the two
curves. The analysis was accepted when the difference between the two
curves is <20%.
were investigated (1.00 ꢁ 10ꢀ4, 2.00 ꢁ 10ꢀ4, 4.00 ꢁ 10ꢀ4, 8.00 ꢁ 10ꢀ4
,
and 1.00 ꢁ 10ꢀ3 M). For the kinetics studies, various concentrations of
the sulfated inhibitors (5.00 ꢁ 10ꢀ4 to 1.00 ꢁ 10ꢀ3 M) and CBS 31.39
(1.25 ꢁ 10ꢀ4 and 2.50 ꢁ 10ꢀ4 M) were used. Under the experimental
conditions, less than 10% of the substrate was consumed in all assays
[ε405nm(p-nitroaniline) = 10.4 mmolꢀ1 L cmꢀ1]. The chromogenic
3
3
assays were carried out at 37 °C in 96-well plates on a microplate reader
(BioTeck Instruments, VT, U.S.) as previous reported.13 Test com-
pounds were diluted in water. Reference inhibitors rivaroxaban (direct
FXa inhibitor), PPACK (direct thrombin inhibitor, D-Phe-L-Pro-L-Arg-
CMK), and LMWH (ATIII/FXa activator) were assayed in the same
conditions.
Activated Protein C Levels. APC was measured after addition of
compound solution (30 μL, saline) to citrated human plasma (300 μL)
using a chromogenic assay (STA protein C chromogen 6007935,
Diagnostica Stago, Roche, Portugal). Protein C was activated by a
specific activator derived from the venom of Agkistrodon c. contortrix
(100 μL), and the quantity of enzyme thus formed was measured by its
amidasic activity on the synthetic substrate CBS 42.46 (200 μL).
Calibration curve was obtained with a linearity range 3ꢀ150% (% C =
546.5365 DO/min ꢀ 3.1556; r2 = 1.000) with STA Unicalibrator
(608035). Normal plasma (STA PreciClot Plus I, Diagnostica Stago,
Roche, Portugal), and pathological plasma (STA PreciClot Plus II,
Diagnostica Stago, Roche, Portugal) were used for checking the accuracy
and reproducibility of results.
Statistics. The statistical significance of the difference between
control and treated samples was calculated by the nonparametric two-
tailed MannꢀWhitney test using GraphPad Prism 5 software. P < 0.05
was considered significant.
’ ASSOCIATED CONTENT
S
Supporting Information. Structural characterization and
Prothrombin Factor Levels. After the addition of compound
solution or water (50 μL) citrated pooled human plasma (100 μL) was
diluted with factor diluent solution (350 μL, IZASA, Portugal). The
b
FXa doseꢀresponse progress curves. This material is available
free of charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
(11) Gandhi, N. S.; Mancera, R. L. Heparin/heparan sulphate-based
drugs. Drug Discovery Today 2010, 15, 1058–1069.
(12) Avci, F. Y.; Karst, N. A.; Linhardt, R. J. Synthetic oligosacchar-
ides as heparin-mimetics displaying anticoagulant properties. Curr.
Pharm. Des. 2003, 9, 2323–2335.
Corresponding Author
*Phone: +351 222078916. Fax: +351 222003977. E-mail: madalena@
ff.up.pt.
(13) Correia-da-Silva, M.; Sousa, E.; Duarte, B.; Marques, F.;
Carvalho, F.; Cunha-Ribeiro, L. M.; Pinto, M. M. M. Flavonoids with
an oligo-polysulfated moiety: a new class of anticoagulant agents. J. Med.
Chem. 2011, 54, 95–106.
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of plant flavonoids on mammalian cells: implications for inflammation,
heart disease, and cancer. Pharmacol. Rev. 2000, 52, 673–751.
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recent developments. Curr. Med. Chem. 2005, 12, 2413–2446.
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overview. Curr. Med. Chem. 2005, 12, 2447–2479.
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derivatives: new insights in biological activities. Curr. Med. Chem. 2005,
12, 2517–2538.
(18) Jiang, D. J.; Dai, Z.; Li, Y. J. Pharmacological effects of
xanthones as cardiovascular protective agents. Cardiovasc. Drug Rev.
2004, 22, 91–102.
’ ACKNOWLEDGMENT
We thank Fundac~ao para a Ci^encia e a Tecnologia (FCT),
)
I&D 4040/2007, FEDER, POCI, FSE, and POPH from QREN
for financial support and for the Ph.D. grant to M.C.-d.-S. (Grant
SFRH/BD/22962/2005). We thank Dr. Bꢀarbara Ribeiro and
IZASA, Portugal, for all the facilities and helpful support in the
multiplate; the technicians of the Coagulation Laboratory from
the Servico de ImunoHemoterapia, Hospital de S. Jo~ao, for
)
technical support; Dr. Elisangela Costa for providing 3,6-dis-
ulfate xanthone; Sara Cravo for microwave assistance; and Bayer
HealthCare for providing rivaroxaban.
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ATIII, antithrombin III;FXa, factor Xa; PFA, platelet function
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