Synthesis of antiplatelet ortho-carbonyl hydroquinones with differential action on platelet aggregation stimulated by collagen or TRAP-6

Article abstract of DOI:10.1016/j.ejmech.2020.112187

Cardiovascular diseases are the leading cause of death in the world. Platelets have a major role in cardiovascular events as they bind to the damaged endothelium activating and forming thrombi. Although some hydroquinone scaffold-containing compounds have known antiplatelet activities, currently there is a lack of evidence on the antiplatelet activity of hydroquinones carrying electron attractor groups. In this work, we evaluate the antiplatelet effect of a series of ortho-carbonyl hydroquinone derivatives on cytotoxicity and function of human platelets, using collagen and thrombin receptor activator peptide 6 (TRAP-6) as agonists. Our structure-activity relationship study shows that gem-diethyl/methyl substitutions and the addition/modifications of the third ring of ortho-carbonyl hydroquinone scaffold influence on the selective index (IC₅₀ TRAP-6/IC₅₀ Collagen) and the inhibitory capacity of platelet aggregation. Compounds 3 and 8 inhibit agonist-induced platelet aggregation in a non-competitive manner with IC₅₀ values of 1.77 ± 2.09 μM (collagen) and 11.88 ± 4.59 μM (TRAP-6), respectively and show no cytotoxicity. Both compounds do not affect intracellular calcium levels and mitochondrial bioenergetics. Consistently, they reduce the expression of P-selectin, activation of glycoprotein IIb/IIIa, and release of adenosine triphosphate and CD63 from platelet. Our findings may be used for further development of new drugs in platelet-related thrombosis diseases.

Full text of DOI:10.1016/j.ejmech.2020.112187

Journal Pre-proof
Synthesis of antiplatelet ortho-carbonyl hydroquinones with differential action on
platelet aggregation stimulated by collagen or TRAP-6
Diego Méndez, Félix A. Urra, Juan Pablo Millas-Vargas, Marcelo Alarcón, Julio
Rodriguez-Lavado, Iván Palomo, Andrés Trostchansky, Ramiro Araya-Maturana,
Eduardo Fuentes
PII:
S0223-5234(20)30154-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112187
EJMECH 112187
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2019
Revised Date: 23 February 2020
Accepted Date: 24 February 2020
Please cite this article as: D. Méndez, Fé.A. Urra, J.P. Millas-Vargas, M. Alarcón, J. Rodriguez-
Lavado, Ivá. Palomo, André. Trostchansky, R. Araya-Maturana, E. Fuentes, Synthesis of antiplatelet
ortho-carbonyl hydroquinones with differential action on platelet aggregation stimulated by
collagen or TRAP-6, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112187.
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SYNTHESIS OF ANTIPLATELET ORTHO-CARBONYL HYDROQUINONES
WITH DIFFERENTIAL ACTION ON PLATELET AGGREGATION STIMULATED
BY COLLAGEN OR TRAP-6
Running title: Hydroquinone derivatives regulate platelet function
By
a
b,f
a
Diego Méndez , Félix A. Urra , Juan Pablo Millas-Vargas c, Marcelo Alarcón ,
d
a
e
Julio Rodriguez-Lavado , Iván Palomo , Andrés Trostchansky , Ramiro Araya-
Maturana a,c,f, Eduardo Fuentes a
10 a Thrombosis Research Center, Medical Technology School, Department of Clinical
11 Biochemistry and Immunohaematology, Faculty of Health Sciences, Universidad
12 de Talca, Talca, Chile.
b
13
Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences,
14 Faculty of Medicine, University of Chile, Santiago, Chile.
c
15
Instituto de Química de Recursos Naturales, Programa de Investigación
16 Asociativa en Cáncer Gástrico (PIA-CG), Universidad de Talca, Talca, Chile.
d
17
Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias
18 Químicas y Farmacéuticas, Universidad de Chile.
e
19
Departamento de Bioquímica and Center for Free Radical and Biomedical
20 Research, Facultad de Medicina, Universidad de la República, Montevideo,
21 Uruguay.
22 f Network for Snake Venom Research and Drug Discovery, Santiago, Chile.
23
24 Correspondence to:
25 Eduardo Fuentes, edfuentes@utalca.cl
26 Ramiro Araya-Maturana, raraya@utalca.cl
27 Félix A. Urra, felixurraf@u.uchile.cl
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28 ABSTRACT
29 Cardiovascular diseases are the leading cause of death in the world. Platelets
30 have a major role in cardiovascular events as they bind to the damaged
31 endothelium activating and forming thrombi. Although some hydroquinone scaffold-
32 containing compounds have known antiplatelet activities, currently there is a lack of
33 evidence on the antiplatelet activity of hydroquinones carrying electron attractor
34 groups. In this work, we evaluate the antiplatelet effect of a series of ortho-carbonyl
35 hydroquinone derivatives on cytotoxicity and function of human platelets, using
36 collagen and thrombin receptor activator peptide 6 (TRAP-6) as agonists. Our
37 structure-activity relationship study shows that gem-diethyl/methyl substitutions
38 and the addition/modifications of the third ring of ortho-carbonyl hydroquinone
39 scaffold influence on the selective index (IC50 TRAP-6/IC50 Collagen) and the
40 inhibitory capacity of platelet aggregation. Compounds 3 and 8 inhibit agonist-
41 induced platelet aggregation in a non-competitive manner with IC50 values of
42 1.77±2.09 µM (collagen) and 11.88±4.59 µM (TRAP-6), respectively and show no
43 cytotoxicity. Both compounds do not affect intracellular calcium levels and
44 mitochondrial bioenergetics. Consistently, they reduce the expression of P-selectin,
45 activation of glycoprotein IIb/IIIa, and release of adenosine triphosphate and CD63
46 from platelet. Our findings may be used for further development of new drugs in
47 platelet-related thrombosis diseases.
