Synthesis and Biological Evaluation of Thio-Derivatives of 2-Hydroxy-1,4-Naphthoquinone (Lawsone) as Novel Antiplatelet Agents

Introduction

Cardiovascular diseases (CVD), one of the leading causes of deaths from non-communicable disease in the world, all have thrombus formation as a common process, with platelet activation at the dysfunctional vessel wall being one of the main steps during thrombosis (ISTH Steering Committee for World Thrombosis Day, 2014; Diamond, 2016).

Platelets are fragments of megakaryocytes circulating within the bloodstream aiding in maintaining hemostasis with both blood components and endothelial cells (Paes et al., 2019). Although platelets have a normal function in primary hemostasis, increased platelet activation has been widely recognized as a key player in the initiation of thrombotic events (Franco et al., 2015). An increase in reactive oxygen species (ROS), [e.g., superoxide and hydrogen peroxide production at the mitochondrial electron transport chain] has been associated with platelet activation (Geisler et al., 2007; Becatti et al., 2013; Caruso et al., 2015). On the other hand, it has been observed that some quinone and hydroquinone compounds protect mitochondrial damage and platelet activation generated by ROS (Kim et al., 2001; Cocheme et al., 2007); in this way, some structure-activity relationship studies with quinone derivatives have been reported (Chang et al., 1997; Cho et al., 1997; Kim et al., 2001). However, given the variability of the mechanisms of action of quinones-hydroquinones it has been suggested that there is a low selectivity on their antiplatelet activity (Bolibrukh et al., 2015; Chang et al., 2019; Mendez et al., 2020). Recent work has shown conflicting results (Fuentes et al., 2018). In this way, 3'-methoxyflavone quinone inhibited arachidonic acid-induced platelet aggregation at low micromolar concentration with an IC₅₀ of ~ 10 μM (Chang et al., 2000). Moreover, the presence of OH groups in addition to their methylation or glycosylation in anthraquinones increases the antiplatelet activity (Yun-Choi et al., 1990; Xu et al., 2014). The available drugs with therapeutic ability to regulate platelet activation are still scarce, thus this research area is the high relevance (Mousa, 1999; Moura et al., 2016).

Lawsone [2- Hydroxy-1,4-naphthoquinone, (1)] or hennotannic acid, is a natural compound from the naphthoquinones family and can be obtained from leaves of henna (Lawsonia spp., family Lythraceae) as a red-orange pigment. The structure of Lawsone (1) has served for the development of new bioactive compounds and its scaffold is present in compounds with several valuable biological activities such as lapachol (2), atovaquone (3), parvaquone (4), and NQ1 (2-OH-3-(2-methyl-trifluorooctyl)-naphthoquinone) (5) (Figure 1) (Jordão et al., 2015). Lawsone (1) and its synthetic derivatives have shown promising biological results with antibacterial, antifungal, antitumor, and antiparasitic effects (Rahmoun et al., 2012; Barani et al., 2018; Al Nasr et al., 2019), their use, or study as antiplatelet agents have not been described, although the antiplatelet activity of other quinones has been reported (Fuentes et al., 2018). The current work aims to evaluate the antiplatelet effects of a series of lawsone thio-derivatives, which were obtained using on-water methodology, which was optimized by microwave irradiation for those cases that yielded poor results with conventional heating.

FIGURE 1

Figure 1. Lawsone and bioactive lawsone derivatives. NQ1: 2-OH-3-(2-methyl-trifluorooctyl)-naphthoquinone.

Materials and Methods

Chemicals, Reagents, General Procedures, and Apparatus

¹H and ¹³C NMR spectra were obtained from a spectrometer operating at either 400.13 MHz (¹H) or 100.61 MHz (¹³C). Measurements were carried out at 300°K. Chemical shifts are reported as ppm downfield from TMS for ¹H NMR. ¹³C NMR spectra were recorded with the NMR spectrometer calibrated with CDCl₃. All melting points are uncorrected and were determined using an Electrothermal 9100 apparatus. IR spectra (KBr discs) were recorded on an FT-IR spectrophotometer; wave numbers are reported in cm⁻¹. High-resolution mass spectra were obtained on an orthogonal time-of-flight (TOF) mass spectrometer (QTOF Micro, Micromass UK) or a Q Exactive Focus (Thermo Scientific, USA). Silica gel 60 (230–400 mesh ASTM) and TLC aluminum sheets silica gel 60 F254 were used for flash-column chromatography and analytical TLC, respectively (Urra et al., 2016; Mendez et al., 2020).

Synthesis of Compounds

General Procedure Using Conventional Heating

A mixture of 2.87 mmol of lawsone and 1.44 mmol of the respective thiol in 15 mL of water was heated at 50°C overnight, and then the mixture was extracted with ethyl acetate (30 mL × 3) the organic phase was dried with anhydrous sodium sulfate and then evaporated under a vacuum.

General Procedure With Microwave Irradiation

In a 100 mL ACE pressure tube with a magnetic stir bar, a mixture of 1.44 mmol of lawsone and 0.72 mmol of the respective thiol in water (6 mL) was heated at 50°C for 20 min under microwave irradiation (CEM Discover, Matthews, NC, USA). Then the mixture was extracted with ethyl acetate (30 mL × 3), and the organic phase dried with anhydrous sodium sulfate and evaporated under a vacuum (Tandon and Maurya, 2009; Tandon et al., 2010). Afterward, lawsone thio-derivatives 1–8, were purified by flash chromatography with ethyl acetate:hexane:methanol 5:2:2 (Table 1).

