Synthesis and evaluation of antioxidant phenolic diaryl hydrazones as potent antiangiogenic agents in atherosclerosis

Accepted Manuscript Synthesis and Evaluation of Antioxidant Phenolic Diaryl Hydrazones as Potent Antiangiogenic Agents in Atherosclerosis Corinne Vanucci-Bacqué, Caroline Camare, Chantal Carayon, Corinne Bernis, Michel Baltas, Anne Nègre-Salvayre, Florence Bedos-Belval PII: DOI: Reference:

S0968-0896(16)30411-4 http://dx.doi.org/10.1016/j.bmc.2016.05.067 BMC 13051

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

11 April 2016 25 May 2016 29 May 2016

Please cite this article as: Vanucci-Bacqué, C., Camare, C., Carayon, C., Bernis, C., Baltas, M., Nègre-Salvayre, A., Bedos-Belval, F., Synthesis and Evaluation of Antioxidant Phenolic Diaryl Hydrazones as Potent Antiangiogenic Agents in Atherosclerosis, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc. 2016.05.067

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Synthesis and Evaluation of Antioxidant Phenolic Diaryl Hydrazones as Potent Antiangiogenic Agents in Atherosclerosis Corinne Vanucci-Bacqué,a,b Caroline Camare, c Chantal Carayon,a,b Corinne Bernis, c Michel Baltas,a,b* Anne Nègre-Salvayre,c,* and Florence Bedos-Belval.a,b* a

Université Paul Sabatier Toulouse III; UMR 5068, Laboratoire de Synthèse et Physico-Chimie

de Molécules d’Intérêt Biologique ; 118, Route de Narbonne, F-31062 Toulouse Cedex 9, France. b

CNRS; UMR 5068,

Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt

Biologique ; 118, Route de Narbonne, F-31062 Toulouse Cedex 9, France. c

INSERM; UMR1048, I2MC, BP 84225, 31432 Toulouse Cedex 4, France

* Corresponding author: Tel.: +33 (0)5 61 55 68 00 ; fax : +33 (0)5 61 55 60 11 E-mail address: [email protected]

[email protected];

[email protected];

Anne.Negre-

Abstract A series of bis-hydrazones derived from diaryl and diaryl ether hydroxybenzaldehyde frames 1 and 2 have been synthesized as potential antioxidant and antiangiogenic agents, two properties required to limit atherogenesis and cardiovascular events. These compounds were evaluated for their ability to neutralize free radical formation, to block endothelial cell-induced low-density lipoprotein oxidation (monitored by the formation of TBARS), an essential step in atherogenesis, and subsequent toxicity, to prevent angiogenesis evoked by low oxidized LDL concentration (monitored by the formation of capillary tubes on Matrigel) and to inhibit intracellular ROS increase involved in the angiogenic signaling. A structure/activity study has been carried out and finally allowed to select the phenolic diaryl ether hydralazine derivative 2a, sharing all these protective properties, as a promising hit for further development.

Keywords: Angiogenesis, antioxidant, atherosclerosis, biphenyl derivatives, polyphenol, hydrazone.

1. Introduction

Atherosclerosis is a multifactorial and inflammatory pathology of large and medium arteries that develops slowly and silently for years in the vascular wall, and may result in acute cardiovascular events (infarction, stroke) due to plaque rupture and thrombosis.1 Among the pathophysiological mechanisms involved in the development of atherosclerotic lesions and their evolution toward advanced stages, the neovascularization of the plaque contributes to its progression by providing nutrients, oxygen and blood cells. Moreover neovascularization increases the risk of plaque instability and rupture, as it is correlated with local inflammation and intraplaque microhemorrhages resulting from neocapillary apoptosis.2,3 Many factors present in atherosclerotic lesions may promote angiogenesis, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ), lipid mediators such as lysophospholipids (sphingosine-1-phosphate or S1P, lysophosphatidic acid or LPA), ischemia, hypoxia, oxidized lipids and oxidized low density lipoprotein (oxLDL).4-8 All these factors are present in atherosclerotic plaques and contribute to generate inflammatory events that are involved in the neovascularization process of the lesions.25

Low density lipoprotein (LDL) are involved in atherogenesis after undergoing oxidation in the intima.9 oxLDL exhibit a huge variety of proatherogenic properties including inflammatory, migratory, mitogenic and apoptotic responses on vascular cells.10-12 The oxLDL implication in angiogenesis has been recently reported in vitro and in vivo in the mouse Matrigel plug assay.13,14 The angiogenic signaling evoked by low oxLDL concentration depends on their content in bioreactive oxidized lipids15,16 and on the subsequent generation of intracellular reactive oxygen species (ROS), as supported by the inhibitory effect of antioxidants such as Trolox or Nacetylcysteine (NAC).13,14,17 Interestingly, higher oxLDL concentration inhibit angiogenesis and trigger apoptosis, suggesting that oxLDL may locally contribute to plaque neovascularization

and destabilization as well, through capillary apoptotic loss.14 Various classes of antioxidants including vitamins C and E and polyphenols, have been used for inhibiting ROS generation, LDL oxidation and early atherogenic events in animal models for atherosclerosis.18-21 However most antioxidants are poorly efficient against the late atherothrombotic events.22-24 Actually, there is a need of agents able to neutralize both ROS generation and oxidized lipid bioreactivity, as they are the main trigger of oxLDL biological properties, including angiogenesis. Ideally these compounds should share both antioxidant, carbonyl scavenger and cytoptotective activities. For example, the antihypertensive drug hydralazine neutralizes aldehydes generated from lipid oxidation and exerts antiatherogenic properties.25 However, hydralazine has poor antioxidant properties, and high concentrations are necessary for reducing LDL oxidation by vascular cells by comparison with classical antioxidants.20,25,26 We recently developed a phenolic biphenyl bis-hydrazone prototype 1a (Fig. 1), exhibiting a hydralazine moiety. This agent inhibits copper- and endothelial cell-induced LDL oxidation and cytotoxicity, as well as the extra- and intra-cellular generation of ROS, and it exhibits antiatherogenic properties in apoE-/- mice that spontaneously develop atherosclerotic lesions.27 In the present study, we have synthesized a series of phenolic bis-hydrazones based on prototype 1a, in order to perform a structure-activity comparison of the different agents to characterize and improve their antioxidant and cytoprotective properties, and check whether they may slow down the angiogenic process evoked by oxLDL. We initially chose to modulate the structure of 1a at the hydrazone scaffold, which is known to induce various biological activities (namely antiinflammatory, cardio protective, anti-platelet and anti-cancer properties).28 Then, an ether link between the two phenolic rings was introduced to figure out the influence of such a spacer. To this aim, we prepared a variety of phenolic bis-hydrazones from two basic dialdehyde patterns, the diaryl linked derivative 1, and the related diaryl ether 2 (Fig. 1).