48
49 Keywords: hydroquinone derivatives; ortho-carbonyl; platelets; thrombosis; small
50 molecules.
51
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52 1. INTRODUCTION
53 Platelets are essentials in hemostasis and thrombosis, both of which are
54 dependent on their activation, occurring in the transition from a functional resting
55 state to a procoagulant and prothrombotic phenotype [1, 2]. This process is
56 triggered by interactions of platelets with adhesive components of the extracellular
57 matrix (e.g. collagen and von Willebrand factor) or by soluble platelet agonists (e.g.
58 adenosine diphosphate [ADP], thromboxane A2 [TXA2] and thrombin, among
59 others) that bind G-protein-coupled receptors [3, 4]. Under stimulation, activated
60 platelets change their morphology resulting in the activation and surface
61 expression of adhesion molecules, procoagulant phosphatidylserine (PS) exposure
62 and secretion of thrombogenic substances from platelet granules. In pathological
63 conditions, this platelet process may induce occlusive thrombosis, resulting in
64 ischemic events such as heart attack and stroke [1, 5], which are the leading
65 causes of death worldwide [6].
66 Besides their well-known antioxidant properties, polyphenols exert other beneficial
67 biological actions [7]. Currently, antiplatelet therapy is still the mainstay in
68 preventing platelet activation and inhibiting thrombus formation [8]. In this line,
69 antiplatelet action both in vitro and in vivo of natural products such as flavonoids
70 [9], curcuminoids [10], catechins [11], terpenoids [12], polyphenols [13, 14], and
71 saponins [15] have been extensively described [16, 17].
72 The antiplatelet effect of several small molecules and FDA-approved drugs have
73 been described by different mechanisms of action resulting from their antioxidant
74 properties [7]. In addition, other mechanisms involve inhibition of oxidative
75 phosphorylation (OXPHOS) through interaction with the electron transport chain
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76 (ETC) [18]. Hydroquinone scaffold-containing compounds exert biological activities,
77 some of them by acting on mitochondrial metabolism [19-22] and activating SIRT 1
78 and AMPK [23]. In this regard, our previous results have shown that the ortho-
79 carbonyl hydroquinone scaffold efficiently crosses the mitochondrial membrane
80 reaching the ETC [19-22], owing to the strong hydrogen bond between the
81 carbonyl group and one of the phenolic hydroxyl groups [24-26]. The structure-
82 activity relationship (SAR) of these hydroquinones has revealed that small
83 structural changes modify the effect on OXPHOS [22]. All these compounds
84 exhibited antioxidant capability, where the intramolecular hydrogen bonding is a
85 relevant feature not only to radical-scavenging activity [27] but also on their
86 oxidation potential [28, 29].
87 The ortho-carbonyl substituted hydroquinone 10 (Figure 1) is a protonophoric
88 uncoupling agent of OXPHOS that causes mitochondrial depolarization,
89 decreasing both intracellular adenosine triphosphate (ATP) and NAD(P)H levels,
90 the GSH/GSSG ratio, and slightly increasing reactive oxygen species (ROS)
91 formation in intact TA3/Ha cancer cells [21, 30]. Compounds 13 and 14 (Figure 1)
92 also inhibit the respiration on the same cell line. Some hydroquinone scaffold-
93 containing compounds with antiplatelet activities are known and their chemical
94 structures are shown in Figure 2. Recently, Chang et al. [31] described that
95 hydroquinone, a natural antioxidant with dermatological uses inhibits TXA2
96 production and suppresses the arachidonic acid (AA)-induced platelet aggregation
97 [31]. Also, arbutin the natural β-glucoside derivative of hydroquinone inhibits
98 platelet aggregation induced by different agonists (ADP, AA, collagen, and
99 thrombin) [32], and their pharmacokinetics showed that it is quickly metabolized in
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100 blood in rats [33]. Besides, 2,5-di-(tert-butyl)-p-hydroquinone inhibits platelet
101 aggregation stimulated by protease-activated receptor (PAR)-1 or PAR-4 agonist
102 peptides (SFLLRN and AYPGKF) and, avarol inhibits platelet aggregation
103 stimulated by AA or A23187 (calcium ionophore) [34].
104 In spite of these reports, the effect of hydroquinone scaffold-containing compounds
105 on platelet function remains scarcely studied [18]. Thus, we decided to synthesize
106 and test a series of ortho-carbonyl hydroquinones (Figure 1) as novel antiplatelet
107 agents.
108 2. RESULTS
109 2.1. Chemistry
110 The synthetic methodology to obtain the studied compounds is depicted in Scheme
111 1. The already reported methodology of obtention of acyl hydroquinones [19, 35]
112 was improved using microwave irradiation at 100°C, using hydroquinone (I) or
113 dimethyl hydroquinone (II) and the corresponding carboxylic acid as starting
114 products, with boron trifluoride dihydrate as the solvent. Compounds 1-14 were
115 synthesized using a methodology previously reported [19, 22, 36-38], involving
116 oxidation of the acylhydroquinones, reaction with enamines and finally a
117 rearrangement in acidic medium. Structural characterization and procedure to
118 synthesize compounds 1-3 and 8-14 were included as supplementary information.
119 2.2. Cytotoxic activity on platelets
120 In order to identify candidate compounds with potential antiplatelet activity (Figure
121 1), we evaluated the effect of synthesized compounds on platelet viability by LDH
122 leakage. The compounds 1, 2, 3, 7, 8, 9, 10, 11, 12, 13 and 14 did not exert LDH
123 release in platelets when incubated at the tested higher concentration (200 µM,
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124 Figure 3). However, under the same experimental conditions, compounds 4, 5 and
125 6 showed cytotoxic activity (p<0.05) (Figure 3). Thus, the following antiplatelet
126 experiments were performed using the non-cytotoxic compounds.
