Synthesis, antiplatelet and antithrombotic activities of resveratrol derivatives with NO-donor properties

Article abstract of DOI:10.1016/j.bmcl.2017.04.007

Resveratrol (RVT) is a stilbene with a protective effect on the cardiovascular system; however, drawbacks including low bioavailability and fast metabolism limit its efficacy. In this work we described new resveratrol derivatives with nitric oxide (NO) re

Full text of DOI:10.1016/j.bmcl.2017.04.007

Accepted Manuscript
Synthesis, antiplatelet and antithrombotic activities of resveratrol derivatives
with NO-donor properties
Luiz Antonio Dutra, Jéssica Frade O. Guanaes, Nadine Johmann, Maria Elisa
Lopes Pires, Chung Man Chin, Sisi Marcondes, Jean Leandro Dos Santos
PII:
S0960-894X(17)30371-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.007
BMCL 24860
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 February 2017
31 March 2017
1 April 2017
Please cite this article as: Dutra, L.A., Guanaes, J.F.O., Johmann, N., Pires, M.E.L., Chin, C.M., Marcondes, S.,
Dos Santos, J.L., Synthesis, antiplatelet and antithrombotic activities of resveratrol derivatives with NO-donor
properties, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.007
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Synthesis, antiplatelet and antithrombotic activities of resveratrol derivatives
with NO-donor properties
Luiz Antonio Dutraa, Jéssica Frade O. Guanaesb, Nadine Johmanna, Maria Elisa
Lopes Piresb, Chung Man China, Sisi Marcondesb, Jean Leandro Dos Santosa,*
aSão Paulo State University (UNESP), School of Pharmaceutical Sciences,
Araraquara, São Paulo, Brazil
bUniversity of Campinas (Unicamp), Faculty of Medical Science, Campinas-SP,
Brazil
Corresponding authors
* e-mail: santosjl@fcfar.unesp.br (J. L. dos Santos), luizdutra_qf@yahoo.com.br (L. A.
Dutra)
Abbreviations List
ASA – acetylsalycilic acid; ADP- adenosine diphosphate; AA - arachidonic acid (AA); NO –
nitric oxide; DNS - Isosorbide dinitrate; RVT - resveratrol.

Abstract
Resveratrol (RVT) is a stilbene with a protective effect on the cardiovascular system;
however, drawbacks including low bioavailability and fast metabolism limit its
efficacy. In this work we described new resveratrol derivatives with nitric oxide (NO)
release properties, ability to inhibit platelet aggregation and in vivo antithrombotic
effect. Compounds (4a-f) were able to release NO in vitro, at levels ranging from
24.1% – 27.4%. All compounds (2a-f and 4a-f) have exhibited platelet aggregation
inhibition using as agonists ADP, collagen and arachidonic acid. The most active
compound (4f) showed reduced bleeding time compared to acetylsalicylic acid (ASA)
and protected up to 80% against in vivo thromboembolic events. These findings
suggest that hybrid resveratrol-furoxan (4f) is a novel lead compound able to prevent
platelet aggregation and thromboembolic events.
Keywords: platelet aggregation inhibition; antithrombotic activity; furoxan; molecular
modification; resveratrol.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (RVT) is a natural non-flavonoid
polyphenolic compound found in several sources including grapes, peanuts, apples,
plums, a variety of berries and products derived therefrom.1 RVT has received
worldwide attention after studies suggesting an inverse correlation between wine
consumption and reduced incidence of coronary heart disease.2 Moreover, animal
studies have shown effects of RVT against several diseases including cancer,
cardiovascular disorders, inflammation and neurodegenerative diseases.3,4
For the cardiovascular diseases, RVT has exhibited anti-hypertensive activity
and in vivo protective effects against stroke, myocardial ischemia and heart failure.5
Multiple targets are implicated in these effects, including sirtuins (e.g. SIRT-1),
nuclear factor-kappa B (NF-κB), AMP-activated protein kinase (AMPK) and nuclear
factor (erythroid-derived 2)-like 2 (Nrf2).5,6 RVT also reduces in vitro platelet
7,8
aggregation induced by ADP, collagen and thrombin
and upregulates eNOS,
enhancing nitric oxide-mediated vasodilation.9 In vivo studies have demonstrated that
resveratrol exhibits protective effect on rats that have undergone portal vein system
thrombosis.10
Interestingly, it was reported that RVT acts synergically with
hypocholesterolemiant drugs (e.g. pravastatin) by down-regulating the enzyme 3-
hydroxy-3-methyl-glutaryl-CoAreductase (HMG-CoA reductase) involved in
cholesterol biosynthesis.11 Hypercholesterolemic mice have exhibited a reduced
atherosclerotic area after systemic administration of this stilbene.12 RVT also
increases the expression of LDL receptors in hepatocytes contributing to the
prevention of atherosclerosis.13 All of these effects, associated with antioxidant/anti-
inflammatory activities, reduction of expression of adhesion molecules (e.g. ICAM,

