Synthesis and pharmacological evaluation of acylhydroquinone derivatives as potent antiplatelet agents

Article abstract of DOI:10.1016/j.bcp.2020.114341

Platelets are the smallest blood cells, and their activation (platelet cohesion or aggregation) at sites of vascular injury is essential for thrombus formation. Since the use of antiplatelet therapy is an unsolved problem, there are now focused and innovative efforts to develop novel antiplatelet compounds. In this context, we assessed the antiplatelet effect of an acylhydroquinone series, synthesized by Fries rearrangement under microwave irradiation, evaluating the effect of diverse acyl chain lengths, their chlorinated derivatives, and their dimethylated derivatives both in the aromatic ring and also the effect of the introduction of a bromine atom at the terminus of the acyl chain. Findings from a primary screening of cytotoxic activity on platelets by lactate dehydrogenase assay identified 19 non-toxic compounds from the 27 acylhydroquinones evaluated. A large number of them showed IC₅₀ values less than 10 μM acting against specific pathways of platelet aggregation. The highest activity was obtained with compound 38, it exhibited sub-micromolar IC₅₀ of 0.98 ± 0.40, 1.10 ± 0.26, 3.98 ± 0.46, 6.79 ± 3.02 and 42.01 ± 3.48 μM against convulxin-, collagen-, TRAP-6-, PMA- and arachidonic acid-induced platelet aggregation, respectively. It also inhibited P-selectin and granulophysin expression. We demonstrated that the antiplatelet mechanism of compound 38 was through a decrease in a central target in human platelet activation as in mitochondrial function, and this could modulate a lower response of platelets to activating agonists. The results of this study show that the chemical space around ortho-carbonyl hydroquinone moiety is a rich source of biologically active compounds, signaling that the acylhydroquinone scaffold has a promising role in antiplatelet drug research.