127 2.3. Structure-antiplatelet activity relationship for the collagen- and TRAP-6-
128 stimulated platelet aggregation
129 Light transmission aggregometry was used to evaluate the activity of non-cytotoxic
130 compounds (1, 2, 3, 7, 8, 9, 10, 11, 12, 13 and 14) on platelet aggregation induced
131 by TRAP-6 or collagen, which are known platelet agonists [39]. The compounds
132 showed different potency of their platelet antiaggregant activity, being more active
133 when inhibition was exerted on platelet aggregation induced by collagen than
134 TRAP-6 (Table 1). Among the compounds tested, compounds 3 and 8 were the
135 most active presenting IC50 of 1.77±2.09 and 11.88±4.59 µM against collagen- and
136 TRAP-6-induced platelet aggregation, respectively (Table 1). Moreover, compound
137 3 exhibited high selectivity toward platelet aggregation induced by collagen
138 signaling. In addition, and according to the selectivity index (S.I., IC50 TRAP-6/IC50
139 collagen), the other compounds had decreasing S.I. as follows: compounds 3 >11
140 > 7~1 >10~9 >12. In platelets stimulated with collagen, the substitution of spiro
141 group by gem-dimethyl moiety in compound 3 produced a moderate decrease
142 (close to 8 folds) of inhibition of platelet aggregation (compound 1). The addition of
143 a third ring to the spiro derivative, compound 3, produced a drop of about 14 folds
144 the activity (compound 9) and simultaneous additions of the third ring and
145 replacement of the spiro moiety resulted in a decrease of about 12 folds the activity
146 (compound 10). On the other hand, only three ortho-carbonyl hydroquinones
147 (compounds 2, 8 and 12) were dual inhibitors, blocking both collagen- and TRAP-
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148 6-stimulated platelet aggregation. From IC50 values, it is observed that a relevant
149 chemical requirement for dual inhibitors (i.e. S.I. values close to 1) was the gem-
150 diethyl substitution (compounds 2 and 8 vs 1 and 10). The regioisomers 11 and 12
151 had evident differences in the S.I. value as well as potencies, and the blocking of
152 the hydroxymethyl group by acylation generated the abolishment of the activity in
153 both stimuli of platelet aggregation (compounds 13 and 14), suggesting that the
154 hydroxymethyl substitution is a relevant structural requirement for inhibition of
155 platelet aggregation.
156 From here, we decided to continue the analysis of the antiplatelet activity by only
157 analyzing the compounds 3 and 8. Both compounds dose-dependently inhibited
158 collagen- and TRAP-6-induced platelet aggregation (Figure 4 A-B). In order to
159 evaluate the type of inhibition exerted by compounds 3 and 8, we induced the
160 aggregation of platelets by using a different collagen (0.1 to 2 µg/mL) or TRAP-6 (1
161 to 10 µM) concentrations in the presence of the vehicle, compound 3 or compound
162 8 (Figure 4C and E). The maximal effect induced by collagen (2 µg/mL) and TRAP-
163 6 (10 µM) was fully inhibited by these compounds, suggesting a non-competitive
164 behavior of both compounds 3 and 8 (Figure 4D and F).
165 2.4. Antiplatelet mechanism of compounds 3 and 8
166 Activated platelets expose PS which acts as a procoagulant stimulus and a signal
167 for phagocytic clearance of apoptotic cells [40]. In this study, we observed that PS
168 was not modified in the presence of compounds 3 and 8 (Figure 5A). When
169 platelets are activated with thrombin and collagen, the intracellular calcium
170 concentration increases locally and transiently [41]. This promotes conformational
171 changes leading to glycoprotein (GP) IIb/IIIa activation [42] in parallel with the
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172 translocation to the platelet surface of P-selectin, an integral membrane GP found
173 in secretory granules [43]. Both P-selectin and GPIIb/IIIa activation were
174 significantly inhibited by compounds 3 and 8 (Figure 5 B-C) (p<0.05). In contrast,
175 no changes in the levels of Ca2+ were observed in resting platelets in the presence
176 of compounds 3 and 8 (Figure 5D). Platelet activation is autocatalytically stimulated
177 by platelet secretion products [44]. In this context, we shown that the secretion of
178 CD63 (Figure 5E) was decreased in TRAP-6-stimulated human platelets only by
179 compound 8 (p<0.05). Meanwhile, ATP secretion was decreased in collagen and
180 TRAP-6-stimulated platelets by both compounds 3 and 8 (Figure 5F).
181 Since it has been reported that the prevention of platelet mitochondrial dysfunction
182 through the control of mitochondrial oxidative stress production or inhibition of
183 respiratory complexes occurs by some compounds [45], we further studied whether
184 compound 3 or 8 exhibited an antiaggregant activity through inhibition of
185 mitochondrial bioenergetics. As shown in Figure 6 A-E, both compounds 3 and 8
186 did not display any effects on mtNADH levels in resting platelets. Similar to the
187 above results this also occurred in maximal mtNADH oxidation upon stimulation of
188 platelets with FCCP and accumulation induced by respiratory complex III inhibition
189 with antimycin A (Figure 6F). Compound 3 lacked an effect on ∆ψm (Fig 6G), ROS
190 production (Figure 6H) and intracellular ATP levels (Figure 6I). In contrast,
191 compound 8 was able to induce a drop in ∆ψm (Figure 6G) in addition to an
192 increase in ROS production (Figure 6H), and a decrease in the ATP levels (Figure
193 6I) at the highest concentrations tested (20 µM).
194
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196 3. DISCUSSION
197 Some hydroquinone scaffold-containing compounds are easily oxidized to
198 quinones, which are known as pan-assay interference compounds (PAINS) with
199 high in vivo toxicity [46, 47], limiting their medical potential. Notably, as previously
200 shown [19, 22, 27, 48], the intramolecular hydrogen bonding of the ortho-carbonyl
201 hydroquinone scaffold and the gem-dimethyl group determine the redox properties
202 and reactivity in biological conditions of these series, reducing the ability to auto-
203 oxidation or interference by interactions with fluorescent assays.
