Improving the thrombin inhibitory activity of glycyrrhizin, a triterpenic saponin, through a molecular simplification of the carbohydrate moiety

Article abstract of DOI:10.1111/cbdd.12204

Glycyrrhizin, a saponin, and its aglycone glycyrrhetinic acid are natural products found in the Liquorice (Glycyrrhiza glabra L.) root extract. This saponin is known for its in vitro and in vivo thrombin inhibitory activity. The design and synthesis of five glycyrrhizin derivatives were carried out to improve the natural product activity. Compound 3b, a phthalic ester derivative of glycyrrhizin, presented a more pronounced thrombin inhibition (IC ₅₀ = 114.4 ± 1.3 μm) than the saponin (IC₅₀ = 235.7 ± 1.4 μm). Molecular docking simulations performed to investigate the molecular interaction between compound 3b and the enzyme indicate that this product is, as previously determined for glycyrrhizin, an allosteric thrombin inhibitor. We report in this article the semisynthesis and the in vitro thrombin inhibitory activity of the glycyrrhetinic acid hemiphthalic ester 3b. This activity was found to be more pronounced than of the saponin glycyrrhizin.

Full text of DOI:10.1111/cbdd.12204

Received Date : 10-Apr-2013
Revised Date : 14-Jun-2013
Accepted Date : 12-Aug-2013
Article type : Research Letter
Improving the thrombin inhibitory activity of Glycyrrhizin, a triterpenic saponin, through a
molecular simplification of the carbohydrate moiety
Fernando T. de Paula1, Petrina Q. Frauches1, Conrado Pedebos2, Markus Berger3, Simone C. B.
Gnoatto4, Grace Gossmann4, Hugo Verli2, Jorge A. Guimarães3 and Cedric S. Graebin1*
1) Laboratório de Diversidade Molecular e Química Medicinal (LaDMol-QM), Departamento de
Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro, Rodovia BR-465,
Km. 7, Postal Code 23897-000, Seropédica, RJ, Brazil;
2) Grupo de Bioinformática Estrutural, Centro de Biotecnologia, Universidade Federal do Rio Grande
do Sul, Porto Alegre, RS, Brazil;
3) Laboratório de Bioquímica Farmacológica, Centro de Biotecnologia, Universidade Federal do Rio
Grande do Sul, Porto Alegre, RS, Brazil;
4) Laboratório de Fitoquímica, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brasil.
* Corresponding author. Tel./Fax number: +55 21 2682-1872. E-mail: cedric@ufrrj.br or
cedric@ladmolqm.com.br
Abstract
Glycyrrhizin, a saponin, and its aglicone Glycyrrhetinic acid are natural products found in the
Liquorice (Glycyrrhyza glabra L.) root extract. This saponin is known for its in vitro and in vivo
Thrombin inhibitory activity. The design and synthesis of five glycyrrhizin derivatives was carried out
in order to improve the natural product activity. Compound 3b, a phthalic ester derivative of
Glycyrrhizin presented a more pronounced Thrombin inhibition (IC50 = 114.4 ± 1.3 µM) than the
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been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12204
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saponin (IC50 = 235.7 ± 1.4 µM). Molecular docking simulations done in order to investigate the
molecular interaction between 3b and the enzyme indicate that this product is, as previously
determined for Glycyrrhiuzin, an allosteric Thrombin inhibitor.
Keywords: Glycyrrhizin, Glycyrrhetinic acid, thrombin, glucoronic acid, saponin.
Abbreviations: MD: Molecular Docking.
Introduction
Pathologies associated with hyper-coagulable states, including myocardial infarction, deep vein
thrombosis and pulmonary embolism, are one of the most important causes of morbidity and
mortality worldwide (1). In spite of the arise of several new synthetic agents in the recent years for
treating such diseases, mainly direct thrombin and fXa inhibitors, concerns still exist for side effects,
specially bleeding (2, 3).
In the recent years the saponin glycyrrhizin (1) was found to be an in vitro and in vivo thrombin
inhibitor, being the first plant-derived natural product with this activity reported in the literature (4,
5). Additionally, the in vitro assays indicate that its observed thrombin inhibitory effect is due to the
selective inhibition of the enzyme’s anion binding exosite 1 (3).
