European Journal of Medicinal Chemistry 234 (2022) 114256

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Chemical synthesis and pharmacological properties of heparin

pentasaccharide analogues
Zhipeng Zhou a, 1, Linlin Zhang b, 1, Xin Wu a, c, Lan Luo a, Jian Wu b, Dan Xu b, **,
Mingyi Wu a, c, *
a
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201,
China
b
Nanjing Chia Tai Tianqing Pharmaceutical Co., Ltd, Nanjing, 210046, China
c
University of Chinese Academy of Sciences, Beijing, 100049, China

a r t i c l e i n f o a b s t r a c t

Article history: The pentasaccharide fondaparinux is a synthetic anticoagulant based on heparin antithrombin-binding
Received 30 January 2022 sequence. Fondaparinux improves safety and predictable pharmacodynamics compared with heparins;
Received in revised form however, it requires a complicate synthesis process which contain more than 50 steps of synthesis.
2 March 2022
Herein, we designed and synthesized four fondaparinux analogues (compounds 1, 2, 3, 4) using a [2þ3]
Accepted 3 March 2022
convergent synthetic method, which greatly simplified the synthetic process, improved the product
Available online 5 March 2022
yield, and curtailed the expenditures. These synthesized compounds showed stronger anticoagulant
activities by factor Xa inhibition (IC50 725e1126 nM vs. 1909 nM for fondaparinux) in the AT-dependent
Keywords:
Heparin
manner. After subcutaneous (s.c.) administration to rats, the compounds displayed long-lasting anti-
Pentasaccharide factor Xa activities and inhibition of thrombin generation ex vivo. Compared with fondaparinux, these
Anticoagulant compounds were slowly eliminated after s.c. administration to rats, the half-lies (t1/2) were more than 2-
Structure-activity relationships fold of that of fondaparinux. These results suggested the pentasaccharide analogues may exhibit better
Synthesis pharmacokinetic and predictable pharmacodynamic characteristics.
© 2022 Elsevier Masson SAS. All rights reserved.

1. Introduction therapy of venous thromboembolism (VTE) and the prevention of

Thrombosis is the leading cause of death worldwide, being
responsible for one in four deaths [1]. The global burden is likely to
increase with the ageing population because thrombosis risk in-
creases with age. Anticoagulants are used to treat a wide variety of
conditions that involve arterial or venous thrombosis, including
prevention of venous thromboembolism and long-term prevention
of ischaemic stroke in patients with atrial fibrillation. There are four
crucial classes of anticoagulants applied to the prevention and
Abbreviations: APTT, activated partial thromboplastin time; AT, antithrombin;
Fpx, fondaparinux sodium; FX, coagulation factor X; HCII, heparin cofactor II;
LMWH, low-molecular-weight heparin; PT, prothrombin time; TT, thrombin time.
* Corresponding author. State Key Laboratory of Phytochemistry and Plant Re-
sources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, China.
** Corresponding author.
E-mail addresses: xiaoxux@gmail.com (D. Xu), wumingyi@mail.kib.ac.cn
(M. Wu).
1
These authors contributed equally to this work.
stroke in patients with atrial fibrillation, namely heparins (paren-
teral), vitamin K antagonists (VKAs; oral), direct thrombin in-
hibitors (DTIs; parenteral and oral) and direct factor Xa (FXa)
inhibitors (oral) [2,3].
The first anticoagulant, heparin (also referred to as unfractio-
nated heparin (UFH)), has been a successful anticoagulant for over
80 years and is on the WHO list of essential drugs [4]. Despite the
availability of other anticoagulants, it remains a major therapeutic
choice especially in surgical interventions. Unfractionated heparin
binds to the protein antithrombin (AT) and markedly increases the
ability of this protein to inhibit factor Xa and thrombin [2e5],
which is currently used for cardiovascular surgery and for the
prevention of venous thromboembolism. Fractionated heparin, in
the form of low-molecular-weight heparins (LMWHs), is intro-
duced to improve the pharmacodynamics. These molecules also
target both factor Xa and thrombin, but their administration results
in a lower incidence of bleeding than does unfractionated heparin
(1.4% for LMWHs versus 2.3% for UFH) [6]. Heparin suffers from
several noteworthy limitations, including heparin-induced

https://doi.org/10.1016/j.ejmech.2022.114256
0223-5234/© 2022 Elsevier Masson SAS. All rights reserved.
Z. Zhou, L. Zhang, X. Wu et al.
thrombocytopenia (HIT), a limb-threatening and life-threatening
severe adverse event in which antibodies form against complexes
of heparin and platelet factor 4 and induce platelet activation and a
prothrombotic state [7,8]. Fortunately, the incidence of heparin-
induced thrombocytopenia is reduced when low molecular-
weight heparins are used [9].
Synthetic pentasaccharides, such as fondaparinux, have been
designed with a structure based on the antithrombin-binding
sequence of heparin [4,5]. Fondaparinux with the sequence D-
GlcNS6S-a-(1,4)-D-GlcA-b-(1,4)-D-GlcNS3,6S-a-(1,4)-L-IdoA2S-a-
(1,4)-D-GlcNS6S-OMe (DEFGH) was identified as the AT-binding
sequence and later has been commercialized since 2002 with the
trade name “Arixtra” (Fig. 1) [10]. Fondaparinux is shown to have
more predictable pharmacodynamics, non-existent risk of HIT, and
better biosafety like those of LMWH [5,8,10,11]. Total synthesis of
fondaparinux has allowed to eliminate inherent limitations of
animal-sourced heparin and LMWHs, and avoided the contami-
nation in naturally occurring heparins that caused several deaths in
2008 [12].
Chemical synthesis of fondaparinux, the most successful
example to date for preparing a synthetic heparin [10,13], entails
more than 50 steps with an overall yield of ~0.1% [14]; as such,
fondaparinux is the most expensive drug among heparins. In
particular, the total synthesis of fondaparinux is very challenging
Fig. 1. Structures and pharmacological properties of fondaparinux, idraparinux and compounds 1e4.
2
European Journal of Medicinal Chemistry 234 (2022) 114256
because of the difficulty in regio- and stereo-selective glycosidic
bonds among the glucosamine, glucuronic acid, and iduronic acid
building blocks, and the strategic installation of OSO 3 and NHSO3