204 The effect of hydroquinone scaffold-containing compounds on platelet function
205 remains scarcely studied, some of them exhibit an antiplatelet effect in platelets
206 stimulated with thrombin, AA, collagen, ADP, A23187 or platelet-activating factor
207 [18], whose structures are shown in Figure 2. There is a lack of evidence about
208 the antiplatelet activity of bi- and tricyclic hydroquinones and of hydroquinones
209 bearing electron attractor substituents. Consequently in our study, we showed that
210 ortho-carbonyl hydroquinone derivatives had differential action on collagen- and
211 TRAP-6-stimulated platelet aggregation, determined by the type of substituent,
212 especially the gem-diethyl/methyl substitutions and the addition/modifications of a
213 third ring influence S.I. values and the inhibitory potencies. The observed SAR for
214 the antiplatelet effects of this series of compounds suggest the involvement of
215 radical intermediates in platelets stimulated with TRAP-6: the greater stability of
216 semiquinone radical, hypothetically formed, the higher antiplatelet activity was
217 observed. Compounds 2 and 8 exhibits closely related structure as compounds 1
218 and 10, replacing the gem-dimethyl group by a gem-diethyl group and the alkyl
219 substitution pattern at the aromatic ring, allowing us to evaluate the effect of
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220 sterical hindrance and electronic effects, on the antiplatelet activity. The higher
221 volume and the conformation of gem-diethyl groups, with their methyl moieties,
222 positioned at above and below the molecular plane [37, 49], overall constituting a
223 structural factor that provides more thermodynamic stability to the semiquinone
224 radical species obtained by hydrogen abstraction atom or by an electrochemical
225 process [29]. These radicals are responsible for the antioxidant or prooxidant
226 activities of hydroquinones. In the same way, the aromatic ring alkylation also
227 stabilizes these intermediate species facilitating their generation [27]. These pieces
228 of evidence may explain that compound 8 was more active than 2 in TRAP-6
229 stimulated platelets, while compounds 1 and 10 were inactive, being this
230 relationship supported by the increased ROS production by 8. On the other hand,
231 the considerable difference between the activities of the regioisomers 11 and 12, in
232 TRAP-6-stimulated platelets, highlights the importance of the hydrogen bonding in
233 the biological activities of this kind of compound. Compound 12 has two hydrogen
234 bonds in their structure, one of them between phenolic hydroxyl proton and the
235 carbonyl oxygen atom and the other, one between the other phenolic proton
236 toward the oxygen of the hydroxymethyl group. Its regioisomer, instead, displays
237 less capability to form hydrogen bonds through the hydroxymethyl group [25], a
238 characteristic that can diminish the interactions inside platelets, triggering a lacking
239 activity in this platelet aggregation assay.
240 Compounds 3 and 8 exhibited lower IC50 against platelet aggregation induced by
241 collagen and TRAP-6 respectively without any cytotoxic effect on platelets. Both
242 compounds showed similar inhibitory behavior on platelet aggregation, expression
243 of molecules at the platelet membrane as well as secretion of granules
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244 components. Since oxidative stress-dependent signaling promotes mitochondrial
245 dysfunction, releasing mitochondrial components are important for both platelet
246 activation and apoptosis [45, 50, 51]. Hydroquinone scaffold-containing
247 compounds inhibit OXPHOS as we have previously described [21-23, 52]. We
248 speculate that the target for the antiplatelet effect of our series of hydroquinone
249 derivatives may be the mitochondria. Notably, compound 3 was more active on
250 collagen-induced platelet activation/secretion/aggregation without affecting
251 mitochondrial bioenergetics. In contrast, compound 8 induced ∆ψm drop,
252 decreased ATP levels and increased ROS production at high concentrations. ROS
253 production may occur at the ETC with leaks at the proton-based electrochemical
254 gradient [53], changing the ∆ψm which can be ascribed to compound 8. However,
255 compound 8 did not display any effects on mtNADH levels and its antiplatelet
256 mechanism is apparently upstream mitochondrial bioenergetic function. This
257 phenomenon is consistent with our previously reported observations in which these
258 compounds did not exhibit effects on mitochondrial respiration and OXPHOS-
259 dependent proliferation using isolated tumor mitochondria and highly oxidative
260 subpopulations of breast cancer cells, respectively [22, 23]. Taken together, our
261 results indicate that both compounds 3 and 8 lack direct effects on mitochondrial
262 bioenergetics and the changes in compound 8-induced mitochondrial parameters
263 may be a consequence of some non-mitochondrial interactions, i.e. modulation of
264 NADPH oxidase activity. Further experiments are required to confirm this point.
265 It has been recently described that small structural changes on resveratrol confer
266 greater oral bioavailability [54] and selective inhibitory action towards a specific
267 platelet aggregation-inducing signaling [9]. Resveratrol inhibits platelet aggregation
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268 induced by agonists such as collagen, U46619, and AA but has a marginal
269 inhibitory effect on ADP-induced platelet activation [55, 56]. Conversely,
270 isorhapontigenin, a methoxylated analog of resveratrol of natural origin [57, 58],
271 selectively inhibits ADP-induced platelet aggregation with an IC50 of 1.85ꢀµM,
272 lacking effects on aggregation induced by other agonists [9]. In line with this
273 observation, herein we report that compound 3, a spiro derivative of ortho-carbonyl
274 hydroquinone, is a selective inhibitor of collagen-stimulated platelet aggregation
275 (IC50 = 1.77±2.09 µM) with marginal effect on TRAP-6-stimulated platelet
276 aggregation. Conversely, compound 8, which exhibits an inhibitory effect on
277 platelet aggregation stimulated by both agonist collagen and TRAP-6, can be
278 considered a dual antiplatelet agent. Considering that there is one patent related to
279 novel spiro compounds useful as selective inhibitors of fibrinogen-dependent
280 platelet aggregation for the prevention of thrombosis (European Patent Office
281 EP0854869B1), our results suggest that compounds 3 and 8 can contribute as
282 promising scaffolds for the design and development of novel antiplatelet agents
283 with differential action on thrombotic diseases.