TABLE 1

Table 1. Synthesis of lawsone thio-derivatives.

2-Hydroxy-3-(phenylthio)naphthalene-1,4-dione (1) (Tandon and Maurya, 2009)

By reaction of lawsone with benzenethiol compound 1 was obtained. ¹H NMR δ (DMSO-d6): 7.2-7.3 (m, 5H), 7.9 (d, J = 7.3 Hz, 1H), 8.0 (t, J = 7.3 Hz, 2H), 8.1 (t, J = 7.4 Hz, 1H).

2-((2-bromophenyl)thio)-3-hydroxynaphthalene-1,4-dione (2)

By reaction of lawsone with 2-bromobenzenethiol, compound 2 was obtained: red solid; mp 270.8 (d); ¹H NMR δ (DMSO-d6): 6.71 (t, J = 7.4 Hz, 1H), 6.78 (d, J = 7.6 Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H); ¹³C NMR δ (DMSO-d6): 105.51, 122.32, 124.45, 126.28, 126.41, 126.66, 127.64, 129.87, 131.19, 134.60, 136.05, 143.29, 172.94, 180.09, 186.86; IR (KBr, cm⁻¹): 3426, 2919, 2850, 1712, 1656, 1646, 1583, 1531, 1276, 744; HRMS (ESI) m/z calculated. for C₁₆H₉BrO₃S M⁺: 359.9456, found: 359.9459.

2-((3-bromophenyl)thio)-3-hydroxynaphthalene-1,4-dione (3)

By reaction of lawsone with 3-bromobenzenethiol, compound 3 was obtained: red solid; mp 231.3°C (d); ¹H NMR δ (DMSO-d6): 6.92 (t, J = 7.8 Hz, 1H), 6.99 (ddd, J = 8.1 Hz, J₂ = J₃ = 1.6 Hz, 1H), 7.02 (ddd J₁ = 7.6Hz, J₂ = J₃ = 1.2 Hz, 1H), 7.10 (dd, J₁ = J₂ = 1.6 Hz, 1H), 7.52 (td, J₁ = 7.4 Hz J₂ = 1 Hz, 1H), 7.65 (td, J₁ = 7.6 Hz J₂ = 1 Hz, 1H), 7.76 (s,1H), 7.89 (d, J = 7.6 Hz, 1H), 8.04 (dd, J₁ = 7.6 Hz, J₂ = 1 Hz, 1H); ¹³C NMR δ (DMSO-d6): 107.34, 116.94, 126.41, 126.94, 127.65, 130.57, 131.12, 131.46, 135.12, 135.72, 138.99, 180.75, 188.11; IR (KBr, cm⁻¹): 3428, 2919, 2850, 1664, 1587, 1529, 1276, 738; HRMS (ESI) m/z calculated. for C₁₆H₉BrO₃S M⁺: 359.9456, found: 359.9474.

2-((4-Bromophenyl)thio)-3-hydroxynaphthalene-1,4-dione (4)

By reaction of lawsone with 4-bromobenzenethiol, compound 4 was obtained: red solid; mp 271.2°C (d); ¹H RMN δ (DMSO-d6): 6.95 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 7.50 (t, J = 7.4 Hz, 1H), 7.53 (s, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.86 (d, J = 7.4 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H); ¹³C RMN δ (DMSO-d6): 116.95, 126.41, 126.95, 127.66, 130.57, 131.12, 131.46, 135.12, 135.71, 138.93, 180.77, 188.14; IR (KBr, cm⁻¹): 3430, 2919, 2850, 1643, 1585, 1536, 1274, 740; HRMS (ESI) m/z calcd. for C₁₆H₉BrO₃S M⁺: 359.9456, found: 359.9479.

Methyl 2-((3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)thio)benzoate (5)

By reaction of lawsone with methyl thiosalicylate, compound 5 was obtained: red solid; mp 235°C (d); ¹H NMR δ (DMSO-d6): 3.84 (s, 3H, CH₃), 6.95 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 7.4, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.70 (t, J = 7.5 Hz, 1H), 7.80(d, J = 7.8 Hz, 1H), 7.91(d, J = 7.6 Hz, 1H), 8.00(d, J = 7.6 Hz, 1H), 8.07(s, 1H);¹³C NMR δ (DMSO-d6): 51.94, 105.71, 122.66, 126.13, 126.15, 126.17, 126.52, 130.94, 131.20, 131.47, 134.49, 136.27, 144.90, 166.70, 173.26, 179.86, 186.42; IR (KBr, cm⁻¹): 3428, 2919, 2850, 1706, 1662, 1587, 1531, 1276, 1251; HRMS (ESI) m/z calcd. for C₁₈H₁₂O₅S: 340.0405, found: 340.0392.

2-((2-Fluorophenyl)thio)-3-hydroxynaphthalene-1,4-dione (6)

By reaction of lawsone with 2-fluorobenzenethiol, compound 6 was obtained: red solid; mp 260.8°C (d); ¹H NMR δ (DMSO-d6): 6.61–6.64 (m, 1H), 6.70-6.80 (m, 2H), 6.84 (t, J = 7.5 Hz, 1H), 7.43-7.51 (m, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 8.03(d, J = 7.5 Hz, 1 H); ¹³C NMR δ (DMSO-d6): 105.00, 114.62 (d, J = 21.8 Hz), 124.05 (d, J = 0.9 Hz), 125.08 (d, J = 7.3 Hz), 126.39, 126.47, 126.70, 127.68, 130.93, 131.21, 134.68, 135.80, 159.22 (d, J = 241.2 Hz), 172.96, 180.70, 187.31; IR (KBr, cm⁻¹): 3428, 2917, 2850, 1673, 1587, 1533, 1469, 1276, 1213; HRMS (ESI) m/z calcd. for C₁₆H₉BrO₃S: 300.0256, found: 300.0270.