groups [15]. Glucosaminylation mainly relies on the anomeric ef-
fect, which is insufficient in curbing production of the unwanted b
isomer. The 1,2-trans glycosylation involving the uronic acid pre-
cursors, although feasible through neighboring group participation,
is confounded by the different C2 functionalizations in the final
product. So far, in order to ameliorate the synthetic efficiency, main
efforts have been made to synthesize heparin-like oligosaccharides
chemically or enzymatically by several groups, including Boons
[16], Huang [17], Hung [18,19], Linhardt [20], Liu [21], Petitou
[14,22], Qin [23], Seeberger [24,25], and Wong [15,26], but the
procedures still encounter problems such as a long stepwise pro-
cess, non-stereoselective glycosylation and low yield and efficiency.
Further researches led to develop a non-glycosaminoglycan
analogue, idraparinux, a synthetic pentasaccharide analogue of
fondaparinux, in which the N-sulfates are replaced by O-sulfates
and the hydroxyl groups are methylated [27e29] (Fig. 1). Although
idraparinux is considerably easier to synthesize than fondaparinux,
it still poses some challenges such as the synthesis of the L-idosyl,
the stereoselectivity of glycosylation between D and E, and the
stereoselectivity of glycosylation between G and H. Since its first
synthesis, several research groups have developed alternate
Z. Zhou, L. Zhang, X. Wu et al.
synthetic routes to idraparinux [27,30e33]. For example, Yu et al.
[27] used DEGþGH coupling strategy in total 51 steps from D-
glucose and methyl D-glucopyranoside. Borba
s et al. used DEþFGH
([2 þ 3]) coupling strategy in 39 steps [30,31] and the L-idose-
containing GH fragment was obtained by a short and straightfor-
ward synthesis whereby a 4,6-cyclic-acetal-protected L-idosyl thi-
oglycoside bearing a C2-nonparticipating group was used as the a-
selective glycosyl donor [33].
Idraparinux binds to antithrombin significantly stronger than
fondaparinux through the additional interaction of the extra sulfate
group at C3 of the H glucose unit as well as through hydrophobic
interactions [34,35]. Due to the increased sulfation, this agent ex-
hibits a 30-fold higher binding affinity to AT than fondaparinux,
and a higher anti-factor Xa potency. Idraparinux exhibits a signifi-
cantly longer elimination half-life of about 120 h in clinical trial,
which allows for once-weekly dosing [35]. Unfortunately, idrapar-
inux did not obtain FDA regulatory approval due to significant
bleeding complications in the clinical trial III [36].
Approaches to establish structure-activity relationships
applying modified heparin-like oligosaccharides have been proved
feasible [5,10,37]. Accordingly, continuous research may lead to
develop more potent pentasaccharide derivatives. According to the
synthesis methods of non-glycosaminoglycan analogues [27e33],
we would expect to obtain some new pentasaccharide analogues
via changing degree of methylation, which might exhibit better
pharmacological properties mostly including proper half-life and
then less bleeding risk. In this work, we adopted a distinctive
[2 þ 3] convergent method to chemically synthesize four fully O-
sulfated, partially O-methylated or O-acetylated analogues (1, 2, 3,
4). Their anticoagulant activities were evaluated by coagulation
time assays, calibrated automated thrombogram (CAT) and
thrombin generation test (TGT). AT-binding affinity and anti-FXa
activity were detected to confirm the action mechanism. Further-
more, their pharmacokinetics and pharmacodynamics were also
investigated in rats. Our results indicated that the pure synthetic
pentasaccharide 3 may be not only much easier to synthesize than
fondaparinux, but also displays excellent anti-FXa activity, a longer
duration of action and lower risk of bleeding.
2. Results
2.1. Synthesis procedure for compounds 1-4
To provide an efficient and concise access to these new heparin
pentasaccharide analogues, we conceived a [2 þ 3] convergent
synthetic route through the building blocks 7, 8, 10 and 11 (Scheme
1), all of which are readily attainable from commercially available
starting materials. The approach followed the formation of the
disaccharide donors 5a, 5b, 5c, 5d and their subsequent coupling
with the reducing end trisaccharide acceptor 6 to generate the fully
protected precursors of the target compounds 1e4 (Fig. 1).
The monosaccharide 11 was synthesized using methyl a-D-glu-
copyranoside as the starting material (Scheme S1). Donor 7a was
derived from compound 11 by the methylation of 5-OH, selective
removal of the methyl group in 1-OH, and reacted with CCl3CN and
DBU, in 59% yield (Scheme S1). The synthesis of donor 7b was also
derived from compound 11 by selective demethylation, acetylation,
and selective deacetylation, etc., in 27% yield (Scheme S2). Donor 7c
was stemmed from 1,2:5,6-di-O-isopropylidene-a-D-glucofuranose
after eleven reactions, in 37% yield. Donor 7d was obtained from
methyl a-D-glucopyranoside with seven steps, in 40% yield.
Acceptor 8 was derived from 22 through five steps with the yield of
71%. According to the strategy of Chen and Yu [27], the L-idopyr-
anose donor 10 was synthesized by the epimerization of 5-OH in a-
3
European Journal of Medicinal Chemistry 234 (2022) 114256
D-glucofuranose via intramolecular epoxide formation, in 38% yield.
The glycosylation between donor 10 and acceptor 11 produced
the protected disaccharide 9, in 46% yield. The yield (46%) is not
good enough because part of donor 10 quickly turned to other
impurities under this condition, which might also be further opti-
mized. We suggested that 1,2-trans a-selective glycosylation may
be due to the formation of 1, 2-cis-nitrilium ions with low steric
resistance from donor 10 to acetonitrile at low temperatures.
Demeter et al. applied a 4,6-acetal-protected L-idose donor without
a C2 participating group to ensure 1,2-trans a-selective glycosyla-
tion [33]. Subsequently, the trisaccharide acceptor 6 (F-G-H) was
synthesized by the glycosylation between 4-OH-glucopyranoside
derivative 9 and donor 7b. Additionally, the disaccharide donor 5
(D-E) was produced by linking donor 7 and acceptor 8, beyond 80%
yield. When TMSOTf was used as a catalyst at low temperature,
donor 7 without neighboring group participation was firstly con-
verted into oxonium ion, and then oxonium ion was attacked by
receptor 8 under the influence of dichloromethane which tended to
form a -D-glycoside [27]. Finally, the target product b-linked pen-
tasaccharide analogues 1e4 were formed by the glycosylation
[2 þ 3] between the trisaccharide 6 and the disaccharide 5 (Scheme
2).
Differing from the traditional synthesis of idraparinux, in this
work, the carboxyl and hydroxyl groups were protected by benzyl
groups, which were stable and susceptible to be removed. As a
result, the conventional two pressurized hydrogenation steps
were simplified to one. And, due to the strong neighboring group
participation of the acetyl group at C2 of E ring, almost no a-iso-
mer was produced during the [D-E] þ [F-G-H] glycosylation, thus
optimizing the impurity control which is very important for in-
dustrial production. When the protected disaccharide 9 were
produced, trichloroacetyl imide lipides were used as a protective
base for C-1 of donor 10, avoiding the use of highly toxic ethyl
mercaptan and very expensive silver trifluoroacetate. In G ring
synthesis, p-methoxy phenyl ether was used to protect the C-1
site, making compounds 46e49 easy to crystallize. By the [2 þ 3]
route, we were also able to avoid the secondary sulfonation and
the mass impurities produced by the de-methyl ester in strong
base.
In the preparation of a-glycosylation between D ring and E ring,
b-isomer is an accompanied by-product (2%e20%), resulting in low
yield of D-E building block. In the conventional [3 þ 2] route [27],
the preparation of D-E-F building block further reduces the yield of
final product, it's also very time-consuming and high-cost. As for
the [1 þ 4] strategy [28,29], it's more complex to synthesize the E-F-
G-H building block, as well as thornier to remove the higher level of
isomer impurities. Additionally, the raw material of iduronic acid
residue (G ring) is derived by conformational transform of other
saccharide units, which is expensive, and its stereo-selective link-
age with other building block can result in further loss [18]. Borba
s
et al. used a [2 þ 3] block synthesis utilizing a 6-O-silyl-protected L-
idose-containing trisaccharide acceptor. The unique strategy
involved triple methylation followed by oxidation of the glucose
and the idose precursors into D-glucuronic and L-iduronic acids in
one step, and provided the target pentasaccharide through a 39-
step synthesis starting from D-glucose and methyl a-D-glucopyr-