284 4. CONCLUSION
285 In this work, we present a SAR study on the inhibition of platelet aggregation by an
286 ortho-carbonyl substituted hydroquinone series. Based on these findings, we
287 identify inhibitors with differential action on collagen- or TRAP-6-stimulated platelet
288 aggregation.
Notably,
gem-diethyl/methyl
substitutions
and
the
289 addition/modifications of the third ring of ortho-carbonyl hydroquinone scaffold
290 influence the S.I. value and inhibitory potency, respectively. A spiro derivative of an
291 ortho-carbonylbicyclic hydroquinone had selectivity toward platelet aggregation
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292 dependent on collagen induced-signaling and a gem-diethyl derivative of tricyclic
293 hydroquinone was a dual agent, inhibiting both collagen- and TRAP-6-stimulated
294 platelet aggregation. These findings may be used for further development of new
295 drugs in platelet-related thrombosis diseases.
296 5. EXPERIMENTAL PROCEDURES
297 5.1. Chemistry
1
298 General methods. H and 13C NMR spectra were obtained from a spectrometer
299 operating at either 400.13 MHz (1H) or 100.61 MHz (13C). Chemical shifts are
300 reported as ppm downfield from TMS for 1H NMR and relative to the central CDCl3
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301 resonance (77.0 ppm) for C NMR. All melting points are uncorrected and were
302 determined using an Electrothermal 9100 apparatus. IR spectra (KBr discs) were
303 recorded on an FT-IR spectrophotometer; wavenumbers are reported in cm-1.
304 High-resolution mass spectra (HRMS) were obtained on an orthogonal time-of-
305 flight (ToF) mass spectrometer (QTof Micro, Micromass UK) or on a Thermo Q
306 exacting focus. Silica gel 60 (230-400 mesh ASTM) and TLC sheets silica gel 60
307 F254 were used for flash-column chromatography and analytical TLC, respectively.
308 Synthesis of acylhydroquinones III-VI. The already reported methodology of
309 obtention of acyl hydroquinones [35] was improved using microwave irradiation as
310 follows. To a 10 mL CEM microwave process vial, equipped with a magnetic stir
311 bar, were added one equivalent of hydroquinone (I) or dimethyl hydroquinone (II),
312 1.5 equivalent of carboxylic acid and 4 mL of Boron trifluoride dihydrate. The
313 mixture was irradiated under microwave for 25 min at 120°C. After completion of
314 the reaction, the mixture was allowed to cool to room temperature (RT) and
315 extracted with ethyl acetate, the organic phase was washed with distilled water and
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316 dried with anhydrous sodium sulfate, then was filtered and concentrated under
317 vacuum. Afterward, acylhydroquinones III-VI were purified by flash
318 chromatography with 6:1 hexane/ ethyl acetate as eluent.
319 Compound III (1-(2,5-dihydroxyphenyl)-1-heptanone). Hydroquinone (600 mg, 5.45
320 mmol), heptanoic acid (96%) (1.21 mL, 8.18 mmol) and 4 mL of boron trifluoride
321 dihydrate react yielding 547.4 mg of III (2.46 mmol, 45% yield). 1H-NMR δ(CDCl3):
322 0.90 (t, 3H, J = 6.7 Hz, 7-CH3); 1.27-1.45 (m, 6H, 3x CH2); 1.73 (p, 2H, J = 7.4 Hz,
323 3-CH2); 2.93 (t, 2H, J = 7.4 Hz , CH2CO); 4,78 (s, 1H, 5’-OH); 6.88 (d, J = 8.9 Hz,
324 1H, 3’-H); 7.02 (dd, 1H, J1 = 8.9 Hz, J2= 3.0 Hz, 4’-H); 7.23 (d, 1H, J = 3.0 Hz, 6’-
325 H); 11.98 (s, 1H, 2’-OH). 13C-NMR δ(CDCl3): 14.04; 22.51; 24.45; 28.97; 31.59;
326 38.48; 114.98; 119.03; 119.29; 142.82; 147.59; 156.45; 206.79. M.P.: 49.7-50.3
327 °C. HRMS (ESI): m/z calcd for C13H18O3 [M+H]+: 223.1289, found: 223.1286
328 Compound IV (1-(2,5-dihydroxyphenyl)-1-octanone). Hydroquinone (600 mg, 5.45
329 mmol), octanoic acid (98%) (1.32 mL, 8.18 mmol) and 4 mL of boron trifluoride
330 dihydrate react yielding 753.7 mg of IV (3.19 mmol, 58% yield). 1H-NMR δ(CDCl3):
331 0.90 (t, 3H, J = 6.9 Hz, 8-CH3); 1.23-1.45 (m, 8H, 4x CH2); 1.74 (p, 2H, J = 7,2 Hz,
332 3-CH2); 2.93 (t, 2H, J = 7.4 Hz, CH2CO); 4.81 (s, 1H, 5’-OH); 6.89 (d, 1H, J = 8.9
333 Hz, 3’-H); 7.03 (dd, 1H, J1 = 8.9 Hz, J2 = 3.0 Hz, 4’-H); 7.23 (d, 1H, J = 3.0 Hz, 6’-
334 H); 11.98 (s, 1H, 2’-OH). 13C-NMR δ(CDCl3): 14.08; 22.62; 24.50; 29.09; 29.26;
335 31.68; 38.48; 114.99; 119.03; 119.28; 124.83; 147.62; 156.43; 206.81. M.P.: 65.2-
336 66.2 °C. HRMS (ESI): m/z calcd for C14H20O3 [M+H]+: 237.1485, found: 237.1479.
