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. 2024 Feb 28;15(4):1198–1209. doi: 10.1039/d4md00038b

Design, synthesis, and evaluation of novel ferrostatin derivatives for the prevention of HG-induced VEC ferroptosis†

Xin-Xin Wang a,‡, Run-Jie Wang b,‡, Hua-Long Ji a,‡, Xiao-Yu Liu c, Nai-Yu Zhang a, Kai-Ming Wang a, Kai Chen c,✉, Ping-Ping Liu d,✉, Ning Meng a,✉, Cheng-Shi Jiang a,✉
PMCID: PMC11042167  PMID: 38665835

Abstract

Ferroptosis is a nonapoptotic, iron-catalyzed form of regulated cell death. It has been shown that high glucose (HG) could induce ferroptosis in vascular endothelial cells (VECs), consequently contributing to the development of various diseases. This study synthesized and evaluated a series of novel ferrostatin-1 (Fer-1) derivatives fused with a benzohydrazide moiety to prevent HG-induced VEC ferroptosis. Several promising compounds showed similar or improved inhibitory effects compared to positive control Fer-1. The most effective candidate 12 exhibited better protection against erastin-induced ferroptosis and high glucose-induced ferroptosis in VECs. Mechanistic studies revealed that compound 12 prevented mitochondrial damage, reduced intracellular ROS accumulation, upregulated the expression of GPX4, and decreased the amounts of ferrous ion, LPO and MDA in VECs. However, compound 12 still exhibited undesirable microsomal stability like Fer-1, suggesting the need for further optimization. Overall, the present findings highlight ferroptosis inhibitor 12 as a potential lead compound for treating ferroptosis-associated vascular diseases.

This work designed and synthesized novel ferrostatin analogs with a benzohydrazide moiety, and identified compound 12 as a promising lead for preventing HG-induced VEC ferroptosis.graphic file with name d4md00038b-ga.jpg

1. Introduction

Vascular endothelial cells (VECs) are the cells that line the inner surface of blood vessels and play a vital role in maintaining vascular homeostasis, regulating blood flow, and preventing thrombosis.1 VEC injury is a condition that affects the function of the endothelium, and thus leads to various diseases, including atherosclerosis, hypertension, diabetes, and sepsis.2,3 In the pathological pathways of VEC-related diseases, high glucose (HG) is considered a key factor due to its involvement in oxidative stress, inflammation, and metabolic disorders.4–6 HG has particularly been identified as a major factor in the development of diabetic vascular disease (DVD), a significant complication of diabetes mellitus that affects millions of people worldwide and causes substantial morbidity and mortality.7 Therefore, finding ways to protect endothelial function from HG is crucial for the prevention and treatment of diabetic-associated vascular diseases.8–11

Ferroptosis is a recently discovered form of regulated cell death characterized by iron-dependent lipid peroxidation. It differs from other forms of cell death, such as apoptosis, necrosis, and autophagy, in terms of morphology, biochemistry, and genetics.12,13 Recent research has demonstrated that HG can induce ferroptosis in VECs, including human umbilical vein endothelial cells (HUVECs), by increasing intracellular iron levels and lipid peroxidation.4,14 Consequently, ferroptosis in VECs can impair the integrity and function of the vascular endothelium, contributing to the development and progression of various cardiovascular diseases including diabetic vascular diseases.15,16 Therefore, inhibiting VEC ferroptosis may hold promise as a strategy for preventing and treating HG-induced vascular diseases.

In recent years, several promising ferroptosis inhibitors with different structures have emerged to prevent iron-induced cell death and associated complications.17,18 One such inhibitor is ferrostatin-1 (Fer-1, Fig. 1), a small derivative of 3,4-diaminobenzoic acid. Fer-1 inhibits ferroptosis by scavenging lipid peroxyl radicals and preventing lipid peroxidation.19 It has demonstrated cytoprotective effects against ferroptosis induced by different stimuli, including erastin, RSL3, and HG.17,20 However, the low potency, poor solubility, and stability impose on the clinic application of Fer-1 which is a great limitation. To overcome these shortcomings, a few improved analogs of Fer-1, such as SRS9-11, SRS9-92, SRS9-86, and UMAC-2148 (Fig. 1), have been designed and synthesized. However, these inhibitors still exhibit low potency or poorly solubility, limiting their in vivo use.21,22 Among the Fer-1-derived analogs, UMAC-3203 has been recently reported as the most promising lead due to its superior properties compared to Fer-1, including improved instability and in vivo efficacy, despite its rapid removal from the bloodstream.22

Fig. 1. Fer-1 and its reported analogs.