Figure 1. Synthetic strategy scope - Chemical structure of compound 1a and dialdehyde precursors 1 and 2.

We have compared the different properties exhibited by the new synthesized agents including their antioxidant activity, either by using the physicochemical DPPH assay, or by testing their ability to block LDL oxidation by endothelial cells (TBARS assay), and their ability to inhibit the generation of intracellular ROS that are involved in the angiogenic process evoked by oxLDL. Indeed, the source, the nature and the mechanism of ROS generation involved in LDL oxidation or in angiogenesis may be different, and it is relevant to evaluate the capacity of the agents to inhibit both intra- and extracellular ROS production. Based on these results, we tested the ability of the different diaryl derivatives to block the formation of microtubes by microvascular endothelial cells (HMEC-1) on Matrigel (a classical model for studying in vitro angiogenesis), upon stimulation by oxLDL.

2. Results and Discussion 2.1. Chemistry The 6,6’-dihydroxy-5,5’-dimethoxybiphenyl-3,3’-dicarbaldehyde (1) was prepared as previously reported.29

Concerning the diaryl ether scaffold synthesis (Scheme 1), we previously demonstrated that Ullmann standard coupling conditions (Cu(0) in refluxing DMF) applied to 4-benzyloxy-3hydroxy-5-methoxybenzaldehyde (3) and 4-benzyloxy-3-bromo-5-methoxybenzaldehyde (4) led to the symmetric dibenzylated diaryl ether 5 as traces (8% yield). A benzyl migration in the phenolic partner is implicated in the poor yield.30 So we envisioned the use of differently Oprotected reactants, with first focus on O-silylated compounds. Unfortunately, regioselective TBS-silylation of 5-hydroxyvanillin failed. Alternatively, commercially available 3,4dimethoxy-5-hydroxybenzaldehyde (6)

and 3-bromo-4,5-dimethoxybenzaldehyde (7) were

reacted in refluxing DMF with Cu(0) affording the expected symmetric diaryl ether 8 in high yield (87%). The optimized regioselective demethylation of the 4-methoxy groups of tetramethoxy ether 8 was achieved by treatment with AlCl3 (6 equiv.) in dichloromethane, cleanly providing the expected diphenol 2 in 81% yield (Scheme 1).

Scheme 1. Synthesis of the diaryl ether core. Reagents and conditions: a) Cu(0), DMF, reflux; b) AlCl3, CH2Cl2, rt.

With the required dialdehydes in hand, we focused on the introduction of the hydrazone functionalities. As shown in Scheme 2, hydrazone-type derivatives were efficiently prepared by condensation of two strict equivalents of various commercially available hydrazines/hydrazides

(1-hydrazinylphthalazine

hydrochloride,

isonicotinic

hydrazide,

3-methyl-1H-pyrazole-5-

carbohydrazide, 2-hydrazinylbenzo[d]thiazole and benzyl hydrazinecarboxylate) on dialdehydes 1 and 2 in refluxing ethanol (75-99% yields).

Scheme 2. Synthesis of bis-hydrazones. Reagents and conditions: a) 1-hydrazinylphthalazine hydrochloride (2 equiv.), EtOH, reflux; b) isonicotinic hydrazide (2 equiv.), EtOH, reflux; c) 3methyl-1H-pyrazole-5-carbohydrazide (2 equiv.), EtOH, reflux; d) 2-hydrazinylbenzo[d]thiazole (2 equiv.), EtOH, reflux; e) benzyl hydrazinecarboxylate (2 equiv.), EtOH, reflux.

2.2. Antiradical activities Radical scavenging potency of the tested compounds was assessed in vitro by the DPPH assay, mostly used for phenol derivatives.

Table 1. Free radical scavenging activity (DPPH), inhibition (TBARS) of cell-induced LDL oxidation and cytoprotective effect (MTT) against oxLDL toxicity of the diaryl derivatives at 1 µM and 10 µM. Compound

DPPH

TBARS (%)c

IC50 (µM)a

1µM

1

382.1±13

103±5

1a

9.7±0.3

1b

10µM

MTT (%)d 1µM

10µM

88±9

19±4

21±3

56±4

8±4

52±5

99±4

38.8±0.3

112±10

92±8

18±4

19±5

1c

29.1±0.3

99±3

71±12

14±2

40±7

1d

27.7±0.3

19±9

7±2

58±6

91±6

1e

59.7±1.4

18±4

5±1

52±4

90±9

2

289.4±6.0

95±10

87±22

32±2

35±6

2a

11.5±0.3

37±7

10±3

56±6

72±9

2b

21.5±0.9

100±2

88±15

42±5

66±4

2c

19.8±2.5

97±4

91±20

41±5

65±6

2d

8.6±0.5b

93±12

12±5

51±2

69±4

2e

23.6±0.1

28±7

6±2

51±4

69±4

57±2

10±6

45±5

68±12

20.1±1.4 Trolox (17.7±0.4)b Control

a

100±5

20±5

Determined by UV-absorption at 517 nm. Results are the means ± SEM (n=3)

b

Determined by EPR.

c

Results are expressed as percent of the control incubated with LDL and HMEC-1 in the

absence of agent. The data are mean ± SEM of 4 separate experiments. p < 0.05. d

The results are expressed as % of the untreated control. Control means with oxLDL without

agent.

The DPPH assay measures the reduction in absorbance of a free stable 2,2′-diphenyl-1picrylhydrazyl (DPPH) radical ethanolic solution at 517 nm,31 in the presence of the tested derivatives. It is noted that in the case of benzothiazol derivative 2d, for which a staining appeared in the presence of the DPPH radical, preventing the reading of the absorbance at 517nm, the measurement was determined using EPR spectroscopy.32,33 Table 1 shows the concentration for 50% of inhibition of DPPH free radical. Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) known for its antioxidant capacity, a water-soluble vitamin E analog, was used as standard reference. It is noteworthy that the phenolic dialdehydes 1 and 2 show no DPPH radical scavenger activity (IC50 values 382.1±13.3 µM and 289.4±6.0 µM respectively). The phenolic moiety is thus not enough in itself to ensure efficient antiradical ability of these compounds. The hydrazone frame is crucial for the antioxidant capacity of studied compounds due to the π-electron delocalization increase.34 Indeed it is observed that compounds 1a, 2a and 2d exhibit antiradical properties twice more effective than Trolox, while 2b, 2c and 2e displays IC50 value in the same range as Trolox (Table 1). All the other hydrazones 1b-e exhibit DPPH scavenging activities (27.7±0.3 ≤IC50≤ 59.7±1.4 µM) positioning them as good radical trapping agents in the DPPH assay compared to the non-antiradical dialdehydes 1 and 2. In most cases, the phenolic diaryl ether

derivatives show better scavenging capacities than the corresponding diaryl ones. However, the DPPH assay gives only an indication of the antioxidant potential of a molecule, as it is carried out in the absence of cells.