337 Compound V (1-(2,5-dihydroxyphenyl)-1-nonanone). Hydroquinone (600 mg, 5.45
338 mmol), nonanoic acid (96%) (1.49 mL, 8.18 mmol) and 4 mL of boron trifluoride
1
339 dihydrate react yielding 627.2 mg of V (2.51 mmol, 46% yield). H-NMR δ(CDCl3):
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340 0.89 (t, 3H, J = 6.9 Hz, 9-CH3); 1.23-1.45 (m, 10H, 5x CH2); 1.73 (p, 2H, J = 7.4
341 Hz, 3-CH2); 2.93 (t, 2H, J = 7.4 Hz, 2-CH2CO); 4.83 (s, 1H, 5’-OH); 6.88 (d, 1H, J =
342 8.9 Hz, 3’-H); 7.02 (dd, 1H, J1 = 8.9 Hz, J2 = 3.0 Hz, 4’-H); 7.23 (d, 1H, J = 3.0 Hz,
13
343 6’-H); 11.98 (s, 1H, 2’-OH). C-NMR δ(CDCl3): 14.11; 22.66; 24.48; 29.16; 29.31;
344 29.39; 31.83; 38.48; 114.93; 119.03; 119.32; 124.73; 147.47; 156.59; 206.61. M.P.:
345 86.2-87.5 °C. HRMS (ESI): m/z calcd for C15H22O3 [M+H]+: 251.1642,
346 found:251.1640.
347 Synthesis of compounds 4-7. The new naphthalenones 4-7 (Scheme 1) were
348 obtained following the reported method, although slight variations were introduced
349 [19]. A mixture of acylhidroquinone (1 equiv) and Ag2O (2.5 equiv) in 20-30 mL of
350 dichloromethane was vigorously stirred for 1-2 h. at RT, yielding the corresponding
351 quinone. This mixture was filtered through celite, and dropwise added, without
352 isolation, to a solution of the 4-(2-methyl-2-propenyl)morpholine at 0°C, allowing it
353 to reach RT in a 30-60 min time off, being monitored by thin-layer chromatography.
354 Then, the solvent was evaporated under reduced pressure. The residue was
355 dissolved in a mixture of ethanol and hydrochloric acid and refluxed by 3 h. Next
356 was poured on an ice/water mixture. The product was extracted with five portions
357 of 20 mL of dichloromethane, which were dried with anhydrous sodium sulfate and
358 then filtered; the solvent was evaporated under reduced pressure and purified by
359 flash chromatography using 6:1 hexane/ethyl acetate as eluent.
360 Compound 4 (5,8-dihydroxy-4,4-dimethyl-2-pentylnaphthalen-1(4H)-one). Two
361 portions of 450 mg (900 mg, 4.05 mmoles) of acylhydroquinone III were oxidized
362 with 1.13 g of Ag2O each one in 40 mL of dichloromethane for 2 h., the filtered
363 solutions
were
put
together
and
dropwise
added
over
4-(2-
15

16
364 methylpropenyl)morpholine (572 mg, 4.05 mmol) for 1.5 h. Evaporation of the
365 solvent and a further reflux of the residue in a mixture of ethanol (20 mL) and
366 hydrochloric acid (1 mL), following the general method, achieved 831 mg of
367 hydroquinone 4 (3.03 mmol, 75% yield). 1H-NMR δ(CDCl3.): 0.91 (t, 3H, J = 6.7Hz,
368 -CH2CH3); 1.30-1,40 (m, 4H, 2x CH2); 1,48-1,57 (m, 2H, CH2); 1.60 (s, 6H, 2x 4-
369 CH3); 2.38 (t, 2H, J = 6,9 Hz, O=C –C-CH2); 4.86 (s, 1H, 5-OH); 6,62 (s, 1H, 3-H);
370 6.78 (d, 1H, J = 8.8 Hz, 6- or 7-H); 6.89 (d, 1H, J = 8.8 Hz, 6- or 7-H); 13.03 (s, 1H,
371 8-OH). 13C-NMR δ(CDCl3): 14.01; 22.45; 24.96; 28.01; 28.88; 31.51; 37.80;
372 115.50; 123.80; 133.69; 135.05; 145.83; 156.29; 157.39; 191.51. M.P.: 114.4-
373 115.5 °C. HRMS (ESI): m/z calcd for C17H23O3 [M+H]+: 275.1647, found: 275.1645.
374 Compound
5
(5,8-dihydroxy-4,4-dimethyl-2-hexylnaphthalen-1(4H)-one). Two
375 portions of 450 mg (900 mg, 3.81 mmol) of acylhydroquinone IV were oxidized with
376 1.1 g of Ag2O each one in 40 mL of dichloromethane for 2 h, the filtered solutions
377 were put together and dropwise added over 4-(2-methylpropenyl)morpholine (565
378 mg, 4.0 mmol) by 1.5 h. Evaporation of the solvent and a further reflux of the
379 residue in a mixture of ethanol (20 mL) and hydrochloric acid (1 mL), following the
380 general method, allowed to obtain 657.2 mg of hydroquinone 5 (2.27 mmol, 60%
1
381 yield). H-RMN δ(CDCl3): 0.89 (t, 3H, J = 7.0 Hz, CH2CH3); 1.20-1.40 (m, 8H, 4x
382 CH2); 1.46-1.57 (m, 2H, CH2); 1.60 (s, 6H, 2x 4-CH3); 2.38 (t, 2H, J = 6.9 Hz, O=C-
383 C-CH2); 4.86 (s, 1H, 5-OH); 6.62 (s, 1H, 3-CH); 6.77(d, 1H, J = 8.8 Hz, 6- or 7-H);
384 6.89 (d, 1H, J = 8.8 Hz, 6- or 7-H); 13.07 (s, 1H, 8-OH). 13C-RMN δ(CDCl3): 22.64;
385 25.11; 28.37; 28.96; 29.12; 29.32; 31.83; 37.52; 115.49; 115.61; 123.72; 133.81;
386 134.98; 145.36; 156.77; 156.96; 191.32. M.P.: 68.5-71.7 °C. HRMS (ESI): m/z
387 calcd for C18H25O3 [M+H]+Calc.: 289.1804, Found: 289.1800.