2-((3-Fluorophenyl)thio)-3-hydroxynaphthalene-1,4-dione (7)

By reaction of lawsone with 3-fluorobenzenethiol, compound 7 was obtained: red solid; mp 268.7°C(d); ¹H NMR δ (DMSO-d6): 6.50-6.77(m, 2H), 6.82 (t, J = 7.0 Hz, 1H), 7.34 (s, 1H), 7.46(t, J = 6.9 Hz, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.84 (d, J = 7.0Hz, 1H), 8.04 (d, J = 7.3 Hz, 1H); ¹³C NMR δ (DMSO-d6): 102.26, 114.87 (d, J = 21.1 Hz), 124.55 (d, J = 2.9 Hz), 124.69 (d, J = 7.3 Hz), 126.21, 126.45, 127.14 (d, J = 2.9 Hz), 127.95 (d, J = 16 Hz), 131.37, 131.61, 134.75, 136.35, 158.92 (d, J = 239.8 Hz), 173.29, 179.13, 185.73; IR (KBr, cm⁻¹): 3430, 2360, 2339, 1664, 1587, 1533, 1469, 1278, 1222; HRMS (ESI) m/z calculated. for C₁₆H₉FO₃S: 300.0256, found: 300.0233.

2-((4-Fluorophenyl)thio)-3-hydroxynaphthalene-1,4-dione (8)

By reaction of lawsone with 4-fluorobenzenethiol, compound 8 was obtained: red solid; mp 268.0°C (d); ¹H NMR δ (DMSO-d6): 6.76 (t, J = 8.6 Hz, 2H), 7.03 (dd, J₁ = 8 Hz, J₂ = 5.5 Hz, 1H), 7.50(t, J = 7.4 Hz, 1H), 7.63(t, J = 7.5 Hz, 1H), 7.79(s, 1H), 7.88(d, J = 7.6 Hz, 1H), 8.0(d, J = 7.6 Hz, 1H); ¹³C NMR δ (DMSO-d6): 106.91, 115.14, (d, J = 21.80 Hz), 126.09, 126.54, 127.40 (d, J = 7.3 HZ), 130.98, 131.29 (d, J = 13.8 Hz), 134.32, 135.51, 136.18, 159.98 (d, J = 241.2 Hz), 172.83, 179.97, 186.60; IR (KBr, cm⁻¹): 3424, 2919, 2850, 2358, 1661, 1587, 1526, 1381, 1273, 1221; HRMS (ESI) m/z calculated. for C₁₆H₉FO₃S: 300.0256, found: 300.0262.

Human Washed Platelets

Platelets were obtained from citrated whole blood of healthy volunteers (two weeks drugs-free) with written informed consent, as previously described (Fuentes et al., 2014a).

Lactate Dehydrogenase (LDH)-Based Cytotoxicity Assay

Washed platelets (200 × 10⁹ platelets/L) were incubated for 10 min at 37°C with DMSO (vehicle) 0.2% or 100 μM of compounds. Then, platelets are centrifuged (800 × g for 8 min), and 50 μL aliquots of the resulting supernatant analyzed by the LDH assay (Cayman Chemical, USA). The reported results correspond to the percentage of total enzyme activity from a control incubation lysed with 0.3% Triton X-100 (Kim et al., 2009; Mendez et al., 2020).

Platelet Aggregation

Platelet aggregation was evaluated using a lumi-aggregometer (Chrono-Log, Haverton, PA, USA) and monitored by light transmission according to Fuentes et al. (Fuentes et al., 2014a; Mendez et al., 2020). Briefly, washed platelets (200 × 10⁹ platelets/L) were pre-incubated for 5 min at 37°C and pH 7.4 with vehicle or different doses of compounds; then platelet aggregation was induced by TRAP-6 (5 μM) or collagen (1 μg/mL) and measured for 5 min at 37°C while stirring.

Flow Cytometry Study

Externalization of phosphatidylserine, glycoprotein (GP) IIb/IIIa activation, P-selectin expression, CD63 release, and ROS levels on platelets were determined by flow cytometry (Ritchie et al., 2000; Fuentes et al., 2014b; Walsh et al., 2014). All results were analyzed in the Accuri C6 flow cytometer (BD, Biosciences, USA).

Statistical Analysis

The data obtained were presented as a mean ± standard deviation (SD) of three independent experiments and analyzed using Prism 8.3 software (GraphPad Inc., San Diego CA, USA). The half-maximal inhibitory concentration (IC₅₀) was calculated from dose-response curves. Differences between samples were analyzed using Tukey or paired t-test (Vaiyapuri et al., 2013). P-values < 0.05 were considered as significant (Fuentes et al., 2014b; Munoz-Gutierrez et al., 2017).