2.3. Antioxidant activity: Protective effect against cell-induced LDL oxidation ROS produced by vascular cells, play a major role in atherogenesis and in the formation of early atherosclerotic lesions, by triggering LDL oxidation in the intima.9,18,35 This mechanism can be mimicked in vitro by incubating vascular cells with LDL.25,27 The LDL oxidation process occurs extracellularly and involves the superoxide anion O2°- that is secreted upon stimulation of NADP(H) oxidase (NOX).36 Antioxidants may inhibit LDL oxidation either via the neutralization of ROS in the extracellular medium, or/and by inhibiting NOX activation.18-23 We checked whether the newly synthesized hydrazones inhibit the oxidation of native LDL by human endothelial cells (HMEC-1). LDL oxidation by HMEC-1 was determined in the absence (control), or in the presence of each agent (tested at 1 and 10 µM final concentration), and was assessed by measuring the generation of thiobarbituric acid reactive substance (TBARS) in the LDL-containing culture medium.37 The results were compared to those obtained with LDL without any agent in presence of the vehicle (DMSO, 0.1% final concentration). The data presented in Table 1 are expressed as percent of TBARS generated in the antioxidant-containing culture medium vs TBARS generated in the absence of antioxidant (100%), using Trolox as reference. These data point out the different efficacy of the synthesized derivatives. First of all, it should be noted that the phenolic dialdehyde precursors 1 and 2 do not exhibit any antioxidant activity by themselves, meaning that other functionalities are required to induce this property, as also

observed in the DPPH assay. Besides, all the tested agents display a better antioxidant activity when tested at 10 µM rather than 1 µM, justifying their use in further experiments at 10 µM. The diaryl phenols 1a, 1d, 1e, 2a, 2d and 2e (TBARS values from 5 to 12%) were as efficient as Trolox (10%) at 10 µM. Interestingly, when used at lower concentration (1µM), compounds 1d, 1e, 2a and 2e, were more efficient than Trolox (1 µM) to block LDL oxidation (TBARS values ranging from 18 to 37%, vs Trolox, 57%). The other agents were either poorly or not efficient. In particular, compounds 1b-2b and 1c-2c exhibiting

isonicotinoyl

hydrazide

or

3-methyl-1H-pyrazole-5-carbohydrazide

moieties

respectively, were surprisingly not active as antioxidants, in spite of their phenolic moieties and of good radical scavenging properties (Table 1). When comparing diaryl and corresponding diaryl ether frames for a same functionalization, it is to note that the oxygen spacer does not play any key role in the ability to block cell-induced LDL oxidation, whereas diaryl ether structures exhibit a better radical scavenging activity (Table 1). This suggests that difference in antioxidant efficacy could result from their ability to neutralize free radicals produced by endothelial cells, but also to modulate the activity of cellular enzymatic systems and signaling pathways involved in free radical production. Since high concentrations of oxidized LDL are toxic for vascular cells,38,39 we checked whether the newly synthesized hydrazones may inhibit this toxicity. The toxicity of oxidized LDL (200 µg mL-1) was studied on HMEC-1, either with the vehicle only (DMSO 0.1%), or in the presence of hydrazones (1 and 10 µM), using Trolox as control. The cell viability was studied using the MTT test 27 and is reported in Table 1. We first tested the self-toxicity of the different newly synthesized hydrazones, in the absence of oxLDL. This was studied by checking whether the different agents added to the culture medium

at 10 µM in the vehicle (0.1%), trigger a loss of cell viability (detected by the MTT assay). In the used conditions, no toxicity was observed for any compounds at 1 and 10 µM concentrations (data in Supporting Information). Regarding their cytoprotective effect, dialdehydes 1 and 2 were found ineffective. Most diaryl hydrazones inhibited oxLDL toxicity, particularly compounds 1a, 1d and 1e exhibiting 90 to 99% of cell viability at 10 µM, higher than Trolox (68%), knowing that the residual viability of HMEC-1, incubated for 36 h with toxic oxLDL, was less than 20%. The latter derivatives were as efficient as Trolox at 1 µM. Furthermore diaryl compounds 1b and 1c have neither cytoprotective effect nor antioxidant activity in this system, which suggests that these agents are either inactive against free radicals generated by endothelial cells, or are unable to counteract the apoptotic signaling of oxLDL . The protective effect of phenol diaryl ether series 2a-e was as efficient as Trolox in preventing oxLDL toxicity at 1 and 10 µM. It is noteworthy that compounds 2b and 2c were cytoprotective though they exert a poor antioxidant activity, as they did not protect LDL from oxidation (Table 1). These results suggest that compounds 2b and 2c could exert their cytoprotective activity by inhibiting the toxic and proapoptotic cell signaling evoked by oxLDL,40 however it is likely that they should not slow down the accumulation of foam cells and fatty streaks which are directly related to the uptake of oxLDL by macrophages,13,41 nor angiogenesis which depends on ROS generation.13,14 Anyhow, our results indicate that the deleterious signaling evoked by oxLDL, oxidized lipids and oxidative stress, may be inhibited either by antioxidants (which also exhibit cytoprotective activity), or by cytoprotective agents devoid of any antioxidant activity.

2.4. Inhibition of capillary formation on Matrigel by HMEC-1, induced by oxidized LDL

We then checked whether the synthetized phenolic diaryl derivatives could inhibit the angiogenicity of oxLDL evidenced by the formation of capillary tubes on Matrigel by HMEC-1, in the presence of low oxLDL concentration (20 µg mL-1).35 Diaryl hydrazones 1a, 1d, 1e and corresponding diaryl ether hydrazones 2a, 2d and 2e were selected as the most efficient compounds against cell-induced LDL oxidation at 10 µM (Table 1). Trolox was used as antioxidant and antiangiogenic control.14

Figure 2. Capillary tube formation by HMEC-1 stimulated by low oxLDL concentration (20 µg mL-1) w/wo diaryl phenol derivatives and Trolox (10 µM each). A) Quantification of formed capillary tubes by HMEC-1 grown on Matrigel. Tube formation was expressed as % of linked cells

to

the

total

cell

number.

