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Abstract

New edaravone derivatives containing a benzylpiperazine moiety are designed and synthesized. The structures are characterized by ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry. The potential neuroprotective activities of the target compounds are evaluated in differentiated rat pheochromocytoma cells (PC12 cells) and in mice subjected to acute cerebral ischemia. Most of the target compounds showed neuroprotective activities both in vivo and in vitro, especially 1-(4-(4-fluorobenzyl) piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone and 1-(4-(4-nitrobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone, which displayed significant protective effects on cell viability against damage caused by H₂O₂, and remarkably prolonged the survival time of mice subjected to acute cerebral ischemia and decreased the mortality rate at all doses. These compounds represent lead compounds for the further discovery of neuroprotective agents for treating cerebral ischemic stroke. Molecular docking studies and basic structure–activity relationships are also presented.

Keywords

anti-ischemic activity antioxidant activity benzylpiperazine edaravone ischemic stroke

New edaravone derivatives containing a benzylpiperazine moiety are designed and synthesized.

Introduction

Ischemic stroke, the most common cerebrovascular disorder, accounts for more than 80% of all strokes and is one of the leading causes of morbidity and mortality worldwide.^1,2 Currently, the treatment of ischemic stroke is accomplished using for thrombolytic therapy in general, which can lead to secondary brain injury, namely, cerebral ischemia–reperfusion injury.^3–5 Oxidative stress is considered as the most important reason among a series of mechanisms related to the pathogenesis of cerebral ischemia–reperfusion injury. Oxidative stress can accelerate the generation of reactive oxygen species (ROS), which significantly exacerbate ischemic brain damage.⁶ However, overproduction of ROS cannot be neutralized under ischemic conditions because the antioxidant defense system in ischemic tissues is interrupted.⁷ Hence, exogenous supplementation of antioxidants with ROS scavenging activity would be a potential therapy for cerebral ischemia–reperfusion injury.

Edaravone (EDA, 3-methyl-1-phenyl-2-pyrazolin-5-one), an acid pyrazoline compound, is a recently developed neuroprotective drug that has been successfully used for treating acute stroke caused by cerebral thrombosis and embolism.⁸ EDA displays potent antioxidant properties, and consequently, it might be useful in managing other pathological processes involving oxidative stress.^9–11

Moreover, there is increasing interest in the synthesis of EDA derivatives designed to potentiate the free-radical scavenging activity of this compound. Some of these derivatives have been reported to present other beneficial properties, such as antioxidant activity, vasodilator activity, anticerebral ischemia activity and antimyocardial ischemia activity.^12–14

The piperazine nucleus is capable of binding to multiple receptors with high affinity. Piperazine derivatives are widely applied in a number of different therapeutic areas, including antitumor, antifungal, antidepressant, and antiviral.^15–17 The piperazine scaffold occupies an important position in the field of medicinal and pesticide chemistry (Figure 1).^18,19 Our previous studies discovered that the benzylpiperazine scaffold often affords neuroprotective activity.²⁰ Given that a number of drug molecules containing piperazine exhibit improved efficacy in several aspects, especially for vasodilation, antihypertension, increasing cerebral blood flow, and neuroprotection,^21–23 we therefore designed and synthesized a new class of compounds obtained by joining EDA through an acetyl linker with benzhydryl piperazines. The obtained edaravane derivatives were evaluated for their antioxidant activities in vitro and anticerebral ischemia activities in vivo. In addition, molecular modeling was employed to further investigate the mechanisms underlying their activities.

Figure 1.

Examples of benzylpiperazine-based drug molecules.

Results and discussion

Synthetic chemistry

The EDA derivatives 8a–l were conveniently synthesized as outlined in Scheme 1. The key intermediate, 1-(4-methoxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (2), which was obtained by cyclization of 4-methoxyphenylhydrazine hydrochloride (1) with ethyl acetoacetate, was transformed into the desired intermediate 1-(4-hydroxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (3), via reaction with a mixture of 40% hydrobromic acid and acetic acid. The synthesis of the other key intermediates, substituted benzylpiperazines 7a–l, commenced with the free-radical bromination reactions of substituted toluenes 4a–l with N-bromosuccinimide (NBS). Subsequent coupling of the obtained benzyl bromides 5a–l with N-Boc-piperazine catalyzed by potassium carbonate, followed by nucleophilic substitution with chloroacetyl chloride, afforded benzylpiperazines 7a–l. The target compounds 8a–l were finally obtained by condensation of substituted 1-(4-benzylpiperazin-1-yl)-2-chloroethanones 7a–l with 1-(4-hydroxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (3).