Full text of DOI:10.1016/j.bcp.2020.114341

Journal Pre-proofs
Synthesis And Pharmacological Evaluation Of Acylhydroquinone Derivatives
As Potent Antiplatelet Agents
Diego Méndez, Viviana Donoso-Bustamante, Juan Pablo Millas-Vargas,
Hernán Pessoa-Mahana, Ramiro Araya-Maturana, Eduardo Fuentes
PII:
DOI:
Reference:
S0006-2952(20)30577-3
https://doi.org/10.1016/j.bcp.2020.114341
BCP 114341
To appear in:
Biochemical Pharmacology
Received Date:
Revised Date:
Accepted Date:
3 September 2020
6 November 2020
9 November 2020
Please cite this article as: D. Méndez, V. Donoso-Bustamante, J. Pablo Millas-Vargas, H. Pessoa-Mahana, R.
Araya-Maturana, E. Fuentes, Synthesis And Pharmacological Evaluation Of Acylhydroquinone Derivatives As
Potent Antiplatelet Agents, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp.2020.114341
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1
SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF
ACYLHYDROQUINONE DERIVATIVES AS POTENT ANTIPLATELET AGENTS
Running title: Antiplatelet activity of acylhydroquinone
By
Diego Méndeza†, Viviana Donoso-Bustamanteb†, Juan Pablo Millas-Vargasb,
Hernán Pessoa-Mahanac, Ramiro Araya-Maturanab, Eduardo Fuentesa
† Both authors contributed equally to this manuscript
a
Thrombosis Research Center, Medical Technology School, Department of
Clinical Biochemistry and Immunohematology, Faculty of Health Sciences,
Universidad de Talca, Talca, Chile.
b
Instituto de Química de Recursos Naturales, Universidad de Talca, Talca,
Chile.
c
Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias
Químicas y Farmacéuticas, Universidad de Chile, Chile.
Correspondence to:
Ramiro Araya-Maturana, raraya@utalca.cl
Eduardo Fuentes, edfuentes@utalca.cl
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ABSTRACT
Platelets are the smallest blood cells, and their activation (platelet cohesion or
aggregation) at sites of vascular injury is essential for thrombus formation. Since
the use of antiplatelet therapy is an unsolved problem, there are now focused and
innovative efforts to develop novel antiplatelet compounds. In this context, we
assessed the antiplatelet effect of an acylhydroquinone series, synthesized by
Fries rearrangement under microwave irradiation, evaluating the effect of diverse
acyl chain lengths, their chlorinated derivatives, and their dimethylated derivatives
both in the aromatic ring and also the effect of the introduction of a bromine atom
at the terminus of the acyl chain. Findings from a primary screening of cytotoxic
activity on platelets by lactate dehydrogenase assay identified 19 non-toxic
compounds from the 27 acylhydroquinones evaluated. A large number of them
showed IC50 values less than 10 µM acting against specific pathways of platelet
aggregation. The highest activity was obtained with compound 38, it exhibited sub-
micromolar IC50 of 0.98±0.40, 1.10±0.26, 3.98±0.46, 6.79±3.02 and 42.01±3.48 µM
against convulxin-, collagen-, TRAP-6-, PMA- and arachidonic acid-induced
platelet aggregation, respectively. It also inhibited P-selectin and granulophysin
expression. We demonstrated that the antiplatelet mechanism of compound 38
was through a decrease in a central target in human platelet activation as in
mitochondrial function, and this could modulate a lower response of platelets to
activating agonists. The results of this study show that the chemical space around
ortho-carbonyl hydroquinone moiety is a rich source of biologically active
compounds, signaling that the acylhydroquinone scaffold has a promising role in
antiplatelet drug research.
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Keywords:
platelets;
antiplatelet;
synthesis;
acylhydroquinone;
acylchlorohydroquinones.
1. INTRODUCTION
Atherothrombosis, characterized by atherosclerotic lesion disruption with
superimposed thrombus formation, is the common underlying process for
numerous progressive manifestations of acute coronary syndromes and
cardiovascular death. Following rupture, fissure, or erosion of an atherosclerotic
plaque; atherothrombotic events are essentially platelet-driven processes [1, 2].
Platelets are the smallest blood cells, numbering 150 to 350ꢀ×ꢀ109/L in healthy
individuals [3]. In addition to being the first step for the maintenance of normal
hemostasis, the ability of platelets to form stable adhesion contacts with other
activated platelets (platelet cohesion or aggregation) at sites of vascular injury is
essential for thrombus formation, myocardial infarction, and stroke [4-7]. Thrombus
development can involve the initial formation and recruitment of reversible platelet
aggregates independent of platelet activation [8].
Antiplatelet therapy represents the cornerstone of the clinical treatment of
atherothrombotic events [9]. Currently, independent of classes of antiplatelet drugs
(e.g. clopidogrel, aspirin, and ticlopidine), these reduce the vascular events by
about 10-12% in secondary prevention [10]. However, it is necessary to further
knowledge on antiplatelet scaffolds and their structural requirements to obtain new
active compounds [11].
Several phenols and FDA-approved drugs exert antiplatelet effects by different
mechanisms resulting from their antioxidant properties [12]. Among them is
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hydroquinone which is a powerful antioxidant used in cosmetics [13] that inhibits
thromboxane A2 (TXA2) production and suppresses arachidonic acid (AA)-induced
platelet aggregation [14]. Besides, some hydroquinone derivatives such as arbutin,
the natural β -glucoside, inhibit platelet aggregation induced by different agonists
(adenosine diphosphate [ADP], AA, collagen, and thrombin) [15], although it is
quickly metabolized in blood in rats [16], 2,5-di-(tert-butyl)-p-hydroquinone also
inhibits platelet aggregation, in this case, it is stimulated by protease-activated
receptor (PAR)-1 or PAR-4 agonist peptides (SFLLRN and AYPGKF) [17], and
avarol, sesquiterpenoid hydroquinone isolated from the sponge Dysidea avara,
inhibits platelet aggregation stimulated by AA and A23187 (calcium ionophore)
(Figure 1) [18].
Some antiplatelet mechanisms involving inhibition of oxidative phosphorylation
(OXPHOS) through the interaction of hydroquinone derivatives with the electron
transport chain (ETC) have been described [19]. Several ortho-
carbonylhydroquinone scaffold-containing compounds affect mitochondrial
bioenergetics, by inhibiting mitochondrial electron transport and/or by uncoupling of
OXPHOS, with consequences on the proliferation of cancer cells [20-24] and the
antioxidant activity of some of them has also been reported [25]. The strong
hydrogen bond between the carbonyl group and one of the phenolic hydroxyl
groups is an important feature regarding these activities [26-28]. Recently, the
effects of the acylhydroquinone scaffold, the simplest ortho-carbonylhydroquinone
structure, against leukemia cell lines, have been reported [29].
Taking into account that ortho-carbonyl hydroquinone moiety is a structural feature
that, besides other activities, confers potent antiplatelet activity via differential
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inhibition of platelet aggregation induced by collagen or thrombin receptor activator
peptide 6 (TRAP-6) [30], also considering that, small structural changes modify
these properties including the effect on OXPHOS [23], and given that, most of the
assessed acylhydroquinones against leukemic cells do not exhibit significative
cytotoxicity, it was decided to assess the antiplatelet activity of a wider series of
acylhydroquinones.
2. MATERIALS AND METHODS
2.1. Chemical Methods
13
1H and C NMR spectra were obtained from a spectrometer (Bruker Avance 400
NMR, Rheinstetten, Germany) operating at either 400.13 MHz (1H) or 100.61 MHz
(13C), or from one operating at 300.13 MHz (1H) or 75.47 MHz (13C). Chemical
shifts are reported as ppm downfield from the tetramethylsilane dissolved in CDCl3
1
(Merck, DA, Germany) for H NMR and relative to the central CDCl3 resonance
13
(77.0 ppm) for C NMR. All melting points are uncorrected and were determined
using an Electrothermal 9100 apparatus. IR spectra (KBr discs) were recorded on
an FT-IR spectrophotometer (Thermo Nicolet Corp, Madison, WI, USA);
wavenumbers reported in cm-1. High-resolution mass spectra (HRMS) were
obtained from an orthogonal time-of-flight (TOF) mass spectrometer
(Waters/Micromass Q-TOF micro, Manchester, UK). Silica gel 60 (230 - 400 mesh
ASTM) (Sigma-Aldrich, DA, Germany) and thin-layer chromatography (TLC) sheets
silica gel 60 F254 (Merck, DA, Germany) were used for flash-column
chromatography and analytical TLC, respectively [30].
2.2. Synthesis of acylhydroquinones and acylchlorohydroquinones
The synthetic methodology for obtaining acylhydroquinones has been already
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reported [29, 30] and was used to synthesize the compounds 13-27 (Table 1).
Briefly, acylhydroquinones 13-27 were obtained using microwave irradiation at
90°C, at a fixed power of 150W as follows. To a 10 mL CEM microwave (CEM
Discovery SP Labmate, NC, USA) process vial equipped with a magnetic bar, a
mixture of one equivalent of hydroquinone was added (1) or dimethyl hydroquinone
(2), 1.5 equivalent of the corresponding carboxylic acid (3-11), and 4 mL of boron
trifluoride dihydrate (Sigma-Aldrich, DA, Germany). The mixture was irradiated
under microwave for 20 min. After that, the mixture was extracted with ethyl
acetate (Panreac Quimica, BCN, Spain), washed with water, and dried with
anhydrous sodium sulfate (Sigma-Aldrich, DA, Germany), then it was filtered and
concentrated under vacuum. Afterward, the corresponding acylhydroquinone was
purified by flash chromatography with hexane/ethyl acetate (Panreac Quimica,
BCN, Spain) 4:1 as eluent. In this manner, the acylhydroquinones 13-27 were
obtained. As shown in Table 1, compounds 28-38 were obtained by chlorination,
with hydrogen chloride (obtained by reaction of concentrated sulfuric acid with
sodium chloride) [31] of the corresponding quinones obtained by oxidation of the
hydroquinones 12-15, 17-22, and 24, with Ag2O (obtained by adding concentrated
sodium hydroxide to a silver nitrate solution) [32] in dichloromethane (Merck, DA,
Germany) [29], these compounds were also purified by flash chromatography with
hexane/ethyl acetate 4:1 as eluent.
The syntheses of the new analogs 23-27, 29, and 38 are described as follows:
Synthesis of 5-bromo-1-(2,5-dihydroxyphenyl)pentan-1-one (23)
Hydroquinone (500 mg, 4.54 mmoles) (Sigma-Aldrich, DA, Germany), 5-
bromopentanoic acid (1.23 g, 6.81 mmoles) (Sigma-Aldrich, DA, Germany) and 3
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7
mL of boron trifluoride dihydrate (Sigma-Aldrich, DA, Germany) react yielding 409
1
mg of 23 (4.54 mmol, 33 % yield). H-NMR (400.13 MHz, CDCl3): 1.87-2.03 (m,
4H, 2xCH2); 2.99 (t, 2H, J = 7.4Hz, CH2-CO); 3.47 (t, 2H, J = 6.4Hz, BrCH2); 4.82
(s, 1H, 5-OH); 6.90 (d, 1H, J = 8.9Hz, 3-H); 7.04 (dd, 1H, J1 = 8.9Hz; J2 = 2.8Hz, 4-
13
H); 7.22 (d, 1H, J = 2.8 Hz, 6-H); 11.86 (s, 1H, 2-OH). C-NMR (100.61 MHz,
CDCl3): 22.71; 32.01; 33.11; 37.24; 114.67; 119.48; 124.90; 147.37; 177.69;
205.24. m.p. 58.7-60.7 °C. IR(KBr) max: 1491.00; 1650.16; 1708.04; 2871.38;
2943.73; 3380.71. HRMS (ESI): m/z calcd for C11H13O3Br M+: 272.0048, found:
272.0048.
Synthesis of 5-bromo-1-(2,5-dihydroxy-3,4-dimethylphenyl)pentan-1-one (24)