Mean

±

SEM

of

4

separate

experiments,

#(oxLDL/control),*(agent/oxLDL) p<0.05. FBS, fetal bovine serum. B) Representative pictures of tube formation on Matrigel in the presence of the different agents.

We recently reported that low oxLDL concentration (20-50 µg/apoB/mL) elicit the formation of capillary tubes by HMEC-1 on Matrigel.14,17 We chose an angiogenic concentration (20 µg/mL)

for the anti-angiogenic effect evaluation of the synthesized agents. As shown in Fig. 2, low oxLDL concentration (20 µg mL-1) stimulated the formation of capillary tubes by HMEC-1, in the same range than the positive control consisting in fetal bovine serum (FBS, 2.5%). The phenolic diaryl derivatives 1a, 1d and 1e display a similar strong efficiency in inhibiting the capillary tube formation by HMEC-1 in the same range as Trolox, whatever their hydrazone moiety nature. On the other hand, the efficiency of the tested phenolic diaryl ether analogues depends on the nature of the hydrazone part: benzothiazolhydrazone 2d exihibits a similar antiangiogenic activity as Trolox, whereas benzyl carboxylate hydrazone 2e is totally inefficient though this agent efficiently inhibited LDL oxidation (Table 1). Gratefully, hydralazine diaryl ether 2a is much more potent than Trolox (less than 5% of residual tube vs 20% respectively). Noteworthy, in this case the ether link promotes the antiangiogenic activity since 2a is four times more potent than the corresponding diaryl analogue 1a. As the angiogenic signaling of oxLDL involves a rapid generation of intracellular ROS,14,17 we checked whether the protective antiangiogenic effect of the selected derivatives is correlated with an inhibition of this intracellular ROS increase in HMEC-1 (detected using the ROS-sensitive probe H2DCFDA-AM).

Figure 3. Effect of phenolic diaryl derivatives on the intracellular ROS increase evoked by oxLDL. ROS were determined in HMEC-1 preloaded with the fluorescent H2DCFDA-AM probe (5 µM), after 40 min incubation with oxLDL (20 µg mL-1), w/wo phenolic diaryl derivatives or Trolox (10 µM each). Mean ± SEM of 4 separate experiments, #(oxLDL/control),*(agent/oxLDL) p<0.05.

As shown in Fig. 3, low angiogenic oxLDL concentration rapidly stimulates the generation of intracellular ROS. This ROS increase involves the oxLDL receptor LOX-1 and the activation of NOX2.13,17 We show here that most tested agents inhibit the intracellular ROS increase, which suggests that they may either directly inhibit NOX activation at the plasma membrane or block the intracellular signaling leading to NOX activation and ROS production. Interestingly, the

diarylether 2e was unable to block the intracellular ROS increase evoked by oxLDL, this being correlated to its lack of inhibitory effect against tube formation on Matrigel (Fig.2), whereas this agent 2e efficiently blocked LDL oxidation (Table 1). These data suggest that 2e is able to neutralize ROS produced in the extracellular medium, but it is probably unable to block the intracell signaling involved in ROS production. These results, together with their potent antiangiogenic effect confer an originality to molecules 1a, 1e, 1d, 2a and 2d against oxLDL atherogenic signaling. 3. Conclusion In conclusion, different phenolic diaryl and diaryl ether derivatives featuring hydrazone functionalities have been synthesized, and their antioxidant and antiangiogenic properties have been evaluated and compared. This study indicates that despite their well-established antioxidant properties, phenol scaffolds are not sufficient to induce this required dual activity, which is mainly modulated by the hydrazone functionalization. The biological data observed with the phenolic hydrazone diaryls 1a, 1d, 1e and diaryl ethers 2a, 2d as promising agents show that they are able to block LDL oxidation elicited by vascular cells, the toxic effect of high oxLDL concentrations for vascular cells, the intracellular ROS increase and the subsequent tube formation characteristic of the proangiogenic oxLDL properties. Particularly, the striking antiangiogenic efficiency of the phenolic hydralazine diaryl ether 2a allows to propose this component as a promising candidate for future studies.

4. Experimental Section 4.1. Chemistry

Melting points (mp) were obtained on a Buchi apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Thermo Nicolet Nexus spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded on Brucker Avance 300 MHz spectrometers. Chemical shifts (δ) were reported in parts per million (ppm) relative to TMS at δ = 0 ppm for 1H NMR spectra and to residual solvent peak signals for 13C NMR spectra. Signals are described as follow: s, singlet; br, broad signal; d, doublet; t, triplet; m, multiplet. High resolution mass spectra (HRMS) were recorded on a Xevo G2 QTOF (Waters) instrument. Compounds 1, 1a, 1b29 and 1d42 have been previously reported and fully characterized. 4.1.1.

5,5'-Oxybis(3,4-dimethoxybenzaldehyde)

(8).

A

mixture

of

3-hydroxy-4,5-

dimethoxybenzaldehyde (6) (576 mg, 3.17 mmol), 3-bromo-4,5-dimethoxybenzaldehyde (7) (2.33 g, 9.51 mmol ) and copper powder (1 g, 15.85 mmol) in dry DMF (6 mL) was heated at reflux temperature under argon atmosphere for 16 h. The reaction mixture was cooled to room temperature, diluted with EtOAc and filtrated over a Celite® pad. Water was added and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtrated and concentrated under vacuum. Purification by silica gel column chromatography (EtOAc/Petroleum ether : 20/80) allowed compound 8 (958 mg, 87%) as a colorless solid. mp : 94-96 °C; IR (neat): υ max = 1693 cm-1 ; 1H NMR (300 MHz, CDCl3,): δ = 3.96 (s, 6H), 3.97 (s, 6H), 7.03 (d, J = 1.8 Hz, 2H), 7.28 (d, J = 1.8 Hz, 2H), 9.79 (s, 2H); 13C NMR (75 MHz, CDCl3): δ = 56.3, 61.1, 107.5, 114.5, 131.7, 145.4, 149.9, 154.3, 190.4; HRMS (DCI, CH4) : m/z [M + H]+calcd for C18H19O7: 347.1131, found: 347.1135. 4.1.2. 5,5'-Oxybis(4-hydroxy-3-methoxybenzaldehyde) (2). To a stirred solution of compound 8 (662 mg, 1.91 mmol) in dry CH2Cl2 (33 mL) was added AlCl3 (1.53 g, 11.5 mmol). The reaction mixture was stirred at room temperature under argon atmosphere for 6 h. Water (24 mL)

was slowly added and the mixture was filtered over a sintered glass funnel. The aqueous layer was extrated with CH2Cl2. EtOAc (30 mL) was added to the collected precipitate. The resulting mixture was stirred at 60 °C for 15 min then cooled to room temperature. After filtration, the filtrate was combined to the CH2Cl2 organic extracts. The organic layers were dried over MgSO4, filtrated and concentrated under reduced pressure. Silica gel column chromatography (EtOAc/petroleum ether: 90/10) afforded pure compound 2 (493 mg, 81%) as a beige solid. Characterization data were identical with that previously reported.30