Glycyrrizin (1) and its aglycone glycyrrhetinic acid (2) are natural products that are abundant in the
Liquorice (Glycyrrhyza glabra L.) root extract. This saponin is used worldwide as a natural sweetener
and employed in many Chinese, Indian and Tibetan traditional preparations. Both compounds are
known in the literature for its wide range of biological activities, and they have been employed as
scaffolds for the semi-synthesis of several derivatives with interesting pharmacological proprieties
(6, 7). “Stronger Neo-Minophagen C”, a glycyrrhizin-containing i.v. formulation, has been used for
more than 30 years in Japan for the treatment of chronic hepatitis diseases (8), being also marketed
in China, Korea, Taiwan, Indonesia, India, and Mongolia (9).
While synthetic derivatives of heparin analogues as fondaparinux are already in therapeutic use (1),
such compounds are difficult and expensive to obtain, which has supported the simplification of the
carbohydrate scaffold by other, nonsaccharidic structures (10). In this context, the current work is
focused in the structural simplification of the carbohydrate moiety of 1, searching for potential new
non-saccharidic scaffolds with improved antithrombotic activity.
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Methods and Materials
Assuming that the carboxylic acid groups present at the carbohydrate units of 1 were crucial to the
interaction with the enzyme’s basic exosite, we devised a simple and straightforward molecular
simplification via the synthesis of hemi-esters employing the triterpene 2 as starting material
(Scheme 1) and cyclic and acyclic anhydrides, leading to the esters 3a (also known as
carbenoxolone), 3b and the 3-O-acetyl derivative 3c (6, 7). We also employed the classical Jones
oxidation protocol (6, 7) to obtain the ketone derivative (4). The reaction of 4 with hydroxylamine
hydrochloride gave its respective oxime 5 in moderate to good yields (Scheme 1). Chemical purity
and properties of all synthesized compounds (melting point, TLC profiles when available and 1H- and
13C-NMR) were compared with previous reports in the literature (3a and 3b (10), 3c (11), 4 (12) and 5
(13)).
Results and Discussion
Since no previous structure-activity relationship was available to guide compound 1 structural
modifications, our first step was to evaluate the importance of the carbohydrate moiety on
thrombin modulation. Accordingly, the saponin 1 and its aglycone 2 were tested in the thrombin-
induced fibrinogen clotting assay While 1 presented an IC50 = 235.7 ± 1.4 µM, compound 2 was
.
found to be inactive on thrombin-induced fibrinogen clotting assay (Fig. 1), pointing to the
importance of the carbohydrate moiety interactions with thrombin.
As previous works from our group described the binding of 1 to thrombin exosite 1, known for its
basic properties, the carbohydrate moiety was substituted by acidic, hydrogen bonding acceptor
groups (Scheme 1) in order to evaluate: i) the influence of the substituent configurational entropy
and flexibility (3a and 3b); ii) the role of the chemical function nature at C-3 upon removal of the
carbohydrate residues (4 and 5); and iii) the importance of the ester group at C-3 (3a-c).
Compounds 4 and 5 presented IC50 values greater than 1000 µM in the thrombin-induced fibrinogen
clotting assay, pointing to an absence of influence of the hydrogen bond characteristics of the group
at C-3, that is, donor or acceptor. Similar results were obtained for 3c, indicating that the ester group
at C-3 has minor or no participation on interactions with thrombin, being unable to recover the
interactions lost from glycan removal.
Compounds 3a and 3b, however, indicates that such carbohydrate moiety is not an absolute
requirement for thrombin binding and inhibition, with the phtallic ester 3b showing an almost
twofold increase in its activity (IC50 = 114.4 ± 1.3 µM) accompanied with a significant simplification of
the molecule structure (Fig. 1A and B). In fact, substitution of carbohydrates by aromatic rings has
been described previously regarding antithrombin activation by heparin mimetics, based on sulfated
flavonoids (14). In spite of a great reduction on the number of hydrogen bond capable groups from 1
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to 3b, there is also a great reduction on configurational entropy lost upon complexation, which may
explain such increase in activity. This possibility correlates with the low activity of compound 3a (IC50
= 398.6 ± 6.9 µM) in comparison to 3b (Figure 1B). Also, compound 3b showed a more pronounced
anticoagulant activity when tested in human plasma. In fact, compound 3b caused a significant
prolongation of thrombin time compared to normal plasma values (Table 1).