anoside [31]. In this work, four new pentasaccharides were syn-
thesized in overall 37 steps from commercially available starting
materials and in 1.2% overall yield over 20 linear steps from methyl
a-D-glucopyranoside (building block D). The [2 þ 3] convergent
synthesis strategy showed excellent stereoselectivity in all glyco-
sylation steps, while avoiding rigorous reaction conditions and
expensive reagents, which could largely benefit the large-scale
industrial production.
Z. Zhou, L. Zhang, X. Wu et al.
Scheme 1. Retrosynthetic analysis of the heparin pentasaccharide analogues 1e4. Ac ¼ acetyl, Bn ¼ benzyl, Ph ¼ benzylidene.
2.2. Anticoagulant activities of the pentasaccharide compounds
in vitro
The anticoagulant activities of synthetic pentasaccharide com-
pounds 1e4 and fondaparinux (Fpx) were evaluated by plasma
clotting assays. These include using activated partial thrombo-
plastin time (APTT), prothrombin time (PT) and thrombin time (TT)
of plasma clotting assays. Such assays are routinely used to deter-
mine the ability of products to inhibit blood clotting through the
intrinsic, extrinsic and common pathways of the coagulation
cascade, respectively [38e40]. The concentrations of these com-
pounds 1e4 needed to double APTT (EC2.0) were 32.47e48.78 mM,
which were slightly lower than that of Fpx (EC2.0, 51.20 mM) while
significantly higher than LMWH (EC2.0, 2.19 mM) (Fig. 2A, Table 1),
indicating that these compounds have potent intrinsic anticoagu-
lant activities. As for PT and TT, no significant influences were
observed in 1e4 and Fpx at their concentrations up to 128 mg/mL
compared with LMWH, which mean that they have no or little ef-
fect on the extrinsic and common coagulation pathways [10,40].
The AT-dependent anti-FXa activities of compounds 1e4 and
their AT binding affinities were evaluated by competitive binding
4
European Journal of Medicinal Chemistry 234 (2022) 114256
assays, and thrombin generation (TG) inhibition activities were also
tested to further study the action mechanism (Fig. 2 and Table 1).
Compounds 1e4 showed potent FXa inhibitory activity in the
presence of AT, with IC50 values of 88.8e120.6 nM, which was
comparable with that of Fpx (IC50, 99.6 nM) (Fig. 2B, Table 1).
Compounds 1e4 could strongly inhibit the binding of FXa to the
immobilized heparin (IC50 values of 725e1126 nM), slightly lower
than that of Fpx (IC50, 1909 nM) (Fig. 2C, Table 1). Additionally,
compounds 1e4 strongly inhibited thrombin generation (TG) in
human plasma in a dose-dependent manner with IC50 values of
11e19 mM (Table 1 and Fig. 2D, Fig. S44), whereas Fpx also inhibited
TG (IC50, 14 mM) as previously described for this compound
[22,34,41]. Taken together, compounds 2e4 exhibited slightly
stronger anticoagulant activity than Fpx.
2.3. Pharmacokinetics and pharmacodynamics of the
pentasaccharide compounds in vivo
After administrating subcutaneously to rates, the plasma con-
centrations of compounds 1e4 and Fpx were determined based on
standard curves between the plasma drug concentration and anti-
Z. Zhou, L. Zhang, X. Wu et al.
Scheme 2. Synthesis of compounds 1e4. Reagents and conditions: a) dichloro-
methane, TMSOTf; b) methanol, palladium carbon; c) N, N-dimethylformamide, sulfur
trioxide trimethylamine complex; d) sodium hydroxide, ammonium acetate. Detailed
reaction conditions are described in Experimental Section.
FXa activity (Fig. 3A), and the pharmacokinetics parameters were
calculated according to noncompartmental procedure model [42]
(Table 2). After administrating each compound at the dose of
100 nmol/kg, the maximum times (Tmax) to reach the maximum
drug concentration (Cmax) were 2.0 ± 0.45 h, 1.0 ± 0.27 h, 1.5 ± 0.29
and 2.0 ± 0.90 h, respectively, and their half-lives (t1/2) were
15.1 ± 1.14 h, 4.8 ± 1.11 h, 6.2 ± 1.10 h and 6.1 ± 1.11 h for compounds
1, 2, 3, and 4, respectively, which were more than 2-fold of that of
Fpx (1.2 ± 0.25 h) (Fig. 3A, Table 2). Moreover, other parameters of
compounds 1e4, including the Cmax and AUC0e32h (the area under
the time-drug concentration profiles within 32 h), displayed much
higher than those of Fpx, while the clearance rates of these com-
pounds were lower compared to Fpx. Above results indicated that
our four synthesized compounds may exhibit the stronger and/or
longer drug exposures than fondaparinux mostly because these
compounds were partially O-methylated or O-acetylated analogues
of Fpx.
The antithrombotic effects of 1e4 (100 nmol/kg) at different
time points were evaluated using the rat model of deep venous
thrombosis induced by stasis and thromboplastin [39]. The results
showed that after 1 h subcutaneously administration, the throm-
bosis inhibition rate of compound 4 was 77.78 ± 8.55%, indicating
the best antithrombotic activity than others compounds, as well as
5
European Journal of Medicinal Chemistry 234 (2022) 114256
compound 3 was comparable to Fpx (56.94 ± 8.12% for 3 vs.
55.66 ± 6.23% for fondaparinux) (Fig. 3B). Notably, compounds 2, 4
and Fpx showed the strongest antithrombotic activity at 1 h, while
1 and 3 were the most potent after injection 3 h. Moreover, at the
same dose, all the four oligosaccharides exhibited stronger
thrombosis inhibitions than Fpx after administration 3 h, consistent
with the results of pharmacokinetics (Fig. 3A). The results of
pharmacodynamics of oligosaccharides showed the linear kinetics,
suggesting the predictable pharmacodynamic characteristics.
2.4. Antithrombotic activity and hemorrhagic risk of the
pentasaccharide compounds in vivo
The dose-antithrombotic activity relationships of compounds
1e4 were further examined using the rat model of deep venous
thrombosis at three doses of 30, 100, 300 nmol/kg after 2h subcu-
taneously administration. The results indicated that all compounds
displayed potent inhibitions of thrombosis the same as Fpx in the
dose-dependent manner (Fig. 4A). At 30 nmol/kg, compound 3
exhibited the highest thrombosis inhibitory activity (inhibition rate
48.58 ± 11.5%), 1 and 2 also showed markedly antithrombotic activity
(33.19 ± 6.52% and 39.48 ± 13.69), whereas 4 and Fpx showed lower
inhibitory activity (less than 15%). At the doses of 100 and 300 nmol/
kg, the four oligosaccharides all showed stronger antithrombotic
activity than Fpx (p < 0.01). Particularly, compound 3 and 4 at
100 nmol/kg had the thrombosis inhibition rates of more than 70%,
moreover at 300 nmol/kg they inhibited thrombosis by
89.54% ± 2.4% and 93.12% ± 1.23%, respectively, whereas the inhi-
bition rate of Fpx was 59.31% ± 6.29%. These results suggested that
our synthesized oligosaccharides exhibited stronger antithrombotic
activities after administration subcutaneously into rats in vivo.
Bleeding risk has been the main problem in the application of
anticoagulants [2,3]. Herein, the bleeding risk of the synthetic
compounds was evaluated using mice tail-cut model [39,42]. Mice
were treated with compounds 1, 2, 3 and 4 at approximately 10
times antithrombosis dose (2000 nmol/kg) (Fig. 4B). The blood loss
of negative control (normal saline) group was 33.7 ± 8.2 mL. In
contrast to the negative control and Fpx treated group, both LMWH
and compound 1 significantly increased the blood loss (P < 0.05).
Compounds 2e4 slightly increased the blood loss, but displayed no
statistical significance (P > 0.05) due to their larger individual
variations. As shown in pharmacokinetic results (Fig. 3A and
Table 2), Tmax for Fpx was 0.6 h, while the compounds were
1.0e2.0 h, which implied that these compounds may exhibit more
exposures than Fpx at 1.5 h in the blood of mice after subcutane-
ously administration. Nevertheless, risk of bleeding for compounds
2e4 are probably acceptable as anticoagulants compared with Fpx
and low molecular weight heparin in clinic. Further investigation
may be required for the hemorrhagic effect of these compounds,
such as more animal groups and detection at different time points
after administration.
3. Discussion and conclusions
Although many anticoagulants are available in clinic, their ap-
plications are still limited out of the safety concern, and great ef-
forts have been made in developing ideal anticoagulant drugs [43].
Fondaparinux has been widely used in treating venous thrombo-
embolic events, deep vein thrombosis, and acute coronary syn-
drome [44,45], etc. Fondaparinux is a highly selective FXa inhibitor
by binding to AT, and as a potent anticoagulant due to it exhibits
fewer side effects and more predictable pharmacodynamics than
heparin or LMWH in clinic [11,45]. However, the high cost of fon-
daparinux treatment results from the high-cost and time-
consuming manufacturing process, limiting its availability.
Z. Zhou, L. Zhang, X. Wu et al. European Journal of Medicinal Chemistry 234 (2022) 114256