VCAM) and ability to inhibit smooth cell migration to atherogenic plate make this
natural product an interesting prototype for drug design.
However, despite the beneficial effects, RVT exhibits unfavorable
pharmacokinetic properties with reduced bioavailability and fast pre-systemic
metabolism.14 Several clinical trials have failed to prove the efficacy of RVT in
humans. Therefore, molecular modifications are necessary to improve its
pharmacokinetic and pharmacodynamic profiles.
Nitric oxide (NO) is one of the crucial components that maintain the
vasculature homeostasis and regulates intracellular signaling pathways.15 NO
decreases endothelial cell adhesion, inhibits platelet aggregation and thereby limits
the thrombotic process.16 The platelet aggregation inhibition effect of NO is attributed
to upregulation of cyclic guanosine monophosphate, leading to a reduced dimerization
of integrin αIIbβ3 and inhibition of von Willebrand factor (VWF)-mediated platelet
adhesion.17,18 Furoxan (1,2,5-oxadiazole-2-N-oxide) is a member of a class of
heterocyclic compounds known for their ability to release nitric oxide19,20,21 with
platelet aggregation inhibition and antithrombotic effects.22,23,24
In this study, molecular modification was used to design new hybrid furoxan-
resveratrol derivatives design as platelet antiaggregant and antithrombotic. First,
bioisosteric replacement of the alkene subunit presents in the resveratrol to N-
acylhydrazone subunit leads to compounds (2a-f) (Figure 1). We have reported
bioisosteric replacement of the double bond present in RVT to the N-acylhydrazone
scaffold.25 Resveratrol derivatives 2a-f were optimized aiming to embed the
phenylsulphonyl subunit as a NO release scaffold resulting in resveratrol derivatives-

furoxan hybrid (4a-f).26 The combination of resveratrol derivatives effects with NO
activity might enhance both platelet aggregation inhibition and antithrombotic effects.
Fig. 1. Design of hybrids RVT derivatives-Furoxan using molecular modifications.
Compounds 2a-f were prepared through a condensation reaction involving
benzhydrazides and aldehydes functionalized in ethanol using acidic catalysis.22,25
(Supplementary material) (Scheme 1). All these compounds were obtained in
excellent yields ranging from 70 – 90%. Compounds 4a-f were synthesized through
reactions of hydroxybenzaldehydes and bis-arylsulfonylfuroxan (Scheme 2). The
intermediates containing the aldehyde function were reacted with functionalized
benzhydrazides in order to obtain N-acylhydrazones.21 The structures of all
1
compounds were established by infrared spectroscopy, and H- and 13C-nuclear
magnetic resonance (1H- and 13C-NMR). The compounds were identified by 1H-NMR
and their spectra showed chemical shifts ranging from 8.32 – 8.36 ppm and 9.62 –
9.98 ppm for H-C=N and N-H, respectively. All of the compounds were analyzed by
high-performance liquid chromatography and their purities were >98.5%.

Scheme 1. Synthesis of resveratrol derivatives (2a-f).
Scheme 2. Synthesis of hybrids resveratrol derivatives- furoxan (4a-f).
The ability of all compounds to release nitric oxide in the presence of cysteine
was evaluated through in vitro assays using Griess reaction.24 In solution, nitric oxide
is quickly oxidized to nitrite, which is measured in the medium. Table 1 summarizes
-
the results expressed as percentage of nitrite (NO2 , mol/mol). All furoxan derivatives
(4a-f) were capable of inducing nitrite formation at levels ranging from 24.1 – 27.4%.
Compounds (2a-f), as well as aspirin, did not generate nitrite in the medium.
Isosorbide dinitrate (DNS), used as control, was able to generate nitrite at levels of
12.1%.