With the purpose of obtaining further insights into the reported activities, compounds 1, 2, 3a and
3b were docked on thrombin exosite I, previously observed as the binding site for glycyrrhizin (4).
Additionally, as the inhibitory effect upon thrombin is expected to modulate the conformation of its
catalytic site, each docking complex was further refined through Molecular Docking (MD)
simulations in order to search for potential allosteric influences of the compounds, in comparison to
non-inhibited thrombin, as reported for the modulation of thrombin by sulfated arabinans (15). A
simulation of uncomplexed thrombin was employed as a negative control and, in addition to the use
of glycyrrhizin as the positive control, another anticoagulant saponin, through thrombin inhibition,
Chikusetsusaponin IVa (β-D-glucopyranosiduronic acid, (3β)-28-(β-D-glucopyranosyloxy)-28-
oxoolean-12-en-3-yl) (6) (16) was also simulated in complex to its target protein. The so obtained
data from the combination of docking and MD is presented in Figure 1 and Table 2.
The MD simulations indicated a progressive reorientation of the docking derived conformations,
further exploring the exosite surface and refining the interactions with the protein along the
simulations (Fig. 1). Regarding compounds 1 and 3b, the dynamics of these products indicate that
the anionic exosite 1 is properly occupied and there are similarities between the binding regions of
both molecules. The less active compounds 2 and 3a appear to populate the site in a more diffuse
manner on the simulations, moving either to the northwest region of the site (2) or to the southern
region (3a). The binding of 1 and 3b to the anionic exosite 1 is important for inhibition because this
site is essential for fibrinogen recognition by thrombin. Thus, MD simulations corroborate our
previous results which showed the ability of compound 1 to displace hirudin (a specific ligand of
thrombin exosite 1) from its binding site (4).
Additionally, MD simulations also indicated that the compounds could modulate the conformation
of thrombin catalytic site. As showed in Table 2, the distance between catalytic triad residues had an
increase after binding of compounds 3b and 6 in comparison with uncomplexed thrombin.
Interestingly, these compounds also had the lowest IC50 values in the thrombin-induced fibrinogen
clotting assay and the most potent anticoagulant activity in plasma. Our hypothesis is that the
binding to exosite 1 could induce these changes in catalytic triad residues via allosteric modulation
and thus contribute to the inhibitory activity. In fact, we recently showed that compound 6 is also
able to inhibit the thrombin-induced hydrolysis of chromogenic substrate S2238 (H-D-Phe-Pip-Arg-p-
nitroanilide), a synthetic peptide designed specifically to the thrombin catalytic site (16). Unlike
fibrinogen, the cleavage of synthetic peptide is not dependent of interaction with exosite 1,
indicating that compound 6 can modulate the active site. Similarly to compound 6, compound 3b
was also able to inhibit hydrolysis of the synthetic peptide while 1 wasn’t (Figure 1C).
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Based on the final frame of thrombin-3b complex, the main interactions are depicted in Figure 2. The
carboxyl group at the aromatic ring contributes with the majority of the interactions, through
hydrogen bonding with Ala113 backbone and Lys110 side chain. Furthermore, the phenyl ring
participates in hydrophobic interactions involving mostly Lys81, Ile82, Lys109 and Ser83 (Figure 2).
These findings suggest that the dual nature of the substituent, comprehending polar and apolar
groups, provides multiple possibilities for the compound binding to the anionic exosite 1 and, most
important, future insights for further compound optimizations.