2,3-Dimethylhydroquinone (500 mg, 3.61 mmol) (Sigma-Aldrich, DA, Germany), 5-
bromopentanoic acid (980 mg, 5.42 mmol) (Sigma-Aldrich, DA, Germany) and 3
mL of boron trifluoride dihydrate (Sigma-Aldrich, DA, Germany) react yielding 233
1
mg of 24 (3.61 mmol, 21 % yield). H-NMR (400.13 MHz, CDCl3): 1.86-2.01 (m,
4H, 2XCH2); 2.21 (s, 3H, Ar-CH3); 2.24 (s, 3H, Ar-CH3); 2.95 (t, 2H, J = 6.6Hz, CO-
CH2); 3.46 (t, 2H, J = 6.4Hz, Br-CH2); 4.53 (s, 1H, 5-OH); 7.02 (s, 1H, 6-H); 12.36
(s, 1H, 2-OH). 13C-NMR (100.61 MHz, CDCl3): 11.58; 13.02; 23.02; 32.07; 33.18;
37.08; 111.16; 115.86; 127.13; 134.17; 145.43; 155.43; 204.98. m.p. 125.2-126.5
°C. IR(KBr) max: 1572.03; 1669.65; 2923.47; 2958.20; 3377.81. HRMS (ESI): m/z
calcd for C13H17O3Br M+: 300.0361 found 300.0359.
Synthesis of 6-bromo-1-(2,5-dihydroxyphenyl)hexan-1-one (25)
Hydroquinone (453 mg, 4.12 mmol) (Sigma-Aldrich, DA, Germany), 6-
bromohexanoic acid (1.2 g, 6.15 mmol) (Sigma-Aldrich, DA, Germany) and 3 mL of
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8
boron trifluoride dihydrate (Sigma-Aldrich, DA, Germany) react yielding 526 mg of
25 (1.83 mmol, 22% yield). 1H-NMR (300.13 MHz, CDCl3): 1.54 (quint, 2H, J = 7.3
Hz; CH2); 1.76 (quint, 2H, J = 7.3 Hz, CH2); 1.92 (quint, 2H, J = 7.3 Hz, CH2); 2.96
(t, J = 7.4 Hz, 2H, COCH2); 3,43 (t, 2H, J = 6.6 Hz, Br-CH2); 5.84 (s, 1H, 5’-OH),
6.88 (d, 1H, J = 8.9 Hz, 3-H); 7.05 (dd, 1H, J1 = 8.9 Hz, J2 = 2.9 Hz, 4-H); 7.23 (d,
13
1H, J = 2.9 Hz, 6-H); 11.92 (s, 1H, 2-OH). C-NMR (75.45 MHz, CDCl3): 23.32;
27.77; 32.49; 33.58; 38.11; 114.75; 118.94; 119.40; 124.87; 147.48; 156.57;
205.86. m.p. 156,1-160,4 °C; IR(KBr) max: 1728.30; 2859.81; 2943.73; 3010.29;
3073.96; 3395.18. HRMS (ESI): m/z calcd for C12H15BrO3 M+: 286.0205 found
286.0205.
Synthesis of 6-bromo-1-(2,5-dihydroxy-3,4-dimethylphenyl)hexan-1-one (26)
2,3-Dimethylhydroquinone (500 mg, 3.61mmol) (Sigma-Aldrich, DA, Germany), 6-
bromohexanoic acid (1.06 g, 5.43 mmol) (Sigma-Aldrich, DA, Germany) and 3 mL
of boron trifluoride dihydrate (Sigma-Aldrich, DA, Germany) react yielding 481mg
of 26 (3.61 mmol, 42 mmol, 42% yield). 1H-NMR (400.13 MHz, CDCl3): 1.48-1.59
(m, 2H, CH2); 1.76 (quint, 2H, J = 7.5Hz, CH2); 1.92 (quint, 2H, J = 6.8Hz, CH2);
2.21 (s, 3H, Ar-CH3); 2.24 (s, 3H, Ar-CH3); 2.93 (t, 2H, J = 7.5Hz, CH2CO); 3.44 (t,
2H, J = 6.6Hz, CH2-Br); 4.59 (s, 1H, 5-OH); 7.02 (s, 1H, 6-H); 12.41 (s, 1H, 2-OH).
13C-NMR (100.61 MHz, CDCl3): 11.58; 13.02; 23.63; 27.83; 32.53; 33.53; 37.95;
111.23; 115.92; 127.08; 134.06; 145.40; 155.41; 205.46. m.p. 125.2-125.9 °C. IR
(KBr) max: 1609.65; 2856.91; 2932.15; 2961.09; 3366.24. HRMS (ESI): m/z calcd
for C14H19BrO3 M+: 314.0518, found 314.0517.
Synthesis of 10-bromo-1-(2,5-dihydroxy-3,4-dimethylphenyl)decan-1-one (27)
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9
2,3-Dimethylhydroquinone (500 mg, 3.61mmol) (Sigma-Aldrich, DA, Germany),
10-bromodecanoic acid (1.36 g, 5.43 mmol) (Sigma-Aldrich, DA, Germany) and 3
mL of boron trifluoride dihydrate (Sigma-Aldrich, DA, Germany) react yielding 228
1
mg of 27 (3.61 mmol, 17% yield). H-NMR (400.13 MHz, CDCl3): 1.27-1.39 (m,
8H, 4xCH2); 1.43 (quint, 2H, J = 7.5Hz, CH2); 1.73 (quint, 2H, J = 7.5Hz, CH2);
1.86(quint, J = 7.0Hz, CH2); 2.21(s, 3H, Ar-CH3); 2.24 (s, 3H, Ar-CH3); 2.90 (t, 2H,
J = 7.5Hz, CO-CH2); 3.41 (t, 2H, J = 7.0Hz, Br-CH2); 4.53 (s, 1H, 5-OH); 7.03(s,
1H, 6-H); 12.45 (s, 1H, 2-OH). 13C-NMR (100.61 MHz, CDCl3): 11.58; 13.00;
24.70; 28.12; 28.68; 29.23; 29.26; 29.28; 32.79; 34.03; 38.27; 111.39; 115.98;
127.01; 133.89; 145.34; 155.42; 206.10. m.p. 97.5-98.1 °C. IR (KBr, cm-1) max
:
1572.03; 1609.65; 2848.23; 2926.37; 3366.24. HRMS (ESI): m/z calcd for
C18H27O3Br M+: 370.1144 found 370.1131.
Synthesis of 1-(2-chloro-3,6-dihydroxy-4,5-dimethylphenyl)ethan-1-one (29)
A mixture of acylhydroquinone 13 (100 mg, 0.55 mmol) and Ag2O (380 mg) in
dichloromethane (6 mL) (Merck, DA, Germany) was stirred for 30 min, after the
mixture was filtered through Celite and dry hydrogen chloride was bubbled through
the filtrate for 5 min. Column flash chromatography on silica gel, allowed the
isolation of 101 mg of 29 (0.47 mmol, 85% yield); m.p. 117.3-118.4 °C; IR max:
1
1615.43; 2926.37; 2628.30; 2544.37; 3453.05. H-NMR (400 MHz, CDCl3): 2.19
(s, 3H, Ar-CH3); 2.30 (s, 3H, Ar-CH3); 2.81 (s, 3H, COCH3); 5.54 (s, 1H, 3-OH);
12.62(s, 1H, 6-OH). 13C-NMR (100.17 MHz, CDCl3): 11.79; 13.72; 33.30; 114.38;
115.60; 126.44; 133.46; 143.03; 155.94; 203.95. HRMS (ESI): m/z calcd for
C10H11ClO3 M+: 214.0397 found 214.0375.
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Synthesis of 5-bromo-1-(2-chloro-3,6-dihydroxy-4,5-dimethylphenyl)pentan-1-one
(38)
A mixture of acylhydroquinone 24 (100 mg, 0.33 mmol) and Ag2O (380 mg) in
dichloromethane (6 mL) (Merck, DA, Germany) was stirred for 30 min, after the
mixture was filtered through Celite and dry hydrogen chloride was bubbled through
the filtrate for 5 min. Column flash chromatography on silica gel allowed the
isolation of 59 mg of 38 (0.18 mmol, 55 % yield). 1H-NMR (400 MHz, CDCl3): 1.86-
2.00(m, 4H, 2xCH2), 2.19 (s, 3H, Ar-CH3); 2.29(s, 3H, Ar-CH3); 3.20(t, 2H, J =
6.5Hz, COCH2); 3.45(t, 2H, J = 6.3Hz, BrCH2); 5.54(s, 1H, 3-OH); 12.23(s, 1H, 6-
OH). 13C-NMR (100.61 MHz, CDCl3): 11.87, 13.70, 23.51, 29.70, 32.16, 33.20,
43.26, 113.43, 115.67, 126.58, 133.15, 143.09, 155.31, 205.80. m.p.: 97.2-98.3 °C.
IR (KBr) max: 1592.28, 1618.33, 2923.47, 2966.88, 3418.33. HRMS (ESI) M+: m/z
calcd for C13H16BrClO3 M+: 333.9971 found 333.9984.
Besides, the monomethyl ethers of some of these hydroquinones (12, 13, 17, 25,
and 26) were obtained by the standard methodology of protecting the phenolic
hydroxyl group [33].
2.3. Isolation of human platelets from whole blood
Venous whole blood was obtained with anticoagulant acid citrate-dextrose (ACD)
(Sigma-Aldrich, MO, USA) from apparently healthy volunteers (during the last 7
days without consumption of non-steroidal anti-inflammatory drugs and absence of
chronic diseases), used at a 1:4 ratio for ACD: venous whole blood [34]. Then the
sample was centrifuged (Eppendorf 5804, CT, USA) for 10 min at 200 g to obtain
platelet-rich plasma (PRP). One part of the PRP (two thirds) was centrifuged for 8
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min at 800 g, the supernatant was removed and the platelet pellet was
resuspended in modified Tyrode's buffer (Sigma-Aldrich, MO, USA) without
calcium. Platelets were centrifuged once more (8 min at 800 g) and used within 3
h. A Hematology analyzer (Hematological counter Mindray BC-3000 Plus, Japan)
was used to count the washed platelets [30]. The study was approved by the
Scientific Ethics Committee from the University of Talca.
2.4. Lactate dehydrogenase (LDH)-based cytotoxicity assay
To assess the cytotoxicity of the compounds under study, LDH Cytotoxicity Assay
Kit (Cayman Chemical, MI, USA) was used [30]. Briefly, washed platelets 300x109
(platelets/L) were used under different conditions: negative control with dimethyl
sulfoxide (DMSO) 0.2% (Duchefa Biochemie, Haarlem, Netherlands), positive
control with triton 10% (Sigma-Aldrich, MO, USA), reactive control and synthesized
compounds (200 µM). All conditions were incubated for 10 min at 37°C and then
centrifuged at 800 g for 8 min at 4°C. After that, 100 µL of the supernatant was
mixed with 100 μL of LDH reaction solution in a 96-well microplate. The microplate
was incubated for 30 min at 37°C under constant orbital shaking and the reaction
was measured at 490 nm in a microplate reader (Microplate Reader Thermo
Scientific Multiskan Go, Finland). Based on positive control, the level of LDH
released was considered as 100%.
2.5. Platelet aggregation and secretion of adenosine triphosphate (ATP)
The real-time analysis of platelet aggregation and secretion of ATP in vitro,
monitored by light transmission and luminescence, was performed using a lumi-
aggregometer (Chrono-Log, Haverton, PA, USA) [34]. Briefly, synthesized
compounds (0.02 to 200 µM) were pre-incubated with washed platelets (300×109
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platelets/L) or DMSO 0.2%, and CaCl2 (2 mM) (Sigma-Aldrich, MO, USA) for 5 min
at 37°C. For ATP secretion, platelets were pre-incubated for 30 s at 37°C with
chrono-lume probe (Chrono-Log, Haverton, PA, USA). Then real-time monitoring of
platelet aggregation and ATP secretion was started by the addition of agonist
(TRAP-6 5 µM [Cayman Chemical, MI, USA], collagen 1 µg/mL [Helena
Laboratories, TX, USA], convulxin 5 ng/mL [Santa Cruz Biotechnology, TX, USA],
phorbol-myristate-acetate [PMA] 100 nM [Cayman Chemical, MI, USA] or
arachidonic acid 30 ng/mL [Helena Laboratories, TX, USA]) for 5 min at 37°C.
Results of platelet aggregation and ATP secretion were determined by the software
AGGRO/LINK (Chrono-Log, Havertown, PA, USA) and expressed as a percentage
of maximal amplitude. The synthesized compounds were solubilized in DMSO
0.2%.
2.6. Flow cytometry assays
Washed platelets (200×109 platelets/L) were pre-incubated for 10 min at 37°C with
synthesized compounds (2 and 20 µM) or DMSO 0.2%, and CaCl2 (2 mM), and
then platelets were stimulated by TRAP-6 5 µM, convulxin 5 ng/mL or PMA 100
nM. Subsequently, 50 µL aliquots were taken per condition to be evaluated: (i)
platelet activation (dense granules and α–granules) were evaluated by CD63PE
and CD62PE antibodies (BD Biosciences, CA, USA), (ii) mitochondrial membrane
potential (∆Ψm) was determined by Accuri C6 flow cytometer (BD Biosciences, CA,
USA), using the potentiometric probe tetramethylrhodamine, methyl ester,
perchlorate (TMRM) 100 nM (Invitrogen, MA, USA), (iii) intraplatelet reactive
oxygen species (ROS) level was determined using the dihydroethidium probe 10
µM (Thermo Fisher Scientific, MA, USA), (iv) intraplatelet calcium levels were
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measured by flow cytometry with the Fluo-3 AM 0.44 µM (Invitrogen, MA, USA)
and (v) externalization of phosphatidylserine on platelet membrane was detected
by FITC Annexin V Apoptosis Detection Kit (BD Biosciences, CA, USA). Finally,
the conditions were incubated with anti-CD61FITC (BD Biosciences, CA, USA) for
30 min at room temperature in the dark and analyzed by Accuri C6 flow cytometer
[30, 34].
2.7. Statistical analysis
Data were obtained from experiments from at least 3 healthy volunteers and
analyzed using Prism 5.0 software (GraphPad Inc., San Diego CA, USA). The half-
maximal inhibitory concentration (IC50) was calculated from the dose-response
curves. Differences between samples were analyzed using ANOVA analysis and
Tukey’s posthoc test. P values <0.05 were considered significant.
3. RESULTS AND DISCUSSION
3.1. Chemistry of synthesized compounds
The chemical structures of acylhydroquinones and acylchlorohydroquinones
studied in this work are depicted in Table 1. The reported methodology for
obtaining acylhydroquinones [29, 30] was used to synthesize the previously
reported compounds 13-27 and 28-37. Meanwhile, compound 12 was obtained
from a commercial source, and compound 13 and 28 were obtained as reported
[35, 36]. Compounds 14-22 were recently synthesized by Fries rearrangement
under microwave irradiation, their chloro-derivatives were also obtained 30-37, and
their antileukemic activity studied [29]. The syntheses of the new analogs 23-27,
29, and 38 were described in the Materials and Methods section.
3.2. Cytotoxicity of synthesized compounds on platelets
13