Scheme 1.

Reagents and conditions: (i) ethyl o, EtOH, reflux, 8 h. (ii) 40% HBr, AcOH, reflux, 10 h. (iii) NBS, AIBN, CCl₄, reflux, 1 h. (iv) N-Boc-piperazine, K₂CO₃, CH₂Cl₂, reflux, 10 h; then CH₂Cl₂, TFA, r.t., 2 h. (v) chloroacetyl chloride, Et₃N, THF, r.t., 6 h. (vi) K₂CO₃, MeCN, reflux, 10 h.

In vivo anti-ischemic activity

First, the target compounds were screened in mice that had undergone bilateral common carotid artery occlusion to study the effects of the prepared compounds on acute cerebral ischemia. As shown in Table 1, most of the target compounds were active in mice intraperitoneally (i.p.) at different doses (200, 100, 50, 25 mg kg⁻¹), among which compounds 8d and 8l at all doses significantly prolonged the survival time of mice subjected to acute cerebral ischemia and showed potent neuroprotective effects. Compounds 8a, 8c, 8f, 8g, 8h, and 8k exhibited activities at high and median doses (200 and 100 mg kg⁻¹). Compound 8b only presented protection at low and extremely low doses (50 and 25 mg kg⁻¹) and showed no effects at high or median doses.

Table 1.

In vivo anti-ischemic effects of compounds 8a–l on the survival time of mice subjected to bilateral common carotid artery ligation.

Compounds	Survival time (min)
Compounds	200 mg kg⁻¹	100 mg kg⁻¹	50 mg kg⁻¹	25 mg kg⁻¹
8a	5.48 ± 1.31*	6.69 ± 2.11*	1.16 ± 0.61	1.56 ± 0.81
8b	3.27 ± 1.12	2.03 ± 1.28	8.57 ± 1.61*	4.42 ± 1.52*
8c	4.47 ± 1.12*	3.95 ± 1.11*	3.53 ± 1.22*	1.33 ± 0.34
8d	7.46 ± 1.65*	7.15 ± 1.81*	6.51 ± 1.68*	4.22 ± 1.02*
8e	2.64 ± 0.98	3.06 ± 1.07	1.52 ± 0.89	2.19 ± 1.17
8f	7.33 ± 1.54*	4.84 ± 1.47*	3.68 ± 0.62*	1.52 ± 0.56
8g	4.23 ± 1.32*	5.53 ± 1.39*	5.37 ± 1.32*	1.09 ± 0.19
8h	6.13 ± 1.59*	6.37 ± 1.67*	1.34 ± 0.63	1.19 ± 0.18
8i	3.45 ± 1.34*	3.06 ± 1.17	3.04 ± 1.41	3.12 ± 1.21
8j	1.64 ± 0.41	2.68 ± 1.07	2.21 ± 1.56	2.14 ± 1.07
8k	7.65 ± 1.34*	5.14 ± 1.33*	4.35 ± 1.26*	3.22 ± 1.01
8l	15.41 ± 1.92*	8.94 ± 1.02*	6.81 ± 1.40*	4.08 ± 1.53*
NS	2.02 ± 0.52
Nimodipine (80 mg kg⁻¹)	3.48 ± 1.01*

NS: normal saline.

p < 0.05 versus NS.

In vitro antioxidant activity related to the structure

In order to study the potential antioxidant activity of the title compounds, another screening was performed to investigate neuroprotection on impairment induced by H₂O₂ in PC12 cells, as evaluated by the MTT assay. We damaged PC12 cells with 10 mM H₂O₂ and assessed the protective actions of 12 EDA derivatives. We found that most of the new compounds exhibited moderate to good protection against H₂O₂-induced cell damage (Table 2).

Table 2.

In vitro neuroprotective effects of compounds 8a–l against H₂O₂-induced neurotoxicity in PC12 cells.