4.2. General procedure for the synthesis of the dihydrazone-type compounds. A mixture of the appropriate hydrazine or hydrazide (2 equiv.) and dialdehyde (1 equiv.) was dissolved in absolute EtOH (30 mL/mmol) and heated under reflux for 18 h. After cooling to room temperature, the precipitated solid was collected by filtration and dried under vacuum to afford the corresponding bis-hydrazone. If no precipitate was observed, the reaction mixture was concentrated under reduced pressure. 4.2.1.

(N',N''E,N',N''E)-N',N''-(6,6'-Dihydroxy-5,5'-dimethoxybiphenyl-3,3'-

diyl)bis(methan-1-yl-1-ylidene)bis(3-methyl-1H-pyrazole-5-carbohydrazide) (1c). Following the general procedure, starting with 1 (50 mg, 0.165 mmol) and 1-hydrazinylphthalazine hydrochloride (46 mg, 0.33 mmol) in ethanol, compound 1c was obtained as a yellow solid (87 mg, 97 % yield). mp >260 °C; IR (neat): υmax = 3426, 3224, 1661, 1602 cm-1; 1H NMR (300 MHz, (CD3)2SO): δ = 2.28 (s, 6H), 3.91 (s, 6H), 6.48 (s, 2H), 7.01 (d, J = 1.4 Hz, 2H), 7.30 (d, J = 1.4 Hz, 2H), 8.39 (s, 2H), 8.92 (br s, 2H), 11.40 (s, 2H), 13.04 (s, 2H);

13

C NMR (75 MHz,

(CD3)2SO): δ = 10.4, 56.0, 104.8, 107.3, 124.3, 125.1, 140.0, 146.0, 146.2, 147.9, 148.2, 158.3; HRMS (ESI+) m/z [M+Na]+ calcd for C26H26N8O6Na: 569.1873, found: 569.1900.

4.2.2. (2E,2'E)-Benzyl 2,2'-(6,6'-dihydroxy-5,5'-dimethoxybiphenyl-3,3'-diyl)bis(methan-1yl-1-ylidene)bis(hydrazinecarboxylate) (1e). Following the general procedure, starting with 1 (50 mg, 0.165 mmol) and benzyl hydrazinecarboxylate (55 mg, 0.33 mmol) in ethanol, compound 1e was obtained as a yellow solid (94 mg, 95 % yield). mp : 135-137 °C; IR (neat): υmax = 3440, 1718, 1622 cm-1 ; 1H NMR (300 MHz, (CD3)2SO): δ = 3.88 (s, 6H), 5.16 (s, 4H), 6.97 (d, J = 1.8 Hz, 2H), 7.21 (d, J = 1.8 Hz, 2H), 7.37-7.41 (m, 10H), 7.95 (s, 2H), 8.92 (br s, 2H), 11.11 (br s, 2H); 13C NMR (75 MHz, (CD3)2SO): δ = 55.9, 65.7, 107.4, 123.4, 124.8, 125.1, 127.9, 128.0, 128.4, 136.7, 144.9, 145.8, 148.0, 153.4; HRMS (ESI+) m/z [M+Na]+ calcd for C32H30N4O8Na: 621.1961, found: 621.1960. 4.2.3.

(E)-6,6'-Oxybis(2-methoxy-4-((E)-(2-(phthalazin-1-yl)hydrazono)methyl)phenol)

dihydrochloride (2a). Following the general procedure, starting with 2 (70 mg, 0.22 mmol) and 1-hydrazinylphthalazine hydrochloride (86.5 mg, 0.44 mmol) in ethanol, compound 2a was obtained as a yellow solid (112 mg, 76 % yield). mp >280°C; IR (neat): υmax = 3361, 3204, 1617, 1595 cm-1 ; 1H NMR (300 MHz, (CD3)2SO): δ = 3.99 (s, 6H), 7.09 (d, J = 1.7 Hz, 2H), 7.70 (d, J = 1.7 Hz, 2H), 8.09-8.19 (m, 6H), 8.88 (br, 2H), 8.99 (br, 2H), 9.05-9.17 (m, 2H), 9.7 (br, 2H), 14.3 (br, 2H);

13

C NMR (75 MHz, (CD3)2SO): δ = 56.4, 106.4, 113.5, 118.9, 123.2,

125.5, 127.8, 128.1, 133.6, 135.8, 141.1, 144.4, 144.6, 147.4, 149.4, 152.9; HRMS (ESI+) m/z [M + H] + calcd for C32H27N8O5: 603.2104, found: 603.2109. 4.2.4.

(N',N''E,N',N''E)-N',N''-(5,5'-Oxybis(4-hydroxy-3-methoxy-5,1-

phenylene))bis(methan-1-yl-1-ylidene)diisonicotinohydrazide (2b). Following the general procedure, starting with 2 (119 mg, 0.37 mmol) and isonicotinic hydrazide (102.5 mg, 0.74 mmol) in ethanol, compound 2b was obtained as a white solid (199 mg, 96 % yield). mp : 230-

231°C; IR (neat): υmax = 3378, 3274,1652 cm-1; 1H NMR (300 MHz, (CD3)2SO): δ = 3.90 (s, 6H), 6.85 (d, J = 1.7 Hz, 2H), 7.14 (d, J = 1.7 Hz, 2H), 7.77 (dd, J = 1.6, 4.4 Hz, 4H), 8.30 (s, 2H), 8.76 (dd, J = 1.6, 4.4 Hz, 4H), 9.48 (br s, 2H), 11.89 (br, 2H);

13

C NMR (75 MHz,

(CD3)2SO): δ = 56.6, 106.3, 110.7, 121.4, 124.4, 140.0, 140.5, 144.8, 149.0, 149.1, 150.2, 161.3; HRMS (ESI+) m/z [M+Na] + calcd for C28H24N6O7Na: 579.1604, found: 579.1601. 4.2.5.