14
The cytotoxicities of acylhydroquinone derivatives were evaluated in human
washed platelets. The LDH is present in numerous cell types, including platelets,
which have high LDH activity [37]. In the LDH assay, cytotoxicity of synthesized
compounds or substances is measured by the release of cytosolic LDH upon
damage of the cell membrane, which is different from platelet activation by
agonists [38]. As a positive control, incubation of membrane detergent triton X-100
with human washed platelets was expressed as 100%. As shown in Figure 2, LDH
assay revealed that the synthesized compounds 36 (p<0.001) > 33 (p<0.001) > 34
(p<0.001) > 32 (p<0.001) > 37 (p<0.001) > 21 (p<0.001) > 35 (p<0.001) > 19
(p<0.01) at 200 µM incubated for 10 min with washed platelets significantly
increased the percentage of LDH release, compared with the control respectively.
In acylhydroquinones, the cytotoxicity of compounds 19 and 21 showed that long
acyl chains, with R1 and R2 = H, confer cytotoxic activity. This was also observed,
although not statistically, in compound 17 (chain with n = 6) which showed a higher
cytotoxicity than the control. While in acylchlorohydroquinones derivatives,
compounds 32, 33, 34, 35, 36, and 37 were toxic with carbon chains of n = 6, 7,
and 8, independently if they had Me or H as R1 substituent. LDH leakage has been
reported to correlate well with the degree of decrease in platelet count, suggesting
that these toxic compounds may decrease platelet count directly through platelet
cytotoxicity [39]. Meanwhile, LDH leakage assay by synthesized compounds 12,
13, 14, 15, 16, 17, 18, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 38 did not
induce human platelet cytolysis and was not significantly different than DMSO-
treated platelets (8.19±1.67%). We continued testing for antiplatelet activity with
these nineteen non-toxic compounds.
14