Compounds	Protection (%)
Compounds	10 μM	1 μM	0.1 μM
8a	46.76 ± 2.34*	36.42 ± 2.22*	19.83 ± 1.46*
8b	9.47 ± 0.88	13.07 ± 1.67	20.14 ± 2.01*
8c	23.38 ± 2.11*	9.28 ± 1.01	14.84 ± 1.13
8d	40.26 ± 2.27*	37.84 ± 1.98*	30.47 ± 2.13*
8e	9.20 ± 1.08	13.02 ± 2.05	9.27 ± 1.88
8f	18.17 ± 2.33*	18.86 ± 2.48*	10.02 ± 2.04
8g	18.93 ± 1.65*	22.08 ± 1.77*	12.93 ± 1.59
8h	38.06 ± 2.31*	27.28 ± 1.27*	26.71± 2.55*
8i	33.04 ± 2.01*	29.53 ± 1.79*	29.07 ± 2.23*
8j	31.39 ± 3.22*	20.11 ± 1.96*	22.77 ± 3.06*
8k	17.69 ± 1.18*	16.80 ± 2.35*	15.97 ± 2.17*
8l	50.28 ± 2.34*	42.77 ± 2.04*	37.43 ± 2.32*
Edaravone	41.03 ± 2.26*	38.76 ± 1.78*	34.18 ± 2.18*

p < 0.05 versus H₂O₂-treated group.

As shown in Table 2, compounds 8d, 8h, 8i, 8j, and 8l showed good antioxidant activity in H₂O₂-induced PC12 cells at all the doses tested (protection >20%). It was found that the cumulative addition of the compounds 8a, 8d, 8h, 8i, 8k, and 8l (0.1–10 μM) caused concentration-dependent neuroprotective effects with the maximum effect observed at 10 μM. Compounds 8c, 8e, 8f, 8g, and 8j showed a pattern of increased protection with increasing concentrations (1.0–10 and 0.1–1.0 μM, respectively) in terms of cell protection. Compound 8b was observed to have the highest protection at the lowest concentrations of 0.1 μM (cell protection: 20.14%). Compounds 8c and 8j were observed to give the highest protection at the highest concentrations of 10 μM (cell protection: 23.38% and 31.39%, respectively).

The derivatives were substituted with different functional groups to study the influence of the substituent effect on the biological activity and find new compounds with better neuroprotective activity. The substituents were considered and selected based on lipophilic and electronic attributes, for example, lipophilic electron–withdrawing groups (NO₂, CN, F, Cl, and Br), hydrophilic electron–donating groups (OCH₂CH₃), and lipophilic electron–releasing groups (COOCH₃ and naphthyl). The general structure–activity relationship (SAR) of these compounds showed that compounds with a 4-substituent on the benzylpiperazine moiety were more active than those with a 2-substitutent (cell protection: 8a > 8c, 8d > 8e, 8h > 8k, 8i > 8g). Most derivatives having electron-withdrawing groups were more active than those having electron-releasing groups. Strong electron-withdrawing group nitro-substituted compound 8l exhibited the best neuroprotective effect, and exhibited a more promising neuroprotective activity than that of the positive control EDA (cell protection: 50.28% vs 41.03% at 10 μM, 42.77% vs 38.76% at 1.0 μM, and 7.43% vs 34.18% at 0.1 μM, respectively). Among the electron-withdrawing donating groups, lipophilic derivatives were more active than a hydrophilic ethoxy derivative (cell protection: 8a > 8k, 8d > 8k, 8h > 8k, and 8l > 8k). However, for the different isoelectronic halogens, a highly electronegative fluoro substituent at the 4-position was more potent and less neurotoxic than the corresponding chloro-substituted derivative (cell protection: 8d > 8j). Similarly, a Br-substituent at the 4-position was more potent and less neurotoxic than the corresponding iodo-substituted derivative (cell protection: 8i > 8f). It was worthwhile to note that the effect of 8i (cell protection: 33.04% at 10 μM, 29.53% at 1.0 μM, and 29.07% at 0.1 μM, respectively) was more pronounced than that of the corresponding chloro-substituted derivative 8j, although compound 8i includes a bromo substituent.