(N',N'',E,N',N'',E)-N',N''-(5,5'-Oxybis(4-hydroxy-3-methoxy-5,1-

phenylene))bis(methan-1-yl-1-ylidene)bis(3-methyl-1H-pyrazole-5-carbohydrazide)

(2c).

Following the general procedure, starting with 2 (119 mg, 0.37 mmol) and 3-methyl-1Hpyrazole-5-carbohydrazide (105 mg, 0.74 mmol) in ethanol, compound 2c was obtained as a white solid (190 mg, 90 % yield). mp >280°C; IR (neat): υmax = 3539, 3497, 3250, 1680, 1652 cm-1; 1H NMR (300 MHz, (CD3)2SO) δ = 2.26 (s, 6H), 3.89 (s, 6H), 6.45 (br s, 2H), 6.73 (br, 2H), 7.07 (br, 2H), 8.29 (s, 2H), 9.38 (br, 2H), 11.37 (s, 2H), 13.02 (s, 2H); 13C NMR (75 MHz, (CD3)2SO) δ = 10.3, 56.1, 104.8, 105.6, 110.8, 125.0, 139.6, 140.0, 144.9, 146.1, 147.3, 149.2, 158.1; HRMS (ESI+) m/z [M+Na] + calcd for C26H26N8O7Na: 585.1822, found: 585.1823. 4.2.6. (E)-6,6'-Oxybis(4-((E)-(2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-2-methoxyphenol) (2d). Following the general procedure, starting with 2 (122 mg, 0.38 mmol) and 2hydrazinylbenzo[d]thiazole (127 mg, 0.76 mmol) in ethanol, compound 2d was obtained as a white solid (195 mg, 83 % yield). mp >280°C; IR (neat): υmax = 3490, 1617, 1578 cm-1 ; 1H NMR (300 MHz, (CD3)2SO): δ = 3.90 (s, 6H), 6.81 (d, J = 1.6 Hz, 2H), 7.04 (td, J = 7.6, 1.1 Hz, 2H), 7.14 (d, J = 1.6 Hz, 2H), 7.25 (td, J = 7.6, 1.1 Hz, 2H), 7.34-7.41 (m, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.97 (s, 2H), 9.37 (br s, 2H), 12.13 (br s, 2H); 13C NMR (75 MHz, (CD3)2SO): δ = 56.0, 101.1, 105.4, 110.2, 117.2, 121.3, 121.4, 124.9, 125.8, 129.2, 139.2, 143.9, 144.8, 149.1, 166.8; HRMS (ESI+) m/z [M + H] + calcd for C30H25N6O5S2: 613.1328, found: 613.1329.

4.2.7. (2E, 2'E)-Benzyl 2,2'-(5,5'-Oxybis(4-hydroxy-3-methoxy-5,1-phenylene))bis(methan1-yl-1-ylidene)bis(hydrazinecarboxylate) (2e). Following the general procedure, starting with 2 (64 mg, 0.20 mmol) and benzyl hydrazinecarboxylate (66 mg, 0.40 mmol) in ethanol, compound 2e was obtained as a white solid (98 mg, 80 % yield). mp (EtOAc) : 133-135 °C; IR (neat): υmax = 3381, 3211, 1699 cm-1 ; 1H NMR (300 MHz, CD3OD): δ = 3.93 (s, 6H), 5.20 (s, 4H), 6.70 (d, J = 1.7 Hz, 2H), 7.32-7.39 (m, 12H), 7.74 (br, 2H); 13C NMR (75 MHz, CD3OD): δ = 56.8, 68.0, 105.8, 113.5, 126.7, 129.1, 129.2, 129.5, 137.8, 140.9, 145.9, 146.7, 150.7, 156.4; HRMS (ESI+) m/z [M + H] + calcd for C32H31N4O9: 615.2091, found: 615.2087.

4.3. DPPH radical scavenging activity To 4 mL of a DPPH• solution in ethanol (concentration 85.65 ± 3.37 µM to obtain a DO = 1) was added 5 to 50 µL of compounds solution in ethanol at different concentrations (4–800 µM). The absorbance was measured at 517 nm, at t = 0 min (A0) with Spectrophotometer UV- Visible Perkin-Elmer lambda 17 and after 60 min incubation at room temperature (A60). Percentage inhibition of DPPH free radical (I%) was calculated in the following way: I% = 100×(A0−A60)/A0. IC50 represents concentration of the tested compound, providing 50% inhibition of DPPH radicals. All experiments were carried out in triplicate. The 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used as antioxidant reference.

4.4. Cell Biology methods 4.4.1. Chemicals. Matrigel was from BD Biosciences (Le-Pont-de-Claix, FR). Corning Transwell Polycarbonate Membrane 24-wells, calcein-AM, MTT, from Sigma-Aldrich. H2DCFDA-acetoxymethyl ester was from Invitrogen, cell culture reagents and other reagents

were from WWR or Sigma.

4.4.2. Cell culture. Human microvascular endothelial cells (HMEC-1) were from the CDC (Dr. Candal, Atlanta, US). HMEC-1 cell line is an immortalized human dermal microvascular endothelial cell line that can be passaged more than 95 times without signs of senescence. These cells express the von Willebrand's Factor specific for endothelial cells, CD31, CD36, adhesion molecules, and form tubes when cultured on Matrigel, thus can be suitably utilized for angiogenesis assay. HMEC-1 may oxidize human LDL, and are suitable for studying the protective effect of antioxidants, as reported.25,27 In this study, HMEC-1 were used at passage 30-36. Cells were grown in MCDB-131 culture medium supplemented with 10% heat inactivated fetal calf serum, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin. The cells were starved in serum-free RPMI medium 24h before the experiments. 4.4.3. LDL isolation and oxidation. LDL from human pooled sera were prepared by ultracentrifugation, dialyzed against PBS containing 100 µmol.L-1 EDTA, as reported.27 Cellinduced LDL oxidation and the protective effect of the diaryl derivatives were studied on HMEC-1 seeded in 12-multiwell plates. Cells were incubated in serum-free RPMI 1640 containing native LDL (100 µg apoB mL-1). The synthesized compounds were solubilized in DMSO, and added to the culture medium at the indicated concentrations (and 0.1 % DMSO final concentration), simultaneously with LDL. At the end of the incubation (14 h at 37 °C), the LDLcontaining medium was used for determining the TBARS content,27 using Trolox as control antioxidant. For angiogenic and toxicity experiments, LDL were mildly oxidized by UV in the absence of the agent or Trolox as described.27 Under standard conditions, UV-oxLDL contained 71-104 nmol lipid hydroperoxide/mg apoB and 6.4-9.7 nmol TBARS/mg apoB.