15
3.3. Antiplatelet activity of non-toxic compounds
To determine the antiplatelet activity of the 19 non-toxic compounds, the platelet
aggregation induced by TRAP-6, collagen, or convulxin was evaluated by light
transmission [40]. TRAP-6 acts as a PAR1 agonist in platelet activation [41].
Platelet GPVI is the major platelet collagen receptor in the activation of the platelet.
Also, integrin α 2 β 1, and CD36 bind to collagen directly, whereas GP Ib α and
integrin α IIb β 3 interact with collagen-bound vWF [42]. Meanwhile, convulxin, a
protein purified from the venom of Crotalusdurissus terrificus, is known to
specifically bind to GPVI [43].
None of the synthesized compounds exhibited proagreggant effect on platelets on
their own. Active compounds that inhibited platelet aggregation in a dose-
dependent manner are summarized with IC50 values in Table 2. A large number of
the compounds studied showed IC50 values less than 10 µM, acting against
specific pathways of platelet aggregation induced by TRAP-6, collagen, or
convulxin. A general view of Table 2 shows that the most active compounds were
38 (IC50 3.98 ±0.46 µM), 17 (IC50 0.04±0.03 µM), and 38 (IC50 0.98±0.40 µM) for
inhibition of platelet aggregation induced TRAP-6, collagen, and convulxin,
respectively. The platelet aggregation, in a dose-dependent manner, was inhibited
by these active compounds, representative curves are shown in Figure 3. Also,
compound 29 showed significant inhibition of platelet aggregation induced by
convulxin with an IC50 of 6.70±0.41 µM. The antiplatelet mechanism of the most
active compounds (17, 29, and 38) was evaluated in platelet aggregation by direct
stimulation of protein kinase C (PKC) (for PMA). Thus in PMA-stimulated platelets,
the inhibitory potential increased in the following order: 17 (IC50 77.96±30.00 µM),
15