Molecular docking

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a principal regulator of the cellular defense system against oxidative stress, promoting the transcription of an expansive set of antioxidants and cytoprotective enzymes.^24,25 Under normal conditions, Nrf2 is kept at low concentrations by the cytosolic repressor protein Kelch-like ECH-associated protein 1 (Keap1). By contrast, under pathological conditions, Nrf2 is released from Keap1.^26–28

In order to further evaluate these promising compounds and to guide further structure–activity relationship (SAR) studies, the interaction effects of the electron-releasing naphthyl-substituted compound 8c and electron-withdrawing nitro-substituted compound 8l with the Keap1-Nrf2 crystal structure (entry 1X2R in the Protein Data Bank) were investigated. The molecular docking was performed by inserting 8c and 8l into the binding site of the Keap1 protein. All docking runs were applied in SurflexDock using Sybyl 1.3. The binding modes of 8c and 8l in Keap1 are depicted in Figures 2 and 3, respectively.

Figure 2.

A 3D plan view of the 8c–Keap1 protein interaction active site (PDB code, 1X2R). Compound 8c is shown as sticks. Hydrogen bonds within 3.0 Å are shown as yellow dashed lines.

Figure 3.

A 3D plan view of the 8l–Keap1 protein interaction active site (PDB code, 1X2R). Compound 8l is shown as sticks. Hydrogen bonds within 3.0 Å are shown as yellow dashed lines.

As shown in Figure 2, the oxygen atom of the carbonyl moiety of 8c forms a hydrogen bond with Ser431, and the oxygen atom of the oxyacetyl moiety forms a hydrogen bond with Arg415. In addition, the nitrogen atom of the piperazine ring forms a hydrogen bond with Arg380. As illustrated in Figure 3, the oxygen atom of the 1H-pyrazol-5(4H)-one moiety of 8l forms two hydrogen bonds with Asn387 and Asp389, and the oxygen atom of the oxyacetyl moiety forms two hydrogen bonds with Asn414 and Ser431, respectively. The presence of these hydrogen bonds shows that compounds 8c and 8l can undoubtedly interact with the catalytic domain of the Keap1 protein. These molecular docking results, along with the biological assay data, indicate that derivatives 8c and 8l are potential inhibitors of the Keap1-Nrf2 protein–protein interaction.

The above research suggests that compounds 8c and 8l can cause dissociation of Keap1 from Nrf2 and the release of Nrf2 from Keap1 through occupation of the Nrf2-binding site, and finally induce the activation of Nrf2.

Conclusion

In summary, 12 novel EDA derivatives 8a–l containing a benzylpiperazine moiety have been designed and synthesized. The in vitro antioxidant activities and the in vivo anti-ischemic activities have also been studied. Most of the prepared derivatives showed neuroprotective activities both in vivo and in vitro, especially 8d and 8l, which displayed significant protective effects on the cell viability against damage caused by H₂O₂, and remarkably prolonged the survival time of mice subjected to acute cerebral ischemia and decreased the mortality rate at all doses. The molecular docking of 8c, and 8l with Keap1 (1X2R) suggested that compounds 8c and 8l are potential inhibitors of the Keap1-Nrf2 protein–protein interaction. The present findings provide a preliminary pharmacological basis to exploit preventive or therapeutic strategies for ischemic stroke and other neurological injuries.

Experimental

Materials and apparatus

All chemicals, reagents, and solvents were commercially available and used without further purification. Melting points were determined on an electrothermal digital apparatus model WRR-401 (Shanghai, China) without correction. The ¹H and ¹³C NMR spectra were recorded on a Bruker ACF-300 MHz instrument (Bruker, Billerica, MA, USA) with CDCl₃ as the solvent and tetramethylsilane as an internal standard (chemical shifts are expressed as δ values, and J in hertz). High-resolution mass spectrometry (HRMS) was performed with a MALDI Micro MX mass spectrometer (Waters, Milford, MA, USA) with electrospray ionization (ESI). The measurements were performed using either positive or negative ionization modes. Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT, USA). Horse serum (HS), penicillin, and streptomycin were obtained from Gibco BRL (Div. of Invitrogen, Gaithersburg, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Co. (St. Louis, MO, USA).