Table 1. Inhibition of platelet aggregation induced by collagen (5 µg/mL),
arachidonic acid (100 µM) and ADP (10 µM) for resveratrol derivatives.
Compounds
Collagen (5 µg/mL)a Arachidonic acid (100 µM)a ADP (10 µM)a % NO2- (mol/mol-1)b
Inhibition (%)
63.4 4.0
Inhibition (%)
28.9 8.0
6.7 4.0*
60.8 4.8*
48.2 3.0*
74.1 4.0*
42.5 2.0*
72.8 3.0*
79.6 3.8*
17.0 1.4*
25.0 3.5
14.3 6.3*
8.5 3.3*
37.3 9.3
20.3 0.5
Inhibition (%) L-Cys 50x10-4M
ASA
3
27.7 1.8
89.8 1.5*
56.0 0.4*
46.1 0.6*
42.4 0.4*
58.2 0.5*
60.3 0.4*
31.3 0.6
-
29.7 4.8*
27.5 3.5*
29.1 7.5*
33.1 10.2*
35.9 3.8*
19.1 10.0*
44.0 10.0*
25.5 5.2*
44.8 9.3*
39.9 9.2*
34.4 10.2*
54.3 3.3*
72.2 3.5*
32.1 1.7
2a
2b
2c
-
-
-
2d
2e
-
-
2f
-
4a
4b
4c
84.2 0.5*
88.4 0.6*
91.9 0.6*
74.3 0.3*
82.2 0.8 *
87.1 0.6*
25.3 1.3
24.1 2.6
27.4 3.1
24.5 2.3
26.2 2.7
25.8 2.4
4d
4e
4f
a
Results are expressed as the mean for n = 3 independent experiments performed in triplicate. All
b
compounds were evaluated at 150 µM. All values are the mean SEM. Determined by Griess
reaction, after incubation for 1 h at 37 °C in pH 7.4 buffered water, in the presence of 1:50 molar
excess of L-cysteine). *P < 0.01 versus the control group ASA (ANOVA followed by Tukey test).
The inhibition of platelet aggregation for all compounds were evaluated in
vitro using platelet-rich plasma in the presence of ADP (10 µM), arachidonic acid
(100 µM) and collagen (5 µg/mL).27 All compounds (2a-f and 4a-f) at 150 µM were
able to inhibit platelet aggregation induced by these agonists. For ADP, for example,
the inhibition ranged from 31.3% to 91.9%. Compound (3) inhibits around 89.8%
platelet aggregation induced by ADP, however, this compound have shown weak
inhibition in the presence of the agonists arachidonic acid or collagen. The presence

of phenylsulfonyl-furoxan subunit increased the antiaggregant effect for compounds
4a-f compared to its parent (2a-f). For compound 4f, for example, the presence of
furoxan subunit increased by 2.8 times the platelet aggregation inhibitory effect
compared to 2f (Table 1). For arachidonic acid (AA) used as agonist, the platelet
aggregation inhibition ranged from 6.7% to 79.6%. Interestingly, the presence of the
phenylsulfonyl-furoxan subunit for compounds 4a-f reduced dramatically the capacity
to inhibit platelet aggregation compared to resveratrol derivatives (2a-f). For collagen
at 5 µg/mL, the platelet aggregation inhibition ranged from 19.1% to 72.2%. The
presence of the phenylsulfonyl-furoxan subunit increased the platelet aggregation
inhibition for compounds 4b, 4c, 4e and 4f compared to their respective parental
compounds.
Bleeding time is an assay used to evaluate platelet function and the capacity of
our body to form a clot. Increased bleeding time is a common adverse effect for all
platelets antiaggregant drugs that limits their clinical use. In this work, the most
promising compound 4f and its parent 2f were evaluated in order to characterize its
ability to alter the bleeding time.28 Both 2f and 4f have exhibited bleeding times of
200 seconds and 380 seconds, respectively. For compound (2f), the values are similar
to those found in the negative control (CMC 0.5%). Acetyl salicylic acid (ASA) has
exhibited bleeding time of around 800 seconds (Figure 2).