Results and Discussion

Synthesis

Several attempts to reproduce the reported yield in the reaction of unsubstituted benzenethiol with lawsone were unsuccessful, even raising the described time to overnight. However, the reaction with fluoro-substituted benzenethiols gave good yields, contrary to Bromo substituted derivatives which gave very low yields. These results show a correlation between yields of the reaction and the solubility of benzenethiols in water. To improve the low yields obtained with the less soluble thiols, microwave irradiation, instead of conventional heating was used, in this way better yields and very reduced reaction times were obtained. The structural assignment of the pure new products relied on the disappearing of the quinone proton of lawsone and the signals reported by the aromatic ring of the thiols, besides high-resolution mass spectrometry. ¹H and ¹³C NMR spectrum of each compound were included as Supplementary Information.

Cytotoxicity Activity on Platelets

Before analyzing the potential antiplatelet activity of the synthesized compounds, we evaluated the cytotoxic activity that lawsone and its derivative may have on platelets. After incubating platelets for 10 min at 37°C with each compound, cytotoxicity detection by LDH leakage showed that compounds 1, 2, 4, 6, 7, and 8 at the higher concentration tested (100 μM) did not exert cytotoxicity on platelets (Table 2). Quinones represent a class of organic compounds that have previously shown that through quite complex mechanisms can have certain levels of cytotoxicity (Bolton et al., 2000; Bolton and Dunlap, 2017). Quinone-based compounds have been regarded as among the most troublesome of all pan-assay interference compounds (PAINS) chemotypes, limiting their medical potential (Baell and Nissink, 2018; Qin et al., 2018). Alternatively, quinones are highly redox-active molecules which can generate oxidative damage in intracellular macromolecules such as lipids, proteins, and DNA (Bolton et al., 2000). In this study, we showed that lawsone and compounds 3 and 5 had a cytotoxic activity with an increase of LDH released by damaged platelets.

TABLE 2

Table 2. Cytotoxic activity of thio-derivatives of 2-hydroxy-1,4-naphthoquinone (lawsone) on washed platelets.

Antiplatelet Activity of New Thio-Derivatives of 2-Hydroxy-1,4-Naphthoquinone

Antiplatelet therapy is called the treatment for the prevention of platelet activation-associated thrombosis (Harrington et al., 2003). Although aspirin and clopidogrel have been the cornerstone in the management of platelet regulation, a high number of patients continued suffering recurrent thrombotic events (Fox et al., 2004; Price et al., 2011). Since the available therapeutic drugs to control non-desired platelet activation remains limited, the search for new antiplatelet agents is necessary (Mousa, 1999; Moura et al., 2016). In this regard, some structure-activity relationship studies with quinone derivatives have shown that the variability of inhibition of platelet activation by these type of compounds is governed by the nature of the substituent (Kim et al., 2001; Fuentes et al., 2018), as explained below: (i) The oxidation of the B-ring of hydroxyl-methoxyisoflavone to methoxyflavone quinone, and the conversion of flavone and isoflavones into their corresponding flavanquinone or isoflavanquinones, result in an increase and potent antiplatelet activity (Chang et al., 2000). (ii) The inhibition of platelet aggregation by 2-alkoxy-1,4-naphthoquinone increases with 2-alkoxy chain length (Lien et al., 2002). (iii) The inclusion of a free amino group in the thiosulfonate moiety of quinone (4-aminobenzenesulfonothioic acid S-[(9,10-dihydro-1,4-dimethoxy-9,10-dioxo2-anthracenyl)methyl] ester) increased its antiplatelet activity (Bolibrukh et al., 2015), (iv) The inhibition of platelet activation by alpha-tocopherol quinone is greater than that of vitamin E (Rao et al., 1981), and (v) 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 (Mendez et al., 2020).

Lawsone is a natural naphthoquinone that is easily obtained from natural sources and is currently commercially available in bulk. It has been used as the starting material for the synthesis of a variety of biologically active compounds and materials with interesting properties in fungi, bacteria, and viruses (Jordão et al., 2015). The synthesis reaction from lawsone described in this article has allowed the development of a variety of lawsone thio-derivatives that could be potentially useful for the treatment of platelets aggregation-associated thrombosis diseases. In this way, we observed that the platelet antiaggregant activity depends on the nature and position of the substituent at the phenyl moiety (Table 3). All tested compounds showed higher effectiveness (lower IC₅₀ values) against platelet aggregation induced by collagen than TRAP-6 (Table 3). It has described that given the variability of quinone in biological activities it is likely that there is a low selectivity in its antiplatelet activity (Ma and Long, 2014). Our work, as shown in Table 3, shows that compound 6 presents a specific inhibition of collagen-induced platelet aggregation. Atherothrombosis is initiated by collagen exposure from endothelial damage to circulating platelets. These platelets, via the GPVI receptor, bind directly to collagen triggering platelet activation (Nieswandt and Watson, 2003). In this context, our results suggest that pretreatment of platelets with compound 6 down-regulates the GPVI-mediated signaling pathway (Jung et al., 2017).

FIGURE 2

Figure 2. Inhibition of platelet aggregation by compound 4. (A) and (B) IC₅₀ of compound 4 was determined from the dose-response curves. (C) Representative curves for platelet aggregation pre-incubated with compound 4 at 2, 20, and 200 μM, and induced by collagen (1 μg/mL) or TRAP-6 (5 μM).

FIGURE 3

Figure 3. Antiplatelet activity of compound 4. (A) Externalization of phosphatidylserine was evaluated by annexin-V binding. (B) Levels of CD63 on the surface of platelets after activation by TRAP-6 (5 μM) or collagen (1 μg/mL). (C) P-selectin expression was measured by flow cytometry in platelets stimulated with TRAP-6 (5 μM) or collagen (1 μg/mL). (D) GPIIb/IIIa activation stimulated by TRAP-6 (5 μM) or collagen (1 μg/mL) was determined by flow cytometry using the PAC-1 antibody. The graph depicts the mean ± SD of n = 3 experiments. The black graph is the control of resting platelets. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001 vs. 0 (vehicle DMSO 0.2%).