1-(4-Hydroxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (3)

A mixture of 4-methoxyphenylhydrazine hydrochloride (1) (100 mmol) and ethyl acetoacetate (140 mmol) in ethanol (50 mL) was refluxed under stirring for 8 h. After cooling, the precipitated crystals were filtered and recrystallized from ethanol to give 1-(4-methoxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (2) as white crystals. Compound 2 (40 mmol) was added to a mixture of 40% hydrobromic acid (50 mL) and acetic acid (50 mL), and the mixture was refluxed under stirring for 10 h. After evaporation of the solvent, aqueous NaHCO₃ solution was added to the residue to adjust the pH to 4, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic layer was dried and concentrated. Recrystallization of the residue from ethanol gave 1-(4-hydroxyphenyl)methyl-1H-pyrazol-5(4H)-one (3).²⁹ White solid was 3.72 g (49%); m.p. was 229.5 °C (dec.). HRMS (ESI): m/z calcd [M + H]⁺ for C₁₀H₁₁N₂O₂: 191.0815; found: 191.0813.

Substituted benzyl bromides 5a–l; general procedure

A mixture of the corresponding commercially available substituted toluene 4a–l (70 mmol), NBS (70 mmol), and azobisisobutyronitrile (AIBN) (7 mmol) in carbon tetrachloride (108 mL) was refluxed under illumination with a 40 W tungsten lamp for 1 h. After cooling, cyclohexane (108 mL) was added to the mixture, and the reaction was stirred for 10 min. After removal of the obtained precipitate by filtration, the filtrate was concentrated and recrystallized from ethanol to give the corresponding substituted benzyl bromide 5a–l.^30,31

Substituted N-benzylpiperazines 6a–l; general procedure

To a stirred solution of N-Boc-piperazine (2 mmol) in CH₂Cl₂ (20 mL) were sequentially added the substituted benzyl bromide 5a–l (1.6 mmol) and K₂CO₃ (7.2 mmol). The resulting mixture was heated at reflux for 10 h. After cooling, the mixture was diluted with EtOAc and poured into water. The organic layer was washed once with brine, dried over Na₂SO₄, and concentrated in vacuo. The resulting crude oil was taken up in CH₂Cl₂/TFA (1:1 v/v, 6 mL total). After the mixture had been stirred for 2 h, the crude product was concentrated in vacuo to give the substituted N-benzylpiperazine 6a–l, which was used without further purification.^32,33

Substituted 1-(4-benzylpiperazin-1-yl)-2-chloroethanones 7a–l; general procedure

A mixture of the corresponding substituted benzylpiperazine 6a–l (15.8 mmol) and triethylamine (23.8 mmol) in dry tetrahydrofuran (THF, 50 mL) was stirred at room temperature under an N₂ atmosphere. Chloroacetyl chloride (17.4 mmol) was then added to the reaction mixture via syringe. The mixture was stirred at room temperature for 16 h, and then concentrated in vacuo. The crude residue was dissolved in CH₂Cl₂ (50 mL) and washed with saturated NaHCO₃ (30 mL × 3). The organic layer was dried and concentrated under reduced pressure to give the substituted 1-(4-benzylpiperazin-1-yl)-2-chloroethanone 7a–l, which was used without further purification.^34,35

EDA derivatives 8a–l; general procedure

A mixture of the corresponding substituted 1-(4-benzylpiperazin-1-yl)-2-chloroethanone 7a–l (6 mmol), 1-(4-hydroxyphenyl)-3-methyl-1H-pyrazol-5(4H)-one (3) (6.6 mmol) and potassium carbonate (9 mmol) in acetonitrile (30 mL) was refluxed under stirring for 10 h. After cooling, the solvent was evaporated to give the crude product, which was purified by column chromatography over silica gel (100–200 mesh) using hexane/ethyl acetate (6:4) as the eluent. The products 8a–l were obtained as crystalline solids.