A 100 B
Fpx 100
90 1
2

FXa activity (% of control)

3 Fpx
80 4 75 1
2
APTT (s)

70 3
50
4

60

50 25

40
0
0 20 40 60 80 100 120 140 0.1 1 10 100 1000
Concentration ( g/mL) Concentration ( g/L)

C 1.2 D 350 Compound 3

AT-heparin binding (% of control)

300
1.0

250
0.8 Fpx
1 Thrombin (nM)
2 200
0.6 3
4 150

0.4
100

0.2
50

0.0 0
10-1 100 101 102 103 104 105 0 10 20 30 40 50
Concentration (nM) Time (min)

Fig. 2. Anticoagulant activities of Fpx and compounds 1e4. Effects of Fpx and compounds 1e4 on human plasma APTT (A), AT-heparin binding (B), FXa activity in the presence of AT
(C), and inhibition of thrombin generation in human plasma (D). Experiments were repeated triplicate, data were expressed as mean ± SD in A-C, and the representative graph was
shown in D. Fpx, fondaparinux; APTT, activated partial thromboplastin time; AT, antithrombin; FXa, activated coagulation factor X.

Table 1
In vitro anticoagulant activities and effects of compounds 1e4 on coagulation factors.

Compd. APTT (EC2.0, mM)a PT (mg/mL)b TT (mg/mL)b Anti-FXa activity (IC50, nM)c Competitive AT-binding (IC50, nM)c TG inhibition (IC50, mM)

1 32.47 >128 >128 88.8 ± 7.00 1126.0 ± 79.18 14.82 ± 1.59

2 33.05 >128 >128 94.7 ± 9.34 778.7 ± 64.71 12.39 ± 0.50
3 48.78 >128 >128 108.1 ± 7.10 787.9 ± 68.30 12.53 ± 0.58
4 40.83 >128 >128 141.08 ± 10.2 724.7 ± 53.34 12.17 ± 1.71
LMWH 2.19 28.4a 1.12a 26.6d >4000 NDe
Fpx 51.20 >128 >128 99.6 ± 10.80 1909.5 ± 290.32 14.51 ± 0.62
a
EC2.0, the activity of agents to prolong APTT, PT or TT is expressed by the concentration of each agent (mM) that is required to double the APTT, PT or TT.
b
No significant influences of compounds on PT or TT were observed at concentration as high as 128 mg/mL.
c
IC50 value, the concentration of each agent required to inhibit 50% of protease activity.
d
Data from ref. 39.
e
ND, not determined.