16
29 (IC50 21.83±2.73 µM), and 38 (IC50 6.79±3.02 µM). Also, the compound 38
inhibited arachidonic acid-induced platelet aggregation with an IC50 of 42.01±3.48
µM. Thus in compound 38, the introduction of a chlorine atom at position 6 on the
aromatic ring and a bromine atom at the terminal carbon of the acyl chain improved
its platelet antiaggregant activity.
In this setting, we continued the study of the antiplatelet mechanism of compound
38. Since the PKC family is a major regulator of platelet granule secretion [44], we
showed that compound 38 at 20 µM inhibited the expression of platelet P-selectin
(CD62) and surface granulophysin (CD63); two markers that represent granules
release (Figure 4A and B). Also, we demonstrated that the antiplatelet activity of
compound 38 was due to its pharmacological action on mitochondrial bioenergetics
in human platelets. In agreement with this, compound 38 reduced resting
mitochondrial membrane potential (depolarization) (Figure 4C). Decreased
membrane potential, in the presence of compound 38 at 20 µM, led to lower ATP
generation (Figure 4D), and caused an increase in ROS generation (Figure 4E)
and intraplatelet calcium (Figure 4F). Conversely, these effects of compound 38 on
mitochondrial bioenergetics did not induce phosphatidylserine exposure on platelet
membrane; confirming the non-cytotoxic effects on platelets (evaluated by annexin
V binding in washed platelets) (Figure 4G). Altogether, these results indicate that
compound 38 decreased a central target in human platelet activation as in
mitochondrial bioenergetics and this could modulate a lower response of platelets
to activating agents [45], which is associated with potent non-selective inhibition of
platelet aggregation induced by TRAP-6, collagen, convulxin, PMA and arachidonic
acid (Figure 5). A similar mechanism has been reported for the antiplatelet activity
16