4-((4-(2-(4-(5-Hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)acetyl)piperazin-1-yl)methyl)-N,N-dimethylbenzenesulfonamide (8a): White solid (1.32 g, 43%). m.p. 167.2–169.3 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.75–6.73 (m, 8H), 5.52 (s, 1H), 4.69 (s, 2H), 3.66 (br s, 2H), 3.56 (s, 2H), 3.44 (br s, 2H), 2.72 (s, 6H), 2.46 (br s, 2H), 2.25 (br s, 2H), 2.25 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.9, 148.4, 129.6, 128.0, 124.8, 115.8, 86.5, 70.2, 61.9, 52.7, 44.9, 37.9, 29.7, 14.4. HRMS (ESI): m/z calcd [M − H]⁻ for C₂₅H₃₀N₅O₅S: 512.1968; found: 512.1978.

Methyl 4-((4-(2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)acetyl)piperazin-1-yl)methyl)benzoate (8b): White solid (1.42 g, 51%). m.p. 209.3–211.1 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.63 (s, 1H), 7.99–6.66 (m, 8H), 5.50 (s, 1H), 4.68 (s, 2H), 3.90 (s, 3H), 3.62 (br s, 2H), 3.51 (s, 2H), 3.40 (br s, 2H), 2.41 (br s, 2H), 2.29 (br s, 2H), 2.23 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 166.9, 164.9, 155.9, 153.3, 148.3, 142.7, 130.0, 129.7, 129.3, 128.9, 124.9, 115.8, 86.4, 69.9, 62.3, 52.8, 52.6, 52.1, 45.1, 42.1, 14.2. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₅H₂₈N₄O₅Na: 487.1957; found: 487.1945.

2-(4-(5-Hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)-1-(4-(naphthalen-1-ylmethyl)piperazin-1-yl)ethanone (8c): White solid (1.42 g, 52%). m.p. 174.0–175.3 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.22–6.73 (m, 11H), 5.50 (s, 1H), 4.68 (s, 2H), 3.92 (s, 2H), 3.64 (br s, 2H), 3.41 (br s, 2H), 2.52 (br s, 2H), 2.37 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.9, 155.7, 153.3, 148.3, 133.9, 132.4, 130.4, 128.6, 128.0, 126.0, 125.8, 125.1, 124.9, 124.4, 115.9, 86.5, 70.0, 60.6, 52.9, 52.7, 45.0, 42.0, 14.3. HRMS (ESI): m/z calcd [M + H]⁺ for C₂₇H₂₉N₄O₃: 457.2240; found: 457.2252.

1-(4-(4-Fluorobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8d): White solid (1.20 g, 47%). m.p. 131.0–132.2 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.43–6.80 (m, 8H), 5.54 (s, 1H), 4.71 (s, 2H), 3.66 (br s, 2H), 3.51 (s, 2H), 3.51 (br s, 2H), 2.48 (br s, 2H), 2.32 (br s, 2H), 2.27 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ 164.9, 163.2 (J = 243.8 Hz), 155.4, 153.3, 148.3, 130.7, 130.6, 130.5, 124.8, 115.8, 115.3, 115.2, 86.5, 70.0, 61.9, 52.7, 52.5, 45.1, 42.1, 14.3. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₃H₂₅N₄O₃FNa: 447.1808; found: 447.1818.

1-(4-(2-Fluorobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8e): White solid (1.04 g, 41%). m.p. 114.7–116.2 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.34–6.70 (m, 8H), 5.50 (s, 1H), 4.68 (s, 2H), 3.65 (br s, 2H), 3.60 (s, 2H), 3.43 (br s, 2H), 2.49 (br s, 2H), 2.39 (br s, 2H), 2.23 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 166.8, 165.0 (J = 244.5 Hz), 157.4, 155.2, 150.3, 133.7, 132.4, 131.5, 126.8, 126.1, 126.0, 117.7, 117.5, 117.3, 88.4, 72.0, 56.9, 54.5, 54.1, 46.8, 43.8, 16.3. HRMS (ESI): m/z calcd [M + H]⁺ for C₂₃H₂₆N₄O₃F: 425.1989; found: 425.2003.

1-(4-(4-Iodobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8f): White solid (1.18 g, 37%). m.p. 204.0–205.9 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.66–6.71 (m, 8H), 5.51 (s, 1H), 4.69 (s, 2H), 3.64 (br s, 2H), 3.42 (br s, 2H), 3.42 (s, 2H), 2.43 (br s, 2H), 2.29 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 166.8, 157.4, 150.3, 139.6, 133.8, 133.2, 126.7, 126.1, 126.0, 117.7, 88.5, 72.0, 63.9, 54.6, 54.4, 16.3. HRMS (ESI): m/z calcd [M − H]⁻ for C₂₃H₂₄N₄O₃I: 531.0893; found: 531.0899.