TABLE 3

Table 3. Inhibition of platelet aggregation by thio-derivatives of 2-hydroxy-1,4-naphthoquinone (lawsone) on TRAP-6- or collagen-stimulated platelets.

Small structural changes led to the synthesis of non-toxic lawsone thio-derivatives with potent antiplatelet activity (Tables 1, 3). Our experimental design led to the synthesis of antiplatelets compounds, being compound 4 the most potent among the non-toxic compounds. Compound 4 presents the lower IC₅₀ values, being 15.03 ± 1.52 μM for TRAP-6, and 5.58 ± 1.01μM for collagen respectively (Figure 2). Thus, we decided to deeply analyze the effects of compound 4 in platelets. In addition to its non-toxic activity (Table 2), we demonstrated that compound 4 was unable to affect phosphatidylserine exposure (Figure 3A), a modification associated with platelet mitochondrial apoptotic-like events (Augereau et al., 2004). In non-activated platelets, CD63 is present on the dense granules and exposed in membranes after platelet activation (Israels and McMillan-Ward, 2005). CD63 is essential for P-selectin function, which is a thrombo-inflammatory molecule that participates in platelet activation and aggregation (Théorêt et al., 2011). As shown in Figure 3B, compound 4 decreased the expression of CD63 on platelets' membrane after activation with either TRAP-6 or collagen. As expected, platelets' P-selectin expression on activated platelets by TRAP-6 or collagen was inhibited by compound 4 at 10 and 40 μM (Figure 3C). In activated platelets, GPIIb/IIIa is very important at the final step of platelet activation leading to aggregation (Chen et al., 2015). We analyzed the activated form of GPIIb/IIIa by studying the binding of FITC-labeled PAC-1 antibody by flow cytometry (Fuentes et al., 2014a). Figure 3D demonstrated that compound 4 inhibited TRAP-6- and collagen-induced PAC-1 binding to platelets at compound levels similar to those that inhibited platelet aggregation (Table 3).

Limitation and Perspectives

The set of experiments presented were designed to demonstrate that thio-derivatives of lawsone are useful novel scaffolds to synthesize antiplatelet compounds. The small number of compounds comprising the series analyzed was limited in most part due to the difficulty in accessing human platelets from a significant number of donors. Besides this fact, we were able to determine that the modification inserted for compound 4 synthesis improved the activity on platelet aggregation. Future studies will be directed toward increasing the series of lawsone-derivatives and comparing their effects in different biological models. Although it was not in the scope of the current work, the lack of testing of cytotoxicity, bio-disponibility, and mechanisms of action on in vivo systems decrease the impact of compound 4 for pharmacological purposes. However, these will be solved in the future with studies in cells and animal models at different pathophysiological conditions.

In conclusion, our study revealed that thio-derivatives of lawsone are useful novel scaffolds to synthesize antiplatelet compounds, with compound 4 being the most potent.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement

The studies involving human participants were reviewed and approved by Scientific ethics committee, Universidad de Talca. The patients/participants provided their written informed consent to participate in this study.

Author Contributions

EF, RA-M, and MM-Ci: conceptualization. MM-Cá and DM: formal analysis. EF, and RA-M: writing—original draft preparation and funding acquisition. AT: writing—review and editing. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by ANID/CONICYT, FONDECYT grants #1180427 (EF) and #1180069 (RA-M), and ANID/REDES #170003 (EF). FONDECYT 11170142 (MM-Ci). AT was funded by Comision Sectorial de Investigación Científica (CSIC)-Uruguay, CSIC-Grupos N°536.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2020.00533/full#supplementary-material

References

Al Nasr, I., Jentzsch, J., Winter, I., Schobert, R., Ersfeld, K., Koko, W. S., et al. (2019). Antiparasitic activities of new lawsone Mannich bases. Archiv. Pharmazie 352:1900128. doi: 10.1002/ardp.201900128

PubMed Abstract | CrossRef Full Text | Google Scholar

Augereau, O., Rossignol, R., DeGiorgi, F., Mazat, J. P., Letellier, T., and Dachary-Prigent, J. (2004). Apoptotic-like mitochondrial events associated to phosphatidylserine exposure in blood platelets induced by local anaesthetics. Thromb. Haemost 92, 104–113. doi: 10.1160/TH03-10-0631

PubMed Abstract | CrossRef Full Text | Google Scholar

Baell, J. B., and Nissink, J. W. M. (2018). Seven year itch: Pan-Assay Interference Compounds (PAINS) in 2017-utility and limitations. ACS Chem. Biol. 13, 36–44. doi: 10.1021/acschembio.7b00903

PubMed Abstract | CrossRef Full Text | Google Scholar

Barani, M., Mirzaei, M., Torkzadeh-Mahani, M., and Nematollahi, M. H. (2018). Lawsone-loaded Niosome and its antitumor activity in MCF-7 breast Cancer cell line: a Nano-herbal treatment for Cancer. Daru 26, 11–17. doi: 10.1007/s40199-018-0207-3