17
of vitamin C [46] and MitoQ [47].
3.4. Structure-activity relationship
The acyl hydroquinone 16 can be considered an analog of compound I [30], with
an acyl chain of five carbons replacing the enone cycle of compound I (Figure 1).
Hydroquinone I is the prototype of ortho-carbonyl hydroquinones whose antiplatelet
activities have been recently reported by us [30]. The structural changes from I to
16 increased activity by five times as an inhibitor of collagen-induced platelet
aggregation (Table 2). Besides, IC50 of compound 16 was only two times lower
than the most active compound, a spirohydroquinone, reported in that study [30].
This promissory result encouraged us to test the effects of the change of the acyl
chain length on the activity of a series of acylhydroquinones as inhibitors of
agonist-induced platelet aggregation.
When acyl chains are shorter than in 16, as in 12 and 14, with two and three
carbons respectively, the activities against collagen-induced aggregation were
similar to 16, but additionally, 12 showed activity against TRAP-6 activation. On the
other hand, when the acyl chain was two carbons longer, as in 17, the activity
against collagen activation increased 76 times higher than 16, with a
submicromolar IC50, and highly selective against collagen-induced platelet
aggregation, the activity against TRAP-6 decreased to half of 12. When a longer
acyl chain was present, with 8 or 9 carbons, compounds 19 and 21 were shown to
be cytotoxic, in the test of leakage of LDH, therefore the antiplatelet activity was
not evaluated.
Substitution by methyl groups at positions 3 and 4 on the aromatic ring was also
evaluated in the test of leakage of LDH. The results showed that the introduction of
17

18
methyl groups in 3,4-dimethylacylhydroquinones 20 and 22 abolished the
cytotoxicity in comparison with compounds 19 and 21. Although these dimethyl
derivatives were less active than 17, they were highly active against collagen-
induced platelet aggregation. Moreover, the activity of 22 against TRAP-6-induced
platelet aggregation was the same as exhibited by the most active ortho-carbonyl
hydroquinone reported previously by us [30]. The effect of the methyl substitution
on the activities against agonist-induced aggregation was dependent on the length
of the acyl chain, increasing or decreasing the activities by variable quantities. On
the other hand, these nine evaluated compounds were inactive against convulxin-
induced platelet aggregation.
The effect of introducing a bromine atom at the terminal carbon of the acyl chain,
hydroquinones 23 to 27, was also evaluated. Compound 23, showed that the
bromine introduction decreased the activity twofold against collagen-induced
platelet aggregation but increased TRAP-6-induced aggregation twofold, when
additionally dimethyl substitution is present, compound 24, the activity against
collagen-induced aggregation increased twenty times, and twice against TRAP-6-
induced platelet aggregation. Also compounds 25 and 27 were strong inhibitors of
the effect of collagen on platelets, but in the former case, high activities against the
three agonists in platelet aggregation were observed, with the same values against
TRAP-6- and convulxin-induced platelet aggregation.
Finally, the introduction of a chlorine atom at position 6 on the aromatic ring was
evaluated
in
compounds
28
to
38.
Among
these
compounds,
acylchlorohydroquinones 32 to 37 showed high cytotoxicity and consequently were
discarded for further studies. Interestingly, the non-cytotoxic chloro-derivatives
18

19
showed high activities against the platelet aggregation induced by TRAP-6,
collagen, and convulxin. It has likewise been reported that chloro-substituted
naphthoquinones inhibit platelet activation [48, 49]. The higher activities were
obtained with compound 38, which exhibited sub-micromolar IC50 inhibitory
activities against convulxin- and collagen-induced platelet aggregation and also a
high inhibitory activity against TRAP-6. In TRAP-6 induced platelet aggregation,
compound 24 showed a similar structure to compound 38, however, the absence
of Cl made this compound about 14 times less active than compound 38.
To assess the plausible role of the semiquinone radicals derived from these
compounds, the monomethyl ethers obtained from compounds 12, 13, 17, 25, and
26 were also evaluated and shown to be inactive in collagen-induced platelet
aggregation (Figure 6). This result suggests the involvement of semiquinone
radicals in the antiplatelet activity of these compounds.
4. CONCLUSION
Due to the key role played by platelets in the pathogenesis of atherothrombotic
events, the use of platelet antiaggregant drugs is essential in the prevention of
thrombus-mediated events. The results of this study showed that the chemical
space around ortho-carbonyl hydroquinone moiety is a rich source of biologically
active compounds, which enabled us to obtain an acylhydroquinone derivative that
decreases the mitochondrial bioenergetics, a central target in human platelet
activation, generating potent non-selective activity against platelet aggregation
induced by TRAP-6, collagen, convulxin, PMA, and arachidonic acid.
Conflict of interest
The authors have no conflicts of interest to disclose.
19

20
Acknowledgements
This research was funded by ANID/CONICYT, FONDECYT grant N° 1180427
(EF), and 1180069 (RAM).
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24
Table 1. Synthesis of acylhydroquinones by Fries rearrangement.
OH
OH
O
OH
OH
O
O
R1
R1
R2
n
R1
R1
R1
R
n
2 1) Ag2O, CH2Cl2
2) HCl(g)
BF3 x 2H2O
R2
n
+
HO
Cl
R
120 °C, 150 W
1
OH
OH
1
2
3 11
-
12 27
28 38
-
: R1 = H
: R1 = Me
-
Compound
12
n
R1
H
Me
H
Me
H
H
Me
H
Me
H
Me
H
Me
H
Me
Me
H
Me
H
Me
H
Me
H
Me
H
Me
Me
R2
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
1
1
2
2
4
6
6
7
7
8
8
4
4
5
5
9
1
1
2
2
6
6
7
7
8
8
4
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
Br
24