1-(4-(2-Bromobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8g): White solid (1.28 g, 44%). m.p. 179.3–180.9 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.56–6.69 (m, 8H), 5.51 (s, 1H), 4.69 (s, 2H), 3.61 (br s, 2H), 3.61 (s, 2H), 3.43 (br s, 2H), 2.52 (br s, 2H), 2.42 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.9, 155.7, 153.3, 148.3, 133.0, 131.1, 130.3, 129.0, 127.4, 124.9, 115.9, 86.5, 70.0, 61.5, 52.8, 52.4, 45.1, 42.1, 29.7, 14.3. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₃H₂₅N₄O₃BrNa: 507.1008; found: 507.1000.

4-((4-(2-(4-(5-Hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)acetyl)piperazin-1-yl)methyl)benzonitrile (8h): White solid (0.78 g, 30%). m.p. 177.6–180.4 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.63–6.70 (m, 8H), 5.52 (s, 1H), 4.70 (s, 2H), 3.64 (br s, 2H), 3.52 (s, 2H), 3.43 (br s, 2H), 2.43 (br s, 2H), 2.28 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 165.3, 155.9, 148.8, 132.7, 130.0, 125.3, 116.2, 86.9, 70.4, 62.5, 59.2, 53.1, 45.4, 44.6, 42.4, 30.1, 14.8. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₄H₂₅N₅O₃Na: 454.1855; found: 454.1845.

1-(4-(4-Bromobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8i): Yellow solid (1.80 g, 62%). m.p. 138.9–140.2 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.67–6.75 (m, 8H), 5.51 (s, 1H), 4.69 (s, 2H), 3.64 (br s, 2H), 3.44 (s, 2H), 3.44 (br s, 2H), 2.44 (br s, 2H), 2.29 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.8, 156.0, 153.2, 148.1, 136.2, 131.4, 130.7, 130.6, 129.8, 124.8, 121.1, 115.7, 86.4, 69.8, 61.8, 52.6, 52.3, 44.9, 41.9, 14.1. HRMS (ESI): m/z calcd [M + H]⁺ for C₂₃H₂₆BrN₄O₃: 485.1188; found: 485.1198.

1-(4-(4-Chlorobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8j): Yellow solid (1.45 g, 55%). m.p. 131.6–133.2 °C. ¹H NMR (300 MHz, CDCl₃): δ 7.31–6.68 (m, 8H), 5.51 (s, 1H), 4.69 (s, 2H), 3.63 (br s, 2H), 3.44 (s, 2H), 3.41 (br s, 2H), 2.42 (br s, 2H), 2.29 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.9, 156.0, 153.1, 148.2, 134.8, 133.2, 130.5, 129.6, 128.4, 124., 115.6, 86.4, 69.7, 61.5, 52.2, 52.1, 44.6, 41.7, 21.0, 14.0. HRMS (ESI): m/z calcd [M + H]⁺ for C₂₃H₂₆ClN₄O₃: 441.1693; found: 441.1704.

1-(4-(2-Ethoxybenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8k): Yellow solid (1.19 g, 44%). m.p. 138.7–140.2 °C. ¹H NMR (300 MHz, dimethyl sulfoxide (DMSO)-d₆): δ 9.50 (s, 1H), 7.48–6.77 (m, 8H), 5.60 (s, 1H), 4.90 (s, 2H), 4.05 (q, 2H, J = 6.9 Hz), 3.50 (s, 2H), 3.44 (br s, 2H), 3.37 (br s, 2H), 2.38 (br s, 4H), 2.10 (s, 3H), 1.35 (t, 3H, J = 6.9 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 164.8, 157.3, 156.0, 153.3, 148.2, 130.9, 129.8, 128.6, 124.9, 115.7, 111.6, 86.4, 69.9, 63.6, 55.9, 52.7, 52.4, 45.0, 42.0, 14.8, 14.2. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₅H₃₀N₄O₄Na: 473.2165; found: 473.2173.