PubMed Abstract | CrossRef Full Text | Google Scholar

Becatti, M., Fiorillo, C., Gori, A. M., Marcucci, R., Paniccia, R., Giusti, B., et al. (2013). Platelet and leukocyte ROS production and lipoperoxidation are associated with high platelet reactivity in Non-ST elevation myocardial infarction (NSTEMI) patients on dual antiplatelet treatment. Atherosclerosis 231, 392–400. doi: 10.1016/j.atherosclerosis.2013.09.030

PubMed Abstract | CrossRef Full Text | Google Scholar

Bolibrukh, K., Polovkovych, S., Khoumeri, O., Halenova, T., Nikolaeva, I., Savchuk, O., et al. (2015). Synthesis and anti-platelet activity of thiosulfonate derivatives containing a quinone moiety. Sci. Pharm. 83, 221–231. doi: 10.3797/scipharm.1411-14

PubMed Abstract | CrossRef Full Text | Google Scholar

Bolton, J. L., and Dunlap, T. (2017). Formation and biological targets of quinones: cytotoxic versus cytoprotective effects. Chem. Res. Toxicol. 30, 13–37. doi: 10.1021/acs.chemrestox.6b00256

PubMed Abstract | CrossRef Full Text | Google Scholar

Bolton, J. L., Trush, M. A., Penning, T. M., Dryhurst, G., and Monks, T. J. (2000). Role of quinones in toxicology. Chem. Res. Toxicol. 13, 135–160. doi: 10.1021/tx9902082

PubMed Abstract | CrossRef Full Text | Google Scholar

Caruso, R., Rocchiccioli, S., Gori, A. M., Cecchettini, A., Giusti, B., Parodi, G., et al. (2015). Inflammatory and antioxidant pattern unbalance in “clopidogrel-resistant” patients during acute coronary syndrome. Mediators Inflamm. 2015:710123. doi: 10.1155/2015/710123

PubMed Abstract | CrossRef Full Text | Google Scholar

Chang, C. Y., Huang, L. J., Wang, J. P., Teng, C. M., Chen, S. C., and Kuo, S. C. (2000). Synthesis and anti-platelet, anti-inflammatory and anti-allergic activities of methoxyisoflavanquinone and related compounds. Chem. Pharm. Bull. 48, 964–73. doi: 10.1248/cpb.48.964

PubMed Abstract | CrossRef Full Text | Google Scholar

Chang, M.-C., Chang, B.-E., Pan, Y.-H., Lin, B.-R., Lian, Y.-C., Lee, M.-S., et al. (2019). Antiplatelet, antioxidative, and anti-inflammatory effects of hydroquinone. J. Cell. Physiol. 234, 18123–18130. doi: 10.1002/jcp.28444

PubMed Abstract | CrossRef Full Text | Google Scholar

Chang, T. S., Kim, H. M., Lee, K. S., Khil, L. Y., Mar, W. C., Ryu, C. K., et al. (1997). Thromboxane A2 synthase inhibition and thromboxane A2 receptor blockade by 2-[(4-cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) in rat platelets. Biochem. Pharmacol. 54, 259–268. doi: 10.1016/S0006-2952(97)00179-2

PubMed Abstract | CrossRef Full Text | Google Scholar

Chen, Z., Mondal, N. K., Ding, J., Koenig, S. C., Slaughter, M. S., Griffith, B. P., et al. (2015). Activation and shedding of platelet glycoprotein IIb/IIIa under non-physiological shear stress. Mol. Cell Biochem. 409, 93–101. doi: 10.1007/s11010-015-2515-y

PubMed Abstract | CrossRef Full Text | Google Scholar

Cho, Y. S., Kim, M. J., Lee, J. Y., and Chung, J. H. (1997). The role of thiols in protecting against simultaneous toxicity of menadione to platelet plasma and intracellular membranes. J. Pharmacol. Exp. Ther. 280, 1335–1340.

PubMed Abstract | Google Scholar

Cocheme, H. M., Kelso, G. F., James, A. M., Ross, M. F., Trnka, J., Mahendiran, T., et al. (2007). Mitochondrial targeting of quinones: therapeutic implications. Mitochondrion 7, S94–S102. doi: 10.1016/j.mito.2007.02.007

PubMed Abstract | CrossRef Full Text | Google Scholar

Diamond, S. L. (2016). Systems analysis of thrombus formation. Circ. Res. 118, 1348–1362. doi: 10.1161/CIRCRESAHA.115.306824

PubMed Abstract | CrossRef Full Text | Google Scholar

Fox, K. A., Mehta, S. R., Peters, R., Zhao, F., Lakkis, N., Gersh, B. J., et al. (2004). Benefits and risks of the combination of clopidogrel and aspirin in patients undergoing surgical revascularization for non-ST-elevation acute coronary syndrome: the Clopidogrel in Unstable angina to prevent Recurrent ischemic Events (CURE) Trial. Circulation 110, 1202–1208. doi: 10.1161/01.CIR.0000140675.85342.1B

PubMed Abstract | CrossRef Full Text | Google Scholar

Franco, A. T., Corken, A., and Ware, J. (2015). Platelets at the interface of thrombosis, inflammation, and cancer. Blood 126, 582–588. doi: 10.1182/blood-2014-08-531582

PubMed Abstract | CrossRef Full Text | Google Scholar

Fuentes, E., Badimon, L., Caballero, J., Padró, T., Vilahur, G., Alarcón, M., et al. (2014a). Protective mechanisms of adenosine 5'-monophosphate in platelet activation and thrombus formation. Thromb. Haemost 111, 491–507. doi: 10.1160/TH13-05-0386