4.4.4. Capillary tube formation. HMEC-1, seeded at 30,000 cells/well on Matrigel in 24 multiwell plates, were grown for 18 h at 37 °C, in 500 µL MCDB-131 medium, containing either 0.1% fetal bovine serum (FBS, negative control) or 2.5 % FBS (positive control) or oxLDL (20 µg mL-1), with or without phenolic diaryl or diaryl ether derivatives (10 µM). Capillary tubes were stained by Calcein (1 µM, 30 min), and photographed (Nikon Coolpix 995 camera) under a fluorescence microscope (λex 496, λem 516 nm). The number of linked cells, (i.e. cells connected to each other by thin microtube network) was counted and reported to the total cell number (linked + not-linked cells).14,17 4.4.5. Intracellular ROS determination. ROS generated in cells treated by oxLDL (20 µg mL1

) were evaluated by measuring the oxidation of 2’, 7’-dichlorodihydrofluorescein diacetate

acetoxymethyl ester (H2DCFDA-AM) at the indicated times.14 The probe was added to the culture medium (5 µM final concentration) 30 min before the end of the experiment. Then, the cells were washed three times with PBS. The fluorescence of the cell homogenate was measured (λex 495, λem 520 nm). The data are expressed as ratio of fluorescence/ fluorescence of the unstimulated control.14 Note that ROS determination could not be performed on Matrigel, because of the adsorption of the probe on this medium. 4.4.6. Evaluation of cytoprotective effect. LDL were mildly oxidized by UV-C irradiation.14,17 Briefly, LDL (2 mg apoB L-1 in dialysis buffer) were irradiated for 1 h and 30 min as a thin film (5 mm) in an open beaker placed 10 cm under the UV-C source (HNS 30W OFR Osram UV-C tube, λmax 254 nm, 0.5 mW cm-2). This method produces mildly UV-oxidized LDL (here referred to as oxLDL) that contained 63-97 nmol lipid hydroperoxide/mg apoB 3, and 5.6-8.3 nmol TBARS/mg apoB. OxLDL were sterilized by filtration and immediately used. For evaluating the protective effect of the synthesized antioxidants, HMEC-1 were seeded in 12

multiwell culture plates, in DMEM supplemented with 10% FBS. At sub-confluency, this medium was discarded and replaced by fresh RPMI medium containing 200 µg mL-1 of UVoxidized LDL, and the different agents at the indicated concentration. After 24 h of contact, the whole cytotoxicity was evaluated by the MTT assay. A control without LDL was done in the same conditions. The results were expressed as percent of the unstimulated control (100% viability). 4.4.7. Statistical analysis. Data are given as mean ± SEM. Estimates of statistical significance were performed by analysis of variance (one way Anova, Tukey test, SigmaStat software).20,27

Acknowledgements The authors acknowledge the financial support from INSERM, CNRS, Université Paul Sabatier, ANR Carina, Synfomia project and the technical support from Institut de Chimie de Toulouse (FR 3599). Supplementary data Supplementary data (NMR spectra and self-toxicity) associated with this article can be found, in the online version, at References and notes 1. Hopkins, P. N. Physiol. Rev. 2013, 93, 1317-542. 2. Carmeliet, P.; Jain, R. K. Nature 2011, 473, 298-307. 3. Michel, J. B.; Virmani, R.; Arbustini, E.; Pasterkamp, G. Eur. Heart J. 2011, 32, 1977-1985. 4. Mulligan-Kehoe, M. J.; Simons, M. Circulation 2014, 129, 2557-2566.

5. Kolodgie, F.D.; Gold, H.K.; Burke, A. P.; Fowler, D. R.; Kruth, H. S.; Weber, D. K.; Farb, A.; Guerrero, L. J.; Hayase, M.; Kutys, R.; Narula, J.; Finn, A. V.; Virmani, R. N. Engl. J. Med. 2003, 349, 2316-2325. 6. Spiegel, S.; Milstien, S. Nat. Rev. Mol. Cell. Biol. 2003, 4, 397-407. 7. Stone, J. R.; Collins, T. Endothelium 2002, 9, 231-238. 8. Ushio-Fukai, M. Cardiovasc. Res. 2006, 71, 226-235. 9. Arai, H. Subcell. Biochem. 2014, 77, 103-114. 10. Pirillo, A.; Norata, G. D.; Catapano, A. L. Mediators Inflamm. 2013, 2013, 152786. 11. Tsimikas, S.; Miller, Y. I. Curr. Pharm. Des. 2011, 17, 27-37. 12. Levitan, I.; Volkov, S.; Subbaiah, P. V. Antioxid. Redox Signal. 2010, 13, 39-75. 13. Dandapat, A.; Hu, C.; Sun, L.; Mehta, J. L. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 24352442. 14. Camaré, C.; Trayssac, M.; Garmy-Susini, B.; Mucher, E.; Sabbadini, R.; Salvayre, R.; NègreSalvayre, A. Br. J. Pharmacol. 2015, 172, 106-118. 15. Harkewicz, R.; Hartvigsen, K.; Almazan, F.; Dennis, E. A.; Witztum, J. L.; Miller, Y. I. J. Biol. Chem. 2008, 283, 10241-10251. 16. Bochkov, V. N.; Philippova, M.; Oskolkova, O.; Kadl, A.; Furnkranz, A.; Karabeg, E. et al. Circ. Res. 2006, 99, 900-908. 17. Camaré, C.; Augé, N.; Pucelle, M.; Saint-Lebes, B.; Grazide, M. H.; Nègre-Salvayre, A.; Salvayre, R. Free Radic. Biol. Med. 2016, 93, 204-216. 18. Heinecke, J. W. Atherosclerosis 1998, 141, 1-15. 19. Stoclet, J. C.; Chataigneau, T.; Ndiaye, M.; Oak, M. H.; El Bedoui, J.; Chataigneau, M.; Schini-Kerth, V. B. Eur. J. Pharmacol. 2004, 500, 299-313.