Conclusions
Although the synthesized compounds are already known in the literature, this is the first report
regarding the synthesis of thrombin-inhibitory Glycyrrhyzin (1) derivatives. Compound 3b, with a
simpler molecular scaffold than 1, presented a greater Thrombin inhibitory activity, prolonged
Thrombin time and, unlike 1, was able to inhibit hydrolysis of the synthetic peptide S2238, indicating
that this ester can modulate the active site. These results, along with the docking and MD
simulations performed, can be used for the design and synthesis of new Glycyrrhyzin derivatives
towards new thrombin inhibitory compounds.
Acknowledgements
F. T. de Paula and acknowledge funding from the Universidade Federal Rural do Rio de Janeiro.
Petrina Q. Frauches and C. S. Graebin acknowledges funding from FAPERJ (Fundação de Amparo à
Pesquisa do Estado do Rio de Janeiro). H. Verli, C. Pedebos and M. Berger acknowledge funding from
CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) and FAPERGS (Fundação de Amparo à Pesquisa do
Estado do Rio Grande do Sul).
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Figures, Figure captions and Tables
Table 1. Pharmacological data on thrombin inhibition by compounds 1-6.
Compound Thrombin Time (s)a
Thrombin IC50 (µM)
86.3 ± 3b
1
235.7 ± 1.4
NAe
191.4 ± 40.0c
2
NTd
62.6 ± 2.1b
107.0 ± 15.4c
118.3 ± 8.0b
282.6 ± 62.2c
NT
3a
392.6 ± 6.9
3b
114.4 ± 1.3
3c
4
927.1 ± 1.5
2,111.5 ± 11.2
1,319.2 ± 3.6
NT
5
NT
92.4 ± 2.7b
240.7 ± 4.5c
45.8 ± 4.8
6
199.4 ± 9.1
-
Controla
a
Thrombin time (TT) was measured by incubation of human plasma in the absence (control) or
presence of inhibitors.
b
Measured at 100 µM.
Measured at 500 µM.
c
d NT = Not tested.
h NA = Not active.
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Table 2. Structural data on thrombin inhibition by compounds 1-6.
Interaction energy
Compound
Distance between catalytic triad residues (nm)a
(kJ/mol)a
His57–Asp102b His57–Ser195c
Sum
0.85
0.97
0.92
1.29
NT
1
0.30 ± 0.03
0.32 ± 0.04
0.31 ± 0.03
0.46 ± 0.15
NTd
0.55 ± 0.10
0.65 ± 0.11
0.61 ± 0.11
0.83 ± 0.34
NT
-136 ± 46
-44 ± 21
-120 ± 42
-147 ± 61
NT
2
3a
3b
3c
4
NT
NT
NT
NT
5
6
NT
NT
NT
NT
0.33 ± 0.10
0.29 ± 0.02
0.76 ± 0.14
0.57 ± 0.12
1.09
0.86
-178 ± 43
-
Controle
a Average values measured after the first 20 ns MD simulations.
b Between atoms ND1 from His and OD2 from Asp.
c Between atoms NE2 from His and OG2 from Ser.
d NT = Not tested.
e Control: simulation of free, uncomplexed thrombin.
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Scheme 1: Reagents and conditions: i) Anhydride (10 equiv.), pyridine, 24-48 h, reflux, 30-90%; ii)
Jones oxidation; iii) Hydroxylamine hydrochloride, pyridine, reflux, 24 h, 38%;
Figure 1. Structural and functional features of thrombin inhibition. A. Conformational ensemble of
the inhibitors at the anionic exosite 1, superimposed at every 10 ns of MD. B. Inhibitory activity of
compounds on thrombin-induced fibrinogen clotting. C. Comparison of thrombin inhibitory activity
using fibrinogen and the synthetic substrate S2238 (H-D-Phe-Pip-Arg-p-nitroanilide) as substrates. All
inhibitors were tested at 500 μM.
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Figure 2. Schematics of thrombin interaction with compound 3b based on the combination of
docking and MD simulation. In the left, a 2D representation based on results obtained from
PoseView Server, where a green line indicates hydrophobic interactions, while a dashed line
indicates hydrogen bonds. In the right, thrombin is represented in a blue surface, while the
highlighted residues (cyan) and compound 3b (green backbone) are represented as sticks.
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