Idraparinux, a synthetic pentasaccharide analogue of fonda- and less bleeding risk, a distinctive [2 þ 3] convergent synthetic
parinux, binds to antithrombin significantly stronger than fonda- strategy was reported to synthesize idraparinux analogues (com-
parinux [34] and is synthesized in shorter steps relative to pounds 1e4). Compared with the conventional [1 þ 4] or [3 þ 2]
fondaparinux [30,31]. Due to the increased sulfation, this agent strategies [19,27,47], the synthetic process was remarkably
exhibits a 30-fold higher binding affinity to AT than fondaparinux, simplified by [2 þ 3] route according to the synthetic process of
and a higher anti-factor Xa potency. Idraparinux exhibits a signifi- idraparinux [30,31]. Particularly, since the acetyl group at E-ring
cantly longer elimination half-life, which allows for once-weekly has a strong neighboring group participation on DEþFGH glucosi-
dosing [34,35]. But in phase III clinical the long-term (more than dation, by-products of isomer impurity were effectively reduced,
6 months) use of idraparinux also led to intracranial bleeding in and almost without alpha product. The expensive G-ring raw ma-
elderly patients and those with renal impairment [36]. terial was less required, and then the product yield increased, thus
In this study, in order to obtain non-glycosaminoglycan fonda- the same products can be obtained by this method in a cost-
parinux analogues as better anticoagulants with moderate half-life effective way. Previously, the structure-activity relationship study

6
Z. Zhou, L. Zhang, X. Wu et al. European Journal of Medicinal Chemistry 234 (2022) 114256

A B 100
Fpx Fpx
2400
1 1

Inhibition of thrombosis (%)

2 2
75 ***
3
Concentration ( g/L)
3
1800 4 4
**
**
**
50
**
1200
* **
25
600

*
0
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25
Time (h) Time (h)

Fig. 3. Pharmacokinetics and pharmacodynamics of the pentasaccharide compounds 1e4 and Fpx (s.c.) in rats. The plasma concentrations (A) and thrombosis inhibition rates (B) of
pentasaccharide compounds at indicated time points, after they were administered sc to rats at the doses of 100 nmol/kg. Data were expressed as mean ± SEM (n ¼ 6). *P < 0.05,
**P < 0.01, ***P < 0.01 vs. control group.

Table 2
Pharmacokinetic parameters of the synthetic pentasaccharide compounds in rats.

Compd. Pharmacokinetics, [Means ± SEM] (n ¼ 4 to 6).

Tmax (h) Cmax (mg/L) t1/2 (h) AUC(0-32) AUC(0-∞) Clearance (L/kg$h) Distribution volume (L/kg)
(h$mg/L) (h$mg/L)

1 2.0 ± 0.45 1141.3 ± 147.98 15.1 ± 1.14 12798.6 ± 2675.16 15057.8 ± 3665.62 0.017 ± 0.004 0.136 ± 0.024
2 1.0 ± 0.27 1247.1 ± 171.97 4.8 ± 1.11 6351.2 ± 1416.71 6754.4 ± 1428.73 0.038 ± 0.011 0.183 ± 0.047
3 1.5 ± 0.29 2016.5 ± 478.28 6.2 ± 1.10 14176.2 ± 4132.44 15536.1 ± 4890.33 0.021 ± 0.010 0.131 ± 0.031
4 2.0 ± 0.90 2041.0 ± 453.78 6.1 ± 1.11 17623.6 ± 4881.77 19053.2 ± 5467.44 0.031 ± 0.020 0.058 ± 0.005
Fpx 0.6 ± 0.12 519.2 ± 131.59 1.2 ± 0.25 1071.1 ± 315.58 1116.4 ± 324.78 0.239 ± 0.077 0.097 ± 0.055

A B *
600
100 *
***
Inhibition of thrombosis (%)

***
Blood loss ( L)

75 *** 450

*** ***
*** **
***
50 300
*** *
** **
** Fpx
25 1 150
2
3
0
4
0
0 50 100 150 200 250 300 Control Fpx LMWH 1 2 3 4
Dose (nmol/kg) Compounds

Fig. 4. Effects of the compounds 1e4 on venous thrombosis and hemostatic function. Dose-antithrombotic activity relationships (A) and bleeding effect (B) of compounds 1e4 after
administrated. (A) The thrombus weight was detected after 2 h administration (s.c.) of each compound to rats. Results are expressed as % thrombus weight (mean ± SEM, n ¼ 10,
**p < 0.01, ***p < 0.001 vs. saline), 100% representing absence of any thrombosis inhibition (thrombus weight in the absence of compound administration). (B) The blood loss was
determined after 1.5 h administration in mice tail-cut model. Data were expressed in mL of blood loss as Mean ± SEM, n  6; compared with the negative control group, *P < 0.05.

demonstrated that sulfate groups of the fondaparinux analogues Anticoagulant activities of synthetic pentasaccharide com-
are essential for biological activity [10,22], whereas the degrees of pounds were tested in contrast to fondaparinux in vitro (Fig. 2 and
methylation are correlateed with elimination half-life [28,37]. Table 1). The results indicated that these pentasaccharide ana-
Accordingly, our synthetic route introduced more sulfuric acid logues 1e4 showed high anti-FXa selectivity by binding to AT and
groups instead of amino groups and appropriate methylated with no obvious anti-thrombin activity, and exhibited APTT pro-
modification (Fig. 1), which enhanced the activities of synthetic longing activities without significant effect on PT and TT. Moreover,
compounds and extended the half-lives, while avoided the use of the synthetic analogues showed comparable anti-FXa activities and
explosive azide groups and further optimized the synthesis of slightly stronger thrombin generation inhibition. Since they exhibit
fondaparinux analogues. strong anticoagulation, including prolonging APTT, anti-FXa