20. Nègre-Salvayre, A.; Coatrieux, C.; Ingueneau, C.; Salvayre, R. Br. J. Pharmacol. 2008, 153, 6-20. 21. Aviram, M. Free Radic. Res. 2000, Suppl:33, S85-97. 22. Münzel, T. L.; Gori,T.; Bruno, R. M.; Taddei, S. Eur. Heart J. 2010, 31, 2741-2748. 23. Brigelius-Flohe, R.; Kluth, D.; Banning, A. Mol. Nutr. Food Res. 2005, 49, 1083-1089. 24. Mitra, S.; Deshmukh, A.; Sachdeva, R.; Lu, J.; Mehta, J. L. Am. J. Med. Sci. 2011, 342, 135142. 25. Galvani, S.; Coatrieux, C.; Elbaz, M.; Grazide, M. H.; Thiers, J. C.; Parini, A.; Uchida, K.; Kamar, N.; Rostaing, L.; Baltas, M.; Salvayre, R.; Nègre-Salvayre, A. Free Radic. Biol. Med. 2008, 45,1457-1467. 26. Zhu, Q.; Sun, Z.; Jiang, Y.; Chen, F.; Wang, M. Mol. Nutr. Food Res. 2011, 55, 1375-1390. 27. Bouguerne, B.; Belkheiri, N.; Bedos-Belval, F.; Vindis, C.; Uchida, K.; Duran, H.; Grazide, M. H.; Baltas, M.; Salvayre, R.; Nègre-Salvayre, A. Antioxid. Redox Signal. 2011, 14, 20932106. 28. Verma, G.; Marella, A.; Shaquiquzzaman, M.; Akhtar, M.; Ali, M. R.; Alam, M. M. J. Pharm. Bioall. Sci. 2014, 6, 69-80. 29. Delomenède, M.; Bedos-Belval, F.; Duran, H.; Vindis, C.; Baltas, M.; Nègre-Salvayre, A. J. Med. Chem. 2008, 51, 3171-3181. 30. Vanucci-Bacqué, C.; Chaabouni, S.; Fabing, I.; Bedos-Belval, F.; Baltas, M. Tetrahedron Lett. 2014, 55, 528-530. 31. a) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. LWT-Food Sci. Technol. 1995, 28, 25-30. b) Molyneux, P.; Songklanakarin, J. J. Sci. Technol. 2004, 26, 211-219.

32. Sanna, D.; Delogu, G.; Mulas, M. ; Schirra, M.; Fadda, A. Food Anal. Methods 2012, 5, 759766. 33. We have checked that comparable IC50 values for Trolox were obtained by UV-absorption or EPR detections (see Table 1). 34. Hernandez-Vazquez, E.; Castaneda-Arriaga, R.; Ramírez-Espinosa, J. J.; Medina-Campos, O. N.; Hernandez-Luis, F.; Pedraza Chaverri , J.; Estrada-Soto S. Eur. J. Med. Chem. 2015, 100, 106-118. 35. Lu, J.; Mitra, S.; Wang, X.; Khaidakov, M.; Mehta, J. L. Antioxid. Redox Signal. 2011, 15, 36. Parthasarathy, S.; Raghavamenon, A.; Garelnabi, M. O.; Santanam, N. Methods Mol. Biol. 2010, 610, 403–417. 37. Yagi, K. Chem. Phys. Lipids 1987, 45, 337-351. 38. Salvayre, R.; Auge, N.; Benoist, H.; Negre-Salvayre, A. Biochim. Biophys. Acta 2002, 1585, 213-221. 39. Napoli, C. Ann. N. Y. Acad. Sci. 2003, 1010, 698-709. 40. Vindis, C.; Elbaz, M.; Escarqueil-Blanc, I.; Augé, N.; Heniquez, A.; Thiers, J. C.; NègreSalvayre, A.; Salvayre, R. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 639-45. 41. Cominacini, L.; Pasini, A. F.; Garbin, U.; Davoli, A.; Tosetti, M. L.; Campagnola, M.; Rigoni, A.; Pastorino, A. M.; Lo Cascio, V.; Sawamura, T. J. Biol. Chem. 2000, 275, 1263312638. 42. Costero, A. M.; Gil, S.; Parra, M. P.; Mancini, M. E.; Kneeteman, M. N.; Quindt, M. I. Tetrahedron Lett. 2015, 56, 3988-3991.

Figure Captions

Figure 1. Synthetic strategy scope - Chemical structure of compound 1a and dialdehyde precursors 1 and 2.

Figure 2. Capillary tube formation by HMEC-1 stimulated by low oxLDL concentration (20 µg mL-1) w/wo diaryl phenol derivatives and Trolox (10 µM each). A) Quantification of formed capillary tubes by HMEC-1 grown on Matrigel. Tube formation was expressed as % of linked cells

to

the

total

cell

number.

Mean

±

SEM

of

4

separate

experiments,

#(oxLDL/control),*(agent/oxLDL) p<0.05. FBS, fetal bovine serum. B) Representative pictures of tube formation on Matrigel in the presence of the different agents.

Figure 3. Effect of phenolic diaryl derivatives on the intracellular ROS increase evoked by oxLDL. ROS were determined in HMEC-1 preloaded with the fluorescent H2DCFDA-AM probe (5 µM), after 40 min incubation with oxLDL (20 µg mL-1), w/wo phenolic diaryl derivatives or Trolox (10 µM each). Mean ± SEM of 4 separate experiments, #(oxLDL/control),*(agent/oxLDL) p<0.05.

Scheme captions

Scheme 1. Synthesis of the diaryl ether core. Reagents and conditions: a) Cu(0), DMF, reflux; b) AlCl3, CH2Cl2, rt.

Scheme 2. Synthesis of bis-hydrazones. Reagents and conditions: a) 1-hydrazinylphthalazine hydrochloride (2 equiv.), EtOH, reflux; b) isonicotinic hydrazide (2 equiv.), EtOH, reflux; c) 3methyl-1H-pyrazole-5-carbohydrazide (2 equiv.), EtOH, reflux; d) 2-hydrazinylbenzo[d]thiazole (2 equiv.), EtOH, reflux; e) benzyl hydrazinecarboxylate (2 equiv.), EtOH, reflux.

Table captions Table 1. Free radical scavenging activity (DPPH), inhibition (TBARS) of cell-induced LDL oxidation and cytoprotective effect (MTT) against oxLDL toxicity of the diaryl derivatives at 1 µM and 10 µM.

Graphical abstract

oxLDL +2a+

oxLDL

Inhibition of capillary tube formation by HMEC1 stimulated by oxLDL

3a