* * *
* * *
* *
1000
800
600
400
200
0
Compounds
Fig. 2. In vivo bleeding time effect from compounds (2f), (4f) and (ASA). **P < 0.001;
***P < 0.0001, ANOVA followed by Tukey test.
In vivo antithrombotic activity was evaluated using mouse pulmonary
thromboembolism model after oral administration in a single dose.29-32 Compound 3
did not exhibit protective effect (not shown). Compounds 2f and 4f have
demonstrated protection against thrombus formation at levels of 40% and 80%,
respectively (Figure 2). ASA, used as control, has protected 40% against these events.
For compounds 2f and ASA the survival rate was 60%, while for 4f this rate was
90%.
Table 2. In vivo antithrombotic effect of compounds (2f), (4f) and acetylsalicylic acid
(ASA).
Paralyseda
Groups
Death
% Protection
animals/total
ASA
2f
6/10
6/10
4
4
40
40
4f
2/10*
1*
80*
Compounds were orally administered at a dose of 100 µmol/10 g body weight (0.1 mL/10 g body
weight). The χ2 test was used to compare the survival rate between the control and treated groups. *P <
0.05 compared with ASA. The vehicle did not exhibit protective effect and no death was observed in
this group. aThe loss of the righting reflex was considered to indicate paralysis.

In conclusion, we synthesized new resveratrol (2a-f) and resveratrol-furoxan hybrids
(4a-f) and evaluated their capacity to prevent thromboembolic events. Compounds
4a-f have shown NO-released properties due to the presence of phenylsulfonyl-
furoxan. Among the series, 4f has inhibited platelet aggregation induced by ADP,
arachidonic acid and collagen. This compound has shown bleeding time 4-fold less
than ASA. Moreover, compound 4f demonstrated potent in vivo antithrombotic effect
using mouse pulmonary thromboembolism model, protecting up to 80% of mice
against thromboembolic events while ASA protection was up to 40%. Based on these
results, compound 4f has emerged as a novel lead compound able to prevent platelet
aggregation and thromboembolic events.
Acknowledgments
This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP) (Ref. Process: 2015/21271-8, 2015/19531-1, 2014/03945-9 and
2011/15520-4) and Conselho Nacional de Desenvolvimento Cientifico e Tecnológico
(CNPq).
Supplementary material
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/. These data include chemical and pharmacological procedures
described in this article.

References
1. Weiskirchen S, Weiskirchen R. Resveratrol: how much wine do you have to drink
to stay healthy. Adv. Nutr. 2016;7:706–718.
2. Renaud S, de Lorgeril M. Wine, alcohol, platelets, and the French paradox for
coronary heart disease. Lancet. 1992;339:1523–1526.
3. Pavan AR, da Silva GDB, Jornada DH, et al. Unraveling the anticancer effect of
curcumin and resveratrol. Nutrients. 2016;8:E628.
4. Smoliga JM, Baur JA, Hausenblas HA. Resveratrol and health – a comprehensive
review of human clinical trials. Mol Nutrition Food Res. 2011;55:1129–1141.
5. Bonnefont-Rousselot D. Resveratrol and cardiovascular diseases. Nutrients.
2016;8:E250
6. Li H, Xia N, Forstermann U. Cardiovascular effects and molecular targets of
resveratrol. Nitric Oxide. 2012;26:102–110.
7. Bertelli A, Giovannini L. Antiplatelet activity of synthetic and natural resveratrol in
red wine. Int J Tissue React. 1994;17:1–3.
8. Wang Z, Zou J, Huang Y, Cao K, Xu Y, Wu JM. Effect of resveratrol on platelet
aggregation in vivo and in vitro. Chin Med J. 2002;115:378–380.
9. Leikert JF. Red wine polyphenols enhance endothelial nitric oxide synthase
expression and subsequent nitric oxide release from endothelial cells. Circulation.
2002;106:1614–1617.
10. Kirimlioglu V, Sozen H, Turkoglu S, Haberal M. Protective effect of resveratrol, a
red wine constituent polyphenol, on rats subjected to portal vein thrombosis. Transpl
Proc. 2008;40:290–292.
11. Cho IJ, Ahn JY, Kim S, Choi MS, Ha TY. Resveratrol attenuates the expression
of HMG-CoA reductase mRNA in hamsters. Biochem Biophys Res Commun.
2008;367:190–194.
12. Zini R, Morin C, Bertelli A, Bertelli AA, Tillement JP. Effects of resveratrol on
the rat brain respiratory chain. Drugs Exp Clin Res. 1999;25:87–97.
13. Yashiro T, Nanmoku M, Shimizu M, Inoue J, Sato R. Resveratrol increases the
expression and activity of the low density lipoprotein receptor in hepatocytes by the