7
Z. Zhou, L. Zhang, X. Wu et al.
activity by AT, binding to AT and TG inhibition, the abilities of these
oligosaccharides to inhibit thrombosis were closely related to their
anticoagulant activities.
Next, the pharmacokinetic results suggested that the fonda-
parinux analogues had longer half-life (t1/2), larger AUC and Cmax
than fondaparinux (Fig. 3A and Table 2). In contrast, fondaparinux
has a short Tmax and higher clearance [34], which consists with its
shorter half-life and AUC. The findings should result from methyl-
ation or acetylation of hydroxyl groups in compounds (Fig. 1),
which may increase their lipid solubility and prolong the metabolic
time in vivo. These pharmacokinetic properties also aligned with
their pharmacodynamics (Fig. 3B). At 3 h after subcutaneously
administration, compounds 1e4 showed stronger thrombosis in-
hibition than fondaparinux, and their inhibition rates could main-
tain high levels for 12 h. Compounds 1 and 3 showed the highest
antithrombotic activity at 3 h. All the compounds showed strong
thrombosis inhibition in rats in dose-dependent manner. At the
same dosage of fondaparinux as is used clinically into rats
(100 nmol/kg), the synthetic pentasaccharide compounds showed
much stronger antithrombotic effects than fondaparinux. The
reduced dose requirements and prolonged effective duration could
further benefit clinical application. In contrast, the half-lives for
compounds 2e4 in rats were much shorter than that for idrapar-
inux (4.8e6.2 h vs 14.7 h) [34], which may imply that their bleeding
risks might less than that of idraparinux. The results of mice tail-cut
model confirmed that risk of bleeding for compounds 2e4 were
low and acceptable as anticoagulants compared with fondaparinux
and low molecular weight heparin in clinic (Fig. 4B). Taken
together, the fondaparinux analogues may exhibit better pharma-
cokinetic and predictable pharmacodynamic characteristics.
In conclusion, the synthesized pentasaccharide compounds may
be potentially superior to fondaparinux and idraparinux, consid-
ering their lower cost, higher yield, stronger antithrombotic activ-
ity, longer half-life, and low bleeding risk. Thus, these compounds
(particularly 3 or 4) may be promising candidates in the treatment
and prevention of thrombotic diseases.
4. Experimental section
4.1. Materials
Fondaparinux sodium injection (Arixtra®, 0.5 mL: 5 mg/mL)
was offered by GSK (Britain). the thromboplastin time (APTT),
prothrombin time (PT), thrombin time (TT) reagents, and standard
human plasma were all from TECO GmbH (Germany). LMWH
(Enoxaparin, 0.4 ml  4000 AXaIU) was from Sanofi-Aventis
(France). AT-dependent anti-factor Xa test kit (Heparin, ANTI-Xa)
and antithrombin (AT) were from HYPHEN BioMed (France).
Normal human plasma was provided by Bedford (France).
Thrombin Generation test kit (Thrombin Calibrator, Flu Ca Kit and
PPP Reagent) were purchased from Stago Co. (Germany). One-time
use of automatic quantitative intravenous blood vessels (sodium
citrate anti-coagulation tube) and blood collection of 4 mL con-
taining sodium citrate were purchased from Wuhan Zhiyuan
Technology Co. Ltd (China). All other chemicals were of reagent
grade and obtained commercially.
4.2. General procedures for compounds 1e4
All reagents were purchased from commercially sources and
used without further purification. All solvents were available
commercially dried or freshly dried and distilled prior to use. Re-
actions were monitored by thin-layer chromatography (TLC) using
silica gel GF254 plates with detection using short wave UV light
(l ¼ 254 nm) and staining with 10% phosphomolybdic acid in EtOH
8
European Journal of Medicinal Chemistry 234 (2022) 114256
or a p-anisaldehyde solution (EtOH/p-anisaldehyde/AcOH/H2SO4,
135:5:4:1.5), followed by heating on a hot plate. Column chroma-
tography was conducted using silica gel (200e300 mesh) with
EtOAc and petroleum ether or EtOAc (or CH2Cl2) and MeOH as
eluent. Procedures for chemical synthesis of 1e4 are complex and
tedious, and the description is very lengthy, thus are shown in
detail in Supporting information.
The structure analysis of compounds 1e4 by NMR analyses were
performed in D2O at 298 K with a Bruker Advance 400 or 800 MHz
spectrometer equipped with a13C/1H dual probe in FT model [28].
All spectra were recorded with HOD suppression by presaturation.
1
H-1H COSY, TOCSY, NOESY, 1H-13C HSQC, and HMBC spectra were
recorded using stateetime proportional phase incrementation for
quadrature detection in the indirect dimension. All chemical shifts
were relative to internal trimethylsilyl-propionic acid sodium (TSP).
All samples were previously dissolved in deuterium oxide (D2O,
99.9% D) and lyophilized three times to replace exchangeable
protons with D2O. The lyophilized samples were then dissolved in
D2O at a concentration of 20 g/L. The desalted, pure oligosaccha-
rides obtained were water-soluble white powders (HPLC purity
>98%) (compounds 1e4).
Methyl [4-O-methyl-2,3,6-tri-O-sulfonato-a-D-glucopyr-
anosyl]-(1 / 4)-[2-O-acetyl-3-O-methyl-b-D-glucopyranosyl
uronate]-(1 / 4)-[2,3,6-tri-O-sulfonato-a-D-glucopyranosyl]-
(1 / 4)-[3-O-methyl-2-O-sulfonato-a-L-idopyranosyluronate]-
(1 / 4)-2,3,6-tri-O-sulfonato-a-D-glucopyranoside sodium salt
(compound 1): 1H NMR (800 MHz, D2O) d 5.57 (dd, J ¼ 9.8, 3.5 Hz,
1H), 5.51 (d, J ¼ 3.7 Hz, 1H), 5.16e5.12 (m, 2H), 4.93e4.81 (m, 1H),
4.60 (dd, J ¼ 11.4, 7.7 Hz, 2H), 4.52e4.46 (m, 2H), 4.42 (dd, J ¼ 9.4,
5.0 Hz, 1H), 4.40e4.28 (m, 5H), 4.26 (t, J ¼ 4.1 Hz, 1H), 4.24 (dd,
J ¼ 10.1, 3.6 Hz, 1H), 4.18 (dd, J ¼ 16.9, 10.5 Hz, 3H), 4.05e3.92 (m,
5H), 3.85 (d, J ¼ 9.7 Hz,1H), 3.78 (d, J ¼ 9.0 Hz, 2H), 3.59 (d, J ¼ 3.2 Hz,