PubMed Abstract | CrossRef Full Text | Google Scholar

Fuentes, E., Caballero, J., Alarcon, M., Rojas, A., and Palomo, I. (2014b). Chlorogenic acid inhibits human platelet activation and thrombus formation. PLoS ONE 9:e90699. doi: 10.1371/journal.pone.0090699

PubMed Abstract | CrossRef Full Text | Google Scholar

Fuentes, M., Araya-Maturana, R., Palomo, I., and Fuentes, E. (2018). Platelet mitochondrial dysfunction and mitochondria-targeted quinone-and hydroquinone-derivatives: review on new strategy of antiplatelet activity. Biochem. Pharmacol. 156, 215–222. doi: 10.1016/j.bcp.2018.08.035

PubMed Abstract | CrossRef Full Text | Google Scholar

Geisler, T., Anders, N., Paterok, M., Langer, H., Stellos, K., Lindemann, S., et al. (2007). Platelet response to clopidogrel is attenuated in diabetic patients undergoing coronary stent implantation. Diabetes Care 30, 372–374. doi: 10.2337/dc06-1625

PubMed Abstract | CrossRef Full Text | Google Scholar

Harrington, R. A., Hodgson, P. K., and Larsen, R. L. (2003). Antiplatelet therapy. Circulation 108, e45–e47. doi: 10.1161/01.CIR.0000083142.13956.D6

PubMed Abstract | CrossRef Full Text | Google Scholar

Israels, S. J., and McMillan-Ward, E. M. (2005). CD63 modulates spreading and tyrosine phosphorylation of platelets on immobilized fibrinogen. Thromb Haemost 93, 311–318. doi: 10.1160/TH.04-08-0503

PubMed Abstract | CrossRef Full Text | Google Scholar

ISTH Steering Committee for World Thrombosis Day (2014). Thrombosis: a major contributor to the global disease burden. J. Thromb. Haemost. 12, 1580–1590. doi: 10.1111/jth.12698

CrossRef Full Text | Google Scholar

Jordão, A. K., Vargas, M. D., Pinto, A. C., da Silva, F. d. C., and Ferreira, V. F. (2015). Lawsone in organic synthesis. RSC Adv. 5, 67909–67943. doi: 10.1039/C5RA12785H

CrossRef Full Text | Google Scholar

Jung, S. H., Han, J. H., Park, H. S., Lee, J. J., Yang, S. Y., Kim, Y. H., et al. (2017). Inhibition of collagen-induced platelet aggregation by the secobutanolide Secolincomolide A from lindera obtusiloba blume. Front. Pharmacol. 8:560. doi: 10.3389/fphar.2017.00560

PubMed Abstract | CrossRef Full Text | Google Scholar

Kim, E. J., Lim, K. M., Kim, K. Y., Bae, O. N., Noh, J. Y., Chung, S. M., et al. (2009). Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia. J. Thromb. Haemost 7, 1172–1183. doi: 10.1111/j.1538-7836.2009.03477.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Kim, S. R., Lee, J. Y., Lee, M. Y., Chung, S. M., Bae, O. N., and Chung, J. H. (2001). Association of quinone-induced platelet anti-aggregation with cytotoxicity. Toxicol. Sci. 62, 176–182. doi: 10.1093/toxsci/62.1.176

PubMed Abstract | CrossRef Full Text | Google Scholar

Lien, J. C., Huang, L. J., Teng, C. M., Wang, J. P., and Kuo, S. C. (2002). Synthesis of 2-alkoxy 1,4-naphthoquinone derivatives as antiplatelet, antiinflammatory, and antiallergic agents. Chem. Pharm. Bull. 50, 672–4. doi: 10.1248/cpb.50.672

PubMed Abstract | CrossRef Full Text | Google Scholar

Ma, W., and Long, Y. T. (2014). Quinone/hydroquinone-functionalized biointerfaces for biological applications from the macro- to nano-scale. Chem. Soc. Rev. 43, 30–41. doi: 10.1039/C3CS60174A

PubMed Abstract | CrossRef Full Text | Google Scholar

Mendez, D., Urra, F. A., Millas-Vargas, J. P., Alarcon, M., Rodriguez-Lavado, J., Palomo, I., et al. (2020). Synthesis of antiplatelet ortho-carbonyl hydroquinones with differential action on platelet aggregation stimulated by collagen or TRAP-6. Eur. J. Med. Chem. 192:112187. doi: 10.1016/j.ejmech.2020.112187

CrossRef Full Text | Google Scholar

Moura, L. A., de Almeida, A. C., da Silva, A. V., de Souza, V. R., Ferreira, V. F., Menezes, M. V., et al. (2016). Synthesis, anticlotting and antiplatelet effects of 1,2,3-triazoles derivatives. Med. Chem. 12, 733–741. doi: 10.2174/1573406412666160502153417

PubMed Abstract | CrossRef Full Text | Google Scholar

Mousa, S. A. (1999). Antiplatelet therapies: from aspirin to GPIIb/IIIa-receptor antagonists and beyond. Drug Discov. Today 4, 552–561. doi: 10.1016/S1359-6446(99)01394-X

PubMed Abstract | CrossRef Full Text | Google Scholar

Munoz-Gutierrez, C., Sepulveda, C., Caballero, J., Palomo, I., and Fuentes, E. (2017). Study of the interactions between Edaglitazone and Ciglitazone with PPARgamma and their antiplatelet profile. Life Sci. 186, 59–65. doi: 10.1016/j.lfs.2017.07.031