1-(4-(4-Nitrobenzyl)piperazin-1-yl)-2-(4-(5-hydroxy-3-methyl-1H-pyrazol-1-yl)phenoxy)ethanone (8l): Yellow solid (1.01 g, 37%). m.p. 190.3–191.6 °C. ¹H NMR (300 MHz, CDCl₃): δ 8.19–6.70 (m, 9H), 5.51 (s, 1H), 4.70 (s, 2H), 3.64 (br s, 2H), 3.55 (s, 2H), 3.42 (br s, 2H), 2.44 (br s, 2H), 2.28 (br s, 2H), 2.24 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.9, 155.8, 153.3, 148.4, 130.1, 129.5, 125.0, 123.6, 115.9, 86.5, 69.9, 61.8, 52.8, 52.7, 45.1, 42.1, 14.3. HRMS (ESI): m/z calcd [M + Na]⁺ for C₂₃H₂₅N₅O₅Na: 474.1753; found: 474.1737.

In vitro antioxidant activity assays

The in vitro antioxidant activities of target compounds 8a–l were evaluated by MTT assays against H₂O₂-induced PC12 cells, with EDA as the positive control.^36–39 PC12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) FBS, 5% (v/v) HS, 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin at 37 °C in a humidified atmosphere of 5% carbon dioxide.

PC12 cells were inoculated in a 96-well microplate (10⁵ cells/well in 100 µL medium) for 24 h. After washing with phosphate-buffered saline (PBS), the PC12 cells were incubated with H₂O₂ (10 mM), or H₂O₂ (10 mM) with compounds 8a–l or EDA (0.1, 1, 10 μM) for 24 h, respectively, at which time MTT (10 μL, 5 mg mL⁻¹) was added to each culture well. After incubation at 37 °C for an additional 4 h, the formazan crystals were dissolved by addition of DMSO (150 μL), and the plates were shaken vigorously to ensure complete solubilization. The formazan absorbance was assessed at 490 nm using an enzyme-linked immunoassay (ELISA) plate reader.⁴⁰

In vivo anti-ischemic activity assays

The in vivo anti-ischemic activity was tested using bilateral common carotid artery occlusion.^41,42 Kunming mice of both sexes were randomly divided into groups (10 mice per group). The title compounds 8a–l were dissolved in aqueous 0.5% sodium carboxy methyl cellulose (CMCNa) solution before use and administered i.p. Nimodipine (80 mg kg⁻¹) was administered i.p. as a positive control. The negative control group received normal saline (NS) in the same volume as other groups. All groups were given drugs twice a day for 3 d. Sixty minutes after the last administration, all mice were anesthetized using ether. All groups underwent the operation for common carotid artery and vagus nerve ligation. Then, the survival time of the mice was recorded.

Molecular modeling

The molecular docking procedure was performed within SYBYLX 1.3 software. The crystal structure of Keap1-Nrf2 (PDB ID: 1X2R) was obtained from the RCSB Protein Data Bank. Keap1-Nrf2 was modified using the Prepare Protein Structure Tool. The designed ligands were constructed and energy minimized using the Tripos force field. The convergence accuracy and the maximum number of iteration steps were 1000 and 0.005 kcal mol⁻¹ Å, respectively. The Keap1 protein was defined as a receptor. Nrf2 was removed, and its site sphere was selected as a reference for inserting 8c and 8l. Other parameters were set as default. After accomplishing the molecular docking procedure, 20 docking poses were scored and selected based on the calculated energy. The best docking pose was prepared using PyMOL, as shown in Figures 2 and 3.

Supplemental Material

sj-docx-1-chl-10.1177_17475198221116827 – Supplemental material for The design and synthesis of benzylpiperazine-based edaravone derivatives and their neuroprotective activities

Supplemental material, sj-docx-1-chl-10.1177_17475198221116827 for The design and synthesis of benzylpiperazine-based edaravone derivatives and their neuroprotective activities by Mengjie Gao, Shuangyan Ma, Tong Xu, Nan Jiang, Yi Xu, Yan Zhong and Bin Wu in Journal of Chemical Research

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Bin Wu

Supplemental material

Supplemental material for this article is available online.

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