8H), 3.55 (s, 3H), 3.46 (d, J ¼ 9.2 Hz, 4H), 2.27 (d, J ¼ 5.7 Hz, 3H). 13C
NMR (201 MHz, D2O) d 176.06, 175.98, 103.95, 102.95, 102.46,
102.43, 102.34, 101.46, 100.03, 99.88, 99.64, 99.46, 99.25, 97.85,
97.50, 97.26, 96.89, 87.92, 87.12, 86.68, 86.64, 86.35, 84.28, 84.05,
83.91, 82.10, 81.35, 80.93, 80.86, 80.55, 80.49, 80.19, 80.13, 80.10,
79.88, 79.57, 79.41, 79.04, 78.87, 78.69, 78.58, 78.33, 78.14, 78.01,
77.92, 77.84, 77.81, 77.77, 77.74, 77.71, 77.66, 77.11, 76.71, 76.57,
76.29, 76.25, 76.21, 75.79, 75.31, 74.89, 74.80, 74.53, 73.31, 72.73,
72.36, 72.30, 72.03, 71.97, 71.88, 71.85, 71.82, 71.58, 71.55, 71.50,
71.36, 69.47, 68.93, 68.83, 68.69, 68.35, 68.29, 68.22, 63.24, 63.19,
62.98, 62.96, 62.86, 62.75, 62.04, 61.96, 61.59, 61.54, 58.12, 58.05,
57.93, 57.26, 23.60, 23.47. MS(ESI): 2018.7 [MþH]þ, 2040.7
[(M þNa]þ, 1960.7 [M-SO3þNa]þ
Methyl [2,3,6-tri-O-sulfonato-a-D-glucopyranosyl]-(1 / 4)-
[3-O-methyl-b-D -glucopyranosyluronate]-(1 / 4)-[2,3,6-tri-O-
sulfonato-a-D-glucopyranosyl]-(1 / 4)-[3-O-methyl-2-O-sulfo-
nato-a-L-idopyranosyluronate]-(1 / 4)-2,3,6-tri-O-sulfonato-a-
1
D-glucopyranoside sodium salt (compound 2): H NMR (800 MHz,
D2O) d 5.66 (d, J ¼ 3.5 Hz, 1H), 5.53 (d, J ¼ 3.5 Hz, 1H), 5.19 (s, 1H),
5.15 (d, J ¼ 3.5 Hz, 1H), 4.64 (d, J ¼ 7.5 Hz, 1H), 4.61 (t, J ¼ 9.2 Hz, 1H),
4.57 (t, J ¼ 9.6 Hz, 1H), 4.52 (s, 1H), 4.49 (d, J ¼ 11.3 Hz, 1H), 4.44 (t,
J ¼ 11.2 Hz, 2H), 4.41e4.34 (m, 4H), 4.33e4.26 (m, 3H), 4.24 (dd,
J ¼ 10.1, 3.5 Hz, 1H), 4.19 (d, J ¼ 10.9 Hz, 1H), 4.03 (q, J ¼ 10.3, 9.6 Hz,
4H), 3.95 (q, J ¼ 10.2 Hz, 2H), 3.88e3.75 (m, 3H), 3.65 (s, 3H), 3.60 (s,
4H), 3.53 (d, J ¼ 8.5 Hz, 1H), 3.46 (s, 3H).13C NMR (201 MHz, D2O)
d 103.92, 100.04, 99.30, 96.64, 88.05, 83.76, 80.88, 80.46, 78.98,
78.67, 78.12, 77.67, 77.64, 77.52, 77.39, 76.25, 75.27, 74.78, 72.70,
72.45, 71.79, 70.81, 68.96, 68.88, 68.71, 62.82, 61.98, 58.13. MS(ESI):
1962.7[MþH]þ, 1984.6 [MþNa]þ
Methyl [3-O-methyl-2,6-di-O-sulfonato-a-D-glucopyr-
anosyl]-(1 / 4)-[3-O-methyl-b-D-glucopyranosyluronate]-
(1 / 4)-[2,3,6-tri-O-sulfonato-a-D-glucopyranosyl]-(1 / 4)-[3-
O-methyl-2-O-sulfonato-a-L-idopyranosyluronate]-(1 / 4)-
Z. Zhou, L. Zhang, X. Wu et al.
2,3,6-tri-O-sulfonato-a-D-glucopyranoside sodium salt (com-
pound 3): 1H NMR (800 MHz, D2O) d 5.59 (d, J ¼ 3.7 Hz, 1H),
5.55e5.49 (m, 1H), 5.32 (s, 1H), 5.15 (d, J ¼ 3.6 Hz, 1H), 4.92 (s, 1H),
4.63 (ddd, J ¼ 36.0, 21.9, 9.4 Hz, 3H), 4.49e4.00 (m, 13H), 3.96e3.77
(m, 4H), 3.66 (s, 4H), 3.62 (s, 3H), 3.62e3.59 (m, 5H), 3.56 (s, 2H),
3.53 (t, J ¼ 8.1 Hz, 1H), 3.47 (s, 3H).13C NMR (201 MHz, D2O)
d 104.53, 103.92, 101.77, 99.99, 99.25, 96.86, 88.57, 86.85, 84.76,
82.80, 82.52, 82.41, 81.49, 79.85, 79.44, 79.40, 79.21, 79.09, 78.76,
78.53, 78.16, 78.04, 77.56, 76.98, 76.55, 76.49, 76.41, 76.15, 76.03,
75.89, 75.83, 75.48, 74.47, 74.08, 72.78, 72.42, 72.38, 71.68, 71.27,
70.93, 69.65, 69.08, 68.90, 68.76, 68.67, 63.32, 63.28, 63.20, 63.13,
62.72, 61.96, 61.94, 60.10, 58.14, 49.53.MS(ESI):913.8[(M  2Na)/2]-
Methyl [2,6-di-O-sulfonato-a-D-glucopyranosyl]-(1 / 4)-[3-
O-methyl-b-D-glucopyranosyluronate]-(1 / 4)-[2,3,6-tri-O-sul-
fonato-a-D-glucopyranosyl]-(1 / 4)-[3-O-methyl-2-O-sulfo-
nato-a-L-idopyranosyluronate]-(1 / 4)-2,3,6-tri-O-sulfonato-a-
1
D-glucopyranoside sodium salt (compound 4): H NMR (800 MHz,
D2O) d 5.62 (d, J ¼ 3.4 Hz, 1H), 5.53 (s, 1H), 5.15 (d, J ¼ 3.2 Hz, 2H),
4.69e4.26 (m, 13H), 4.20e4.11 (m, 2H), 4.12e3.72 (m, 6H),
3.71e3.42 (m, 15H).13C NMR (201 MHz, D2O) d 103.87, 101.61,
100.04, 99.04, 96.70, 88.52, 84.63, 83.61, 82.69, 80.34, 80.09, 79.08,
79.05, 78.71, 78.55, 78.11, 77.63, 77.50, 77.31, 76.76, 76.44, 76.29,
75.74, 75.21, 74.97, 73.87, 73.04, 72.69, 72.47, 72.43, 71.82, 71.78,
71.52, 69.50, 68.98, 68.72, 68.65, 62.97, 61.99, 59.88, 58.13. MS(ESI):
1904.6 [Mþ2Na-H]-.
4.3. Determination of anticoagulant activities in vitro
APTT, PT, and TT were determined with a coagulometer (TECO
MC-4000, Germany) using APTT, PT and TT reagents and standard
human plasma as previously described [39,46]. In brief, 5 mL sample
and 45 mL plasma were pipetted to cuvette; incubated at 37  C for
2 min, then 50 mL APTT reagent was added; after incubating for
3 min, 50 mL CaCl2 reagent was added, and clotting time was
recorded. The compound plasma concentrations clotting times
were fitted linearly by Origin 2017 software (OriginLab, USA), and
the EC2.0 (concentration required for doubling clotting time) were
calculated from the fitting curves. Determinations were performed
in duplicate.
4.4. Inhibition of factor Xa by AT
Antithrombin and anti-factor Xa activities in the presence of AT
were measured with Biophen Heparin Anti-IIIa kits and Biophen
Antithrombin 2.5 kits. A mixture of 30 mL sample and 30 mL 1 IU/mL
AT was incubated at 37  C for 2 min. Then 30 mL of 24 NIH/mL
thrombin (or 8 mg/mL bovine factor Xa) was added. After incubation
for 2 min (or 1 min for factor Xa), the residual thrombin or factor Xa
activity was measured by the addition of 30 mL of 1.25 mM
thrombin chromogenic substrate (CS-11(65)) or 1.2 mM factor Xa
chromogenic substrate SXa-11. The absorbance of the reaction
mixture was read at 405 nm and were tested in twice. The reaction
was stopped 7 min later. The percentage of inhibition was then
calculated as [% Inhibition ¼ 100  (Optical Density [OD] Blank - OD
Sample)/OD Blank]. The activity per milligram of each compound
was determined by comparison with a calibrated standard (FXa)
using an Origin 2017 progress function (OriginLab, USA).
4.5. Thrombin generation assay
The thrombin generation (TG) methodology was adapted from
Hemker as previously reported [47]. TG was triggered by PPP-