PubMed Abstract | CrossRef Full Text | Google Scholar

Nieswandt, B., and Watson, S. P. (2003). Platelet-collagen interaction: is GPVI the central receptor? Blood 102, 449–461. doi: 10.1182/blood-2002-12-3882

PubMed Abstract | CrossRef Full Text | Google Scholar

Paes, M. A. A., Gaspar, R. S., Fuentes, E., Wehinger, S., Palomo, I., and Trostchansky, A. (2019). Lipid metabolism and signaling in platelet function. Adv. Exp. Med. Biol. 1127, 97–115. doi: 10.1007/978-3-030-11488-6_7

PubMed Abstract | CrossRef Full Text | Google Scholar

Price, M. J., Angiolillo, D. J., Teirstein, P. S., Lillie, E., Manoukian, S. V., Berger, P. B., et al. (2011). Platelet reactivity and cardiovascular outcomes after percutaneous coronary intervention: a time-dependent analysis of the Gauging Responsiveness with a VerifyNow P2Y12 assay: Impact on Thrombosis and Safety (GRAVITAS) trial. Circulation 124, 1132–1137. doi: 10.1161/CIRCULATIONAHA.111.029165

PubMed Abstract | CrossRef Full Text | Google Scholar

Qin, X., Peng, Y., and Zheng, J. (2018). In vitro and in vivo studies of the electrophilicity of physcion and its oxidative metabolites. Chem. Res. Toxicol. 31, 340–349. doi: 10.1021/acs.chemrestox.8b00026

PubMed Abstract | CrossRef Full Text | Google Scholar

Rahmoun, N. M., Boucherit-Otmani, Z., Boucherit, K., Benabdallah, M., Villemin, D., and Choukchou-Braham, N. (2012). Antibacterial and antifungal activity of lawsone and novel naphthoquinone derivatives. Médecine et Maladies Infectieuses 42, 270–275. doi: 10.1016/j.medmal.2012.05.002

PubMed Abstract | CrossRef Full Text | Google Scholar

Rao, G. H., Cox, C. A., Gerrard, J. M., and White, J. G. (1981). Alpha tocopherol quinone (alpha TQ): a potent inhibitor of platelet function. Prog. Lipid Res. 20, 549–552. doi: 10.1016/0163-7827(81)90097-7

PubMed Abstract | CrossRef Full Text | Google Scholar

Ritchie, J. L., Alexander, H. D., and Rea, I. M. (2000). Flow cytometry analysis of platelet P-selectin expression in whole blood–methodological considerations. Clin. Lab. Haematol. 22, 359–363. doi: 10.1046/j.1365-2257.2000.00339.x

PubMed Abstract | CrossRef Full Text | Google Scholar

Tandon, V. K., and Maurya, H. K. (2009). “On water:” unprecedented nucleophilic substitution and addition reactions with 1,4-quinones in aqueous suspension. Tetrahedron Lett. 50, 5896–5902. doi: 10.1016/j.tetlet.2009.07.149

CrossRef Full Text | Google Scholar

Tandon, V. K., Maurya, H. K., Verma, M. K., Kumar, R., and Shukla, P. K. (2010). “On water” assisted synthesis and biological evaluation of nitrogen and sulfur containing hetero-1,4-naphthoquinones as potent antifungal and antibacterial agents. Eur. J. Med. Chem. 45, 2418–2426. doi: 10.1016/j.ejmech.2010.02.023

PubMed Abstract | CrossRef Full Text | Google Scholar

Théorêt, J.-F., Yacoub, D., Hachem, A., Gillis, M.-A., and Merhi, Y. (2011). P-selectin ligation induces platelet activation and enhances microaggregate and thrombus formation. Thrombosis Res. 128, 243–250. doi: 10.1016/j.thromres.2011.04.018

PubMed Abstract | CrossRef Full Text | Google Scholar

Urra, F. A., Cordova-Delgado, M., Lapier, M., Orellana-Manzano, A., Acevedo-Arevalo, L., Pessoa-Mahana, H., et al. (2016). Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation. Toxicol. Appl. Pharmacol. 291, 46–57. doi: 10.1016/j.taap.2015.12.005

CrossRef Full Text | Google Scholar

Vaiyapuri, S., Moraes, L. A., Sage, T., Ali, M. S., Lewis, K. R., Mahaut-Smith, M. P., et al. (2013). Connexin40 regulates platelet function. Nat. Commun. 4:2564. doi: 10.1038/ncomms3564

PubMed Abstract | CrossRef Full Text | Google Scholar

Walsh, T. G., Berndt, M. C., Carrim, N., Cowman, J., Kenny, D., and Metharom, P. (2014). The role of Nox1 and Nox2 in GPVI-dependent platelet activation and thrombus formation. Redox Biol. 2, 178–186. doi: 10.1016/j.redox.2013.12.023

PubMed Abstract | CrossRef Full Text | Google Scholar

Xu, K., Wang, P., Wang, L., Liu, C., Xu, S., Cheng, Y., et al. (2014). Quinone derivatives from the genus Rubia and their bioactivities. Chem. Biodivers 11, 341–363. doi: 10.1002/cbdv.201200173

PubMed Abstract | CrossRef Full Text | Google Scholar

Yun-Choi, H. S., Kim, J. H., and Takido, M. (1990). Potential inhibitors of platelet aggregation from plant sources, V. Anthraquinones from seeds of Cassia obtusifolia and related compounds. J. Nat. Prod. 53, 630–633. doi: 10.1021/np50069a014

PubMed Abstract | CrossRef Full Text | Google Scholar