proteolytic activation of the sterol regulatory element-binding proteins.
Atherosclerosis. 2012;220:369–374.
14. Wenzel E, Somoza V. Metabolism and bioavailability of trans-resveratrol. Mol
Nutr Food Res. 2005;49:472–481.
15. Tousoulis D, Kampoli AM, Tentolouris C, Papageorgiou N, Stefanadis C. The
role of nitric oxide on endothelial function. Curr Vasc Pharmacol. 2012;10:4–18.
16. Vilahur G, Segaleés E, Salas E, Badimon L. Effects of a novel platelet nitric oxide
donor (LA816), aspirin, clopidogrel and combined therapy in inhibiting flow- and
lesion-dependent thrombosis in the porcine ex vivo model. Circulation.
2004;110:1686–1693.
17. Sylman JL, Lantvit SM, Vedepo MC, Reynolds MM, Neeves KB. Transport
limitations of nitric oxide inhibition of platelet aggregation under flow. Ann Biomed
Eng. 2013;41:2193–2205.
18. Roberts W, Michno A, Aburima A, Naseem KM. Nitric oxide inhibits von
Willebrand factor-mediated platelet adhesion and spreading through regulation of
integrin alpha(IIb)beta(3) and myosin light chain. J Thromb Haemost. 2009;7:2106–
2115.
19. Scatena R, Bottoni P, Pontoglio A, Giardina B. Pharmacological modulation of
nitric oxide release: new pharmacological perspectives, potential benefits and risks.
Curr Med Chem. 2010;17:61–73.
20. Fernandes GFS, de Souza PC, Marino LB, et al. Eur. Synthesis and biological
activity of furoxan derivatives against Mycobacterium tuberculosis. J Med Chem.
2016;123:523–531.
21. Dutra LA, De Almeida L, Passalacqua TG, et al. Leishmanicidal activities of
novel synthetic furoxan and benzofuroxan derivatives. Antimicrob Agents Chemother.
2014;58:4837–4447.
22. Rosseto LA, Pires ME, Melchior AC, et al. Synthesis and Preliminary Evaluation
of N-Oxide Derivatives for the Prevention of Atherothrombotic Events. Molecules.
2015;20:18185–18200.

23. Turnbull CM, Cena C, Fruttero R, Gasco A, Rossi AG, Megson IL. Mechanism of
action of novel NO-releasing furoxan derivatives of aspirin in human platelets. Br J
Pharmacol. 2006;148:517–526.
24. Sorba G, Medana C, Fruttero R, Cena C, Di Stilo A, Galli U, Gasco A. Water
soluble furoxan derivatives as NO prodrugs. J Med Chem. 1997;40:463–469.
25. Reis JS, Corrêa MA, Chung MC, Dos Santos JL. Synthesis, antioxidant and
photoprotection activities of hybrid derivatives useful to prevent skin cancer. Bioorg
Med Chem. 2014;22:2733–2738.
26. Dos Santos JL, Lanaro C, Chelucci RC, et al. Design, synthesis and
pharmacological evaluation of novel hybrid compounds to treat sickle cell disease
symptoms. part II: furoxan derivatives. J Med Chem. 2012;55:7583–7592.
27. Born GV, Cross MJ. The aggregation of blood platelets. J Physiol. 1963;168:178–
195.
28. Dejana E, Callioni A, Quintana A, de Gaetano G. Bleeding time in laboratory animals.
II - A comparison of different assay conditions in rats. Thromb Res. 1979;15:191–197.
29. DiMinno G, Silver MJ. Mouse antithrombotic assay: a simple method for the
evaluation of antithrombotic agents in vivo. Potentiation of antithrombotic activity by
ethyl alcohol. J Pharmacol Exp Ther. 1983;225:57–60.
30. Lima LM, Frattani FS, Dos Santos JL, et al. Synthesis and anti-platelet activity of novel
arylsulfonate--acylhydrazone derivatives, designed as antithrombotic candidates. Eur J
Med Chem. 2008;43:348–356.
31. Hong Q, Ma ZC, Huang H, et al. Antithrombotic activities of ferulic acid via
intracellular cyclic nucleotide signaling. Eur J Pharmacol. 2016;777:1–8.
32. Lee W, Lee J, Kulkarni R, et al. Antithrombotic and antiplatelet activities of small-
molecule alkaloids from Scolopendra subspinipes mutilans. Sci Rep. 2016;6:21956.