Reagent contains a mixture of phospholipids and tissue Factor in
human platelet-poor plasma. Aliquots were taken every 15 s and
the concentration of thrombin was measured using the CAT
9
European Journal of Medicinal Chemistry 234 (2022) 114256
method (Thrombinoscope BV®, Maastricht, Netherlands). The total
amount of thrombin generated was quantified by computing the
area under the TG curve (AUC). The TGT was performed in normal
control plasma by the CAT method (ThrombinoscopeBV®, Maas-
tricht, Netherlands), the thrombinoscope® software was used to
construct the curve of time (min) versus thrombin concentration
(nM) and to calculate the TG parameters. The formation of this
curve could be observed kinetically on the computer screen. The
following TG parameters were measured and analyzed: (A) lag time
(min), which is the time between addition of the trigger and the
initiation of the TG; (B) peak (nM) refers to the maximum of
thrombin concentration generated; (C) the endogenous thrombin
potential (ETP) (nM  min) represents the amount of thrombin
formed over 60 min. The total amount of free thrombin produced is
referred to the area under the curve (AUC) or ETP, as described by
Wolberg and Campbell [48].
4.6. Antithrombotic properties and bleeding effects in vivo
4.6.1. Animals
Sprague Dawley male rats (of body mass 250e300 g) from
Hunan Shrek Jingda Experimental Animal Co., Ltd (Hunan, PR
China) were used for animal experiments. Rats were caged and fed
a regular diet for at least 1 week before use. These experiments
were reviewed and approved by the Animal Ethics Committee of
Kunming Institute of Botany, Chinese Academy of Sciences.
4.6.2. Stasis-induced venous thrombosis
Thrombus formation was induced by promoting a combination
of stasis and hypercoagulability using a modification of the method
of Vogel et al. [39,46,49]. Normal saline, compounds 1e4 and fon-
daparinux sodium were administered dorsally and subcutaneously
at 100 nmol/kg. At the indicated time (1, 3, 12 and 24 h, respec-
tively), rats were anesthetized by 10% chloral hydrate (0.3 mL/kg).
Two loose sutures were prepared about 2.0 cm apart on the inferior
vena cava and all the collateral veins were ligated. Rabbit powder
leaching agent (2%) was injected intravenously. At a time of 20 s
following the injection, stasis was established by tightening the
two sutures: the proximal and then the distal. The abdominal cavity
was provisionally closed and stasis was maintained for 20 min. The
cavity was then reopened, the ligated segment was opened longi-
tudinally, and the thrombus formed was removed, rinsed, blotted
on filter paper, dried for 24 h at 50  C, and weighed.
To test the dose-antithrombotic activity relationship, three
different doses 30, 100, 300 nmol/kg were administered for 2 h. For
each group (n ¼ 8), the mean thrombus mass was determined and
then expressed as a percentage of thrombus mass. The absence of
any inhibition of thrombus formation (compared to the mean
thrombus mass in the group administered normal saline) was
represented as 100%.
4.7. Ex vivo anti FXa activities in rats
Three Sprague-Dawley rats were anesthetized by chloral hy-
drate (10%, 0.3 mL/kg; intraperitoneal). Arterial blood samples
(4 mL) were withdrawn in a 3.8% trisodium citrate solution (1/9, v/
v). Each blood sample was centrifuged (1800 g  10 min) and the
platelet-poor plasmas were mixed in equal volume. Gradient con-
centration of Fpx or compounds 1e4 were mixed with plasmas (1/
9, v/v), then the anti FXa activities of mixtures were measured and
prepared the standard curve.
Sprague-Dawley rats were randomly divided into 6 groups
(n ¼ 5e8% group), and treated by subcutaneous of Fpx or com-
pounds 1e4 at 100 nmol/kg, and the normal saline was injected as
control. The venous blood (100 mL/time) were harvested in 15 min
Z. Zhou, L. Zhang, X. Wu et al.
before injection, and 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 24 h,
32 h after injection. The anti-FXa activities were examined and each
concentration of compounds in the blood was calculated according
to the preceding curve.
4.8. Statistical analysis
The results of the coagulation assays were calculated as sigmoid
or linear regressions using an Origin 2017 progress function (Ori-
ginLab, USA). The results of the venous thrombosis and bleeding
effect experiments were expressed as means ± SD and were
analyzed by SPSS statistics version 16.0 (IBM, USA), and the sta-
tistical significance of results was determined using one-way
analysis of variance (ANOVA), followed by Levene's test or the
chi-square test. Data were considered significantly different when:
*p < 0.05, **p < 0.01 or ***p < 0.001.
Credit author contribution statement
Zhipeng Zhou: Data curation, Investigation, Writing-Original
draft; Linlin Zhang: Methodology, Data curation, Writing-
Reviewing and Editing; Xin Wu:Data curation, Investigation,
Writing-Reviewing and Editing; Lan Luo: Investigation, Writing-
Reviewing and Editing; Jian Wu: Investigation; Dan Xu and Min-
gyi Wu: Conceptualization, Supervision, Writing-Reviewing and
Editing, Funding acquisition, Data curation.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported in part by the National Natural Science
Foundation of China (NO. 81872774 and 81930097), Youth Inno-
vation Promotion Association CAS (Y2021104), CAS "Light of West
China" Program and the Ten-thousand Talents Program of Yunnan
Province (YNWR-QNBJ-2018-271), and Yunnan Fundamental
Research Projects (grant NO. 202101AS070292 and
202101AT070152).
Appendix B. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2022.114256.
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