16

17
388 Compound 6 (5,8-dihydroxy-4,4-dimethyl-2-heptylnaphthalen-1(4H)-one). Acyl
389 hydroquinone V (500 mg, 2 mmol) was oxidized with Ag2O (1.16 g) in accordance
390 with the general methodology. Then the filtrated solution was added over a solution
391 of 4-(2-etylbutenil)morpholine (337.6 mg, 2 mmol). After evaporation of the solvent,
392 the residue was refluxed in ethanol HCl mixture yielding hydroquinone 6 (284.5
393 mg, 48% yield). 1H-NMR δ(CDCl3): 0,89 (t, 3H, J = 6.9 Hz, CH2CH3); 1.17-1,42 (m,
394 10H, 5x CH2); 1.46-1.57 (m, 2H, CH2); 1.59 (s, 6H, 2x 4-CH3); 2.38 (t, 2H, J = 6.9
395 Hz, O=C-C-CH2); 5.29 (s, 1H, 5-OH); 6.62 (s, 1H, 3-CH); 6.67 (d, 1H, J = 8.8 Hz, 6-
396 or 7-H); 6.90 (d, 1H, J = 8.8 Hz, 6- or 7-H); 13.07 (s, 1H, 8-OH). 13C-NMR
397 δ(CDCl3): 14.06; 22.51; 25.16; 28.06; 28.55; 28.94; 29.17; 31.56; 31.69; 37.49;
398 115.64; 115.78; 123.67; 133.48; 134.93; 145.18; 156.75; 157.10; 191.22. M.P.:
399 74.3-76.5 °C. HRMS (ESI): m/z calcd for C19H64O3 [M-H]+ : 303.1960, found:
400 303.4227.
401 Compound 7 (5,8-dihydroxy-4,4,6,7-tetramethylnaphthalen-1(4H)-one). 2’,5’-
402 dihydroxy-3’,4’-dimethylacetophenone (400 mg, 2.2 mmol) [35], was oxidized
403 according to general methodology with Ag2O (1.28 g). After filtering the
404 solution was added over a solution of 4-(2-methylpropenil)morfoline (314 mg,
405 2.2 mmol), after evaporation under vacuum a viscous residue was obtained.
406 451 mg of this residue was refluxed in a mixture of ethanol and hydrochloric
407 acid by 1 h. Following the same procedure as described before, 256 mg of
1
408 compound 7 (1.1 mmol, 78% yield) was obtained. H-NMR δ(CDCl3): 1,61 (s, 6H,
409 2x 4-CH3); 2.23 (s, 3H, Ar-CH3); 2.26 (s, 3H, Ar-CH3); 4.55 (s, 1H, 5-OH); 6.23 (d,
13
410 1H, J = 10 Hz, 2-H), 6.82 (d, 1H, J = 10 Hz, 3-H), 13.20 (s,1H, 8-OH). C-NMR
411 δ(CDCl3): 11.56; 13.04; 25.17; 38.03; 112.68; 123.43; 124.04; 131.51; 131.64;
17

18
412 143.52; 155.12; 160.73; 191.11. M.P.: 200.5-201.6 °C. HRMS (ESI): m/z calcd for
413 C14H16O3 [M+1]+: 233.1178, found: 233.1166.
414 Synthesis of compounds 1-3 and 8-14. Compounds 1-3 and 8-14 were
415 synthesized as previously reported [19, 22, 36-38]. Structural characterization and
416 procedure to synthesize compounds 1-3 and 8-14 were included as supplementary
417 information.
418 5.2. Biological assays
419 5.2.1. Preparation of human platelets
420 Platelets were obtained from the human blood of healthy volunteers (two weeks
421 drug-free) with written informed consent, as we have previously described [59].
422 Briefly, whole blood was collected with acid-citrate-dextrose (ACD) 4:1 v/v and
423 centrifuged at RT for 10 min x 240 g (low acceleration and deceleration) to obtain
424 platelet-rich plasma (PRP). PRP was then centrifuged at RT for 8 min x 800 g and
425 platelets’ pellet was resuspended in calcium-free Tyrode’s buffer: ACD (9:1 v/v)
426 and washed by centrifugation at RT for 8 min x 800 g. Platelets were counted with
427 hematology analyzer (Hematological counter Mindray BC-3000 Plus, Japan) and
428 used within 3 h.
429 5.2.2. Cytotoxic activity
430 Washed platelets (3x108 platelets/mL) were incubated for 10 min at 37°C with the
431 highest concentration of compounds tested (200 µM). Then, platelets were
432 centrifuged at 800 x g for 8 min and the resulting supernatant (100 µL) was
433 analyzed with lactate dehydrogenase (LDH) cytotoxicity assay kit (Cayman
434 Chemical, USA). The absorbance of the reaction was measured at 490 nm in a
435 microplate reader (Microplate Reader Thermo Scientific Multiskan Go, Finland)
18

19
436 with 10% Triton X-100 was used as a positive control.