25
Table 2. Antiplatelet activity of synthesized compounds.
Compound
IC50 (µM) (mean ± SD)
Collagen (1 µg/mL) Convulxin (5 ng/mL)
TRAP-6 (5 µM)
31.25±3.31*
97.96±25.65
131.48±11.54
64.04±5.052
94.14±16.57
61.91±11.26
53.07±5.39
28.10±11.67
13.45±4.41
59.63±7.06
59.21±16.88
10.44±2.98
61.31±5.84
90.62±6.64 a
34.77±11.55
8.80±1.64
12
13
14
15
16
17
18
20
22
23
24
25
26
27
28
29
30
5.95±1.10
0.51±0.15
3.17±1.94
1.93±0.69
3.03±0.64
0.04±0.03
0.33±0.27
0.82±0.27
1.14±0.21
6.36±1.03
0.15±0.06
0.16±0.02
7.06±2.27
0.38±0.18
8.54±2.48
6.17±0.59
9.79±3.24
4.48 ± 1.12
1.10±0.26
0.57±0.19
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
10.09± 1.17
>20
>20
14.86± 2.11
6.70± 0.41
12.79± 0.74
8.99± 0.58
0.98± 0.40
1.45±0.40
31.41±11.94
9.44 ± 0.387
3.98 ±0.46
31
38
Ticagrelor
0.77±0.51
Mean ± SD (standard deviation) of three independent determinations and IC50 (μM)
interpolated from the dose-response curves. * Maximum inhibition to 200 µM.
Thrombin Receptor Activator Peptide 6: TRAP-6.
25

26
Figure legends
Figure 1. Chemical structure of avarol, arbutin, 2,5-di-(tert-butyl)-p-hydroquinone,
compounds 16 (reported in this article) and I [30].
Figure 2. Cytotoxicity activity of acylhydroquinone and acylchlorohydroquinones
derivatives. The results are shown as the mean ± SD (standard deviation) of n=3
experiments. Differences between the vehicle (DMSO 0.2%) and compounds were
analyzed using ANOVA analysis and Tukey’s posthoc test. **p<0.01 and
***p<0.001.
Figure 3. Representative dose-response curves of inhibition of platelet aggregation
by compounds 17 and 38. A) The half-maximal inhibitory concentration (IC50) was
calculated from the dose-response curve in platelet aggregation stimulated by
TRAP-6, collagen, and convulxin. B) Representative curves of platelet aggregation
(5 min at 37°C) pre-incubated with compounds 17 and 38 (0.02, 0.2, 2, 20 and 200
µM), and then stimulated by TRAP-6, collagen or convulxin.
Figure 4. Antiplatelet mechanism of compound 38. A) and B) Platelet activation of
α and dense granules were evaluated by P-selectin (CD62PE) and CD63PE, and
analyzed by Accuri C6 flow cytometer.C) Mitochondrial membrane potential (∆Ψm)
was determined by flow cytometry, using tetramethylrhodamine, methyl ester,
perchlorate (TMRM). D) Platelet ATP secretion was determined with chrono-lume
reagent (firefly luciferin-luciferase) in a lumi-aggregometer. E) Intraplatelet ROS
level was determined using dihydroethidium probe in a flow cytometer. F)
Intraplatelet calcium levels were measured by flow cytometry with the FLUO-3 AM.
G) Externalization of phosphatidylserine on the platelet membrane was detected by
FITC Annexin V Apoptosis Detection Kit BD Pharmingen and activated with TRAP-
26

27
6 5 µM plus collagen 1 µg/ml. AA: antimycin A, FCCP: carbonyl cyanide-4-
(trifluoromethoxy)phenylhydrazone, PMA: phorbol 12-myristate 13-acetate, TRAP-
6: thrombin Receptor Activator Peptide 6. The results are shown as the mean ±
SD (standard deviation) of n=3 experiments. For A), B) and D) differences between
absence of compound (0) and compounds (2 and 20 µM), and for C), E), F) and G)
differences between control and compounds (2 and 20 µM) were analyzed using
ANOVA analysis and the Tukey’s post-hoc test. *p<0.05, **p<0.01 and ***p<0.001.
Figure 5. Scheme of antiplatelet mechanism of compound 38. This compound
decreased a central target in human platelet activation as the mitochondrial
function and therefore exerted a potent non-selective inhibition of platelet
aggregation induced by TRAP-6 (3.98±0.46), collagen (1.10±0.26), convulxin
(0.98±0.40 µM), PMA (IC50 6.79±3.02 µM) and arachidonic acid (IC50 42.01±3.48
µM). ATP: adenosine triphosphate, GP: glycoprotein, PAR1: protease-activated
receptor 1, PMA: phorbol 12-myristate 13-acetate, PKC: protein kinase C, TP:
thromboxane A2 receptor, TRAP-6: thrombin Receptor Activator Peptide 6, ∆Ψm:
mitochondrial membrane potential.
Figure 6. Effect of monomethyl ether of hydroquinones 12, 13, 17, 25 and 26 on
inhibition of platelet aggregation. A) Chemical structures of evaluated compounds.
B) Platelets were stimulated with collagen 1 µg/mL and compounds at 20 µM.
OMe: monomethyl ether. The results are shown as the mean ± SD (standard
deviation) of n=3 experiments and were analyzed using ANOVA analysis and the
###
Tukey’s post-hoc test. ***p<0.001 vs., control,
(monomethyl ether).
p<0.001 vs., structural analog
27

28
28

29
29