437 5.2.3. Antiplatelet activity
438 Platelet aggregation. Platelet aggregation was evaluated using
a
lumi-
439 aggregometer (Chrono-Log, Haverton, PA, USA) and monitored by light
440 transmission [59]. Washed platelets (3x 108 platelets/mL) were pre-incubated for 5
441 min with CaCl2 (2 mM) plus compounds (0.2 to 200 µM). Platelet aggregation was
442 induced with TRAP-6 (5 µM) or collagen (1 µg/mL) and transmittance followed for 6
443 min. DMSO 0.2% was used as a vehicle.
444 Phosphatidylserine externalization. The externalization of PS in platelets was
445 determined by flow cytometry [60]. Washed platelets (3x108 platelets/mL) were
446 pre-incubated for 5 min with CaCl2 (2 mM) plus compounds. Then, 50 µL of the
447 sample was diluted with 150 µL of annexin V binding buffer and incubated in the
448 dark with annexin V-FITC and anti-CD61-PE for 25 min. The samples were
449 acquired and analyzed in the Accuri C6 flow cytometer (BD, Biosciences, USA).
450 Platelet activation markers. The GPIIb/IIIa activation and P-selectin expression,
451 considered as platelet activation markers [61] were evaluated by flow cytometry in
452 washed platelets incubated with PAC‐1 antibody or anti-CD62-FITC for 25 min at
453 RT in the dark. Platelets were identified with anti-CD61-PE and the samples were
454 acquired and analyzed in the Accuri C6 flow cytometer [62].
455 Platelet secretion (intracellular and extracellular ATP, and dense granules).
456 Extracellular ATP was determined by luminescence detection using Chrono-lume
457 (Chrono-Log, Haverton, PA, USA) and monitored in a lumi-aggregometer [59].
458 Intraplatelet ATP levels were determined with the CellTiter-Glo Luminescent Cell
459 Viability Assay kit (Promega, USA) according to the manufacturer’s instructions.
19

20
460 Dense granules secretion was determined by Accuri C6 flow cytometer using anti-
461 CD63-PE.
462 Intracellular calcium levels. Washed platelets (5x107 platelets/mL) were mixed
463 with Fluo-3-AM (0.4 µM) and incubated at RT for 30 min followed by 5 min
464 incubation with vehicle (DMSO 0.2%) or different concentrations of compounds.
465 After
collecting
data
for
15
seconds,
carbonylcyanide
p‐
466 triflouromethoxyphenylhydrazone (FCCP,1 µM) was added and the data were
467 obtained for further 60 s. The effect of compounds on cytosolic calcium
468 mobilization was calculated with respect to the control.
469 Reactive oxygen species. ROS production was determined in washed platelets at
470 5x107 platelets/mL using 10 µM dihydroethidium (DHE) in the presence of
471 compounds for 20 min at 37°C. ROS formation was analyzed by the Accuri C6 flow
472 cytometer (BD, Biosciences, USA) [63].
473 Mitochondrial membrane potential. Mitochondrial membrane potential (∆Ψm)
474 was evaluated using the cell-permeant dye tetramethylrhodamine methyl ester
475 perchlorate (TMRM) as previously described by us [23]. Washed platelets were
476 loaded with TMRM (100 nM) and then incubated with DMSO 0.2% (Control),
477 compounds or FCCP (1 µM) at 37°C for 20 min. Samples were then analyzed by
478 the Accuri C6 flow cytometer (BD, Biosciences, USA).
479 Intracellular NAD(P)H levels. Intracellular NAD(P)H levels were measured by
480 auto-fluorescence using specific excitation and emission wavelengths of 340/428
481 nm as described [23].
482 Statistical analysis
483 The data obtained were presented as the mean ± standard error of the mean
20

21
484 (SEM) of three individual experiments and analyzed using Prism 6.0 software
485 (GraphPad Inc., San Diego CA, USA). The half-maximal inhibitory concentration
486 (IC50) was calculated from the dose-response curves. Differences between
487 samples were analyzed using a paired t-test [64]. P values <0.05 were considered
488 significant.
489 Conflict of interest
490 The authors have no conflicts of interest to disclose.
491 Acknowledgments
This work was supported by CONICYT/FONDECYT grants #1180427 (EF),
#1180069 (RAM), CONICYT/REDES #170003 (EF) and #170002 (IP),
FONDECYT postdoctoral fellowships #3170813 (FAU) and # 3170264 (JR-L),
CONICYT/PCI #REDBIO0027 (FAU and RAM) and Programa de Investigación
Asociativa en Cáncer Gástrico #PIA-CG, RU2107, Universidad de Talca (RAM).
CSIC Grupos N°536 Comisión Sectorial de investigación Científica (CSIC)-
Uruguay (AT).
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715
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717
718
719
720
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737
738
Table 1. Inhibitory effect of ortho-carbonyl hydroquinones on collagen- and
739
TRAP-6-stimulated platelet aggregation.
Compounds
IC50 µM
S.I.
(IC50 TRAP-6/IC50 Collagen)
Collagen
TRAP-6
(5 µM)
(1 µg/mL)
14.30±5.64
35.34±0.96
1.77±2.09
11.97±0.93
17.12±2.87
25.51±9.29
20.58±5.82
4.38±3.32
11.50±1.45
>100
1
>100
31.95±6.31
>100
7.00
0.90
56.50
8.35
0.69
3.92
4.86
22.83
1.37
n.d.
2
3
7
>100
8
11.88±4.59
>100
9
10
>100
11
>100
12
15.78±1.78
>100
13
14
>100
>100
n.d.
Ticagrelor (positive control)
2.0±1.26
4.0±2.43
2.00
740 The IC50 values are presented as the mean ± SEM of three independent
741 experiments. S.I. (selectivity index) is the calculated ratio IC50 TRAP-6/IC50
742 collagen. n.d.: not determined.
743
744
745
746
27

28
747
748
749
750 Figure 1. Chemical structures of ortho-carbonyl hydroquinones studied in this
751 work.
752
753
754
755
756
757
758
28