Fig. 1

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Highly potent and specific ferrostatins could benefit greatly the numerous therapies for ferroptosis-driven disease.18 To develop novel inhibitors with increased potency, a project involving structural modification of Fer-1 was undertaken. Based on previous studies highlighting the significance of the benzyl group at N-3,21,22 a series of twenty novel ferroptosis inhibitors derived from Fer-1 were designed (Fig. 2). In this design, the ester fragment was replaced with a benzohydrazide moiety, taking into consideration the presence of benzohydrazide in various bioactive compounds.23 In addition, the benzohydrazide moiety was introduced to replace the ester fragment in Fer-1, mainly based on the hypothesis that: first, the benzohydrazide moiety can act as a scavenger similar to the arylamine group; second, the hydrazide can enhance hydrogen bond interaction with water and the carbonyl group of lipid peroxides, which probably enhance its solubility and bioavailability. Herein, we will describe the design, synthesis, and evaluation of these Fer-derived benzohydrazide analogs, as well as their protective effects against HG-induced HUVEC injury, antioxidant ability, and microsomal stability.

Fig. 2. The structural modification of Fer-1 resulting in new ferroptosis inhibitors.

Fig. 2

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2. Results and discussion

2.1. Chemical synthesis

Scheme 1 outlines the synthetic route for the target Fer-1 analogs 7–26. Initially, compound 2 was synthesized by reacting the starting material 1 with methanol mediated by SOCl2.24 Compound 3 was obtained by the reaction of 2 and cyclohexylamine in DMSO, catalyzed by potassium carbonate.21 Subsequently, compound 4 was synthesized through the reduction of 3 using iron powder and ammonium chloride.25 Compound 5 was then obtained by treating 4 and benzyl bromide in DMF. The key intermediate 6 was prepared by reacting 5 with hydrazine hydrate under refluxing conditions. Finally, the condensation of 6 with various aldehydes under microwave conditions resulted in the corresponding analogs 7–26,26 whose structures are presented in Table 1. The 1H, 13C NMR and MS spectra of 7–26 can be found in the ESI.†

Scheme 1. Synthetic route for Fer-1 analogs 7–26. Reagents and conditions: (a) thionyl chloride, MeOH, 60 °C, 6 h; (b) cyclohexylamine, K2CO3, DMSO, 80 °C, 12 h; (c) Fe, NH4Cl, THF : MeOH : H2O = 4 : 2 : 1, 80 °C, 8 h; (d) benzyl bromide, K2CO3, DMF, 70 °C, 8 h; (e) NH2NH2·H2O, MeOH, 80 °C, reflux 10 h; (f) corresponding aldehydes, acetic acid, MeOH, MW = 100 W, 80 °C, 0.5 h.

Scheme 1

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Anti-ferroptosis activity and solubility of selected compounds.

Compd R EC50 (μM) for erastin lethality suppression Solubilitya (μM) clog Pb
8 4-Cl 0.73 ± 0.09 28.6–33.3 6.7
11 4-OH 0.79 ± 0.15 28.6–33.3 7.2
12 4-OCH2Ph 0.50 ± 0.17 20.0–25.0 6.0
16 3-OCH2Ph 0.68 ± 0.05 20.0–25.0 7.2
17 3-CN 0.61 ± 0.14 22.2–25.0 6.0
18 3-NO2 0.69 ± 0.17 20.0–22.2 6.1
19 2-OCH3 0.65 ± 0.35 25.0–28.6 6.2
Fer-1 0.60 ± 0.12 >200 3.6
SRS9-92 6.2–12.5 5.3
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a

Final test compound concentration ranges between 3.125 μM and 200 μM [4 μL DMSO solution in 196 μL buffer solution (10 mM PBS pH 7.4)].

b

Online ADMETlab 2.0.27

2.2. Anti-ferroptosis activity assay

The anti-ferroptosis activity of compounds 7–26 was initially screened for their ability to prevent ferroptotic HUVEC death induced by erastin, a classical ferroptosis inducer. The SRB assay results revealed that cell viability decreased to 75% after 24 h of incubation with 10 μM erastin. However, all the tested compounds, except 13, 20, 21, and 24, significantly inhibited the decline in cell survival induced by erastin. Interestingly, compounds 11 and 12 exhibited better inhibitory effects compared to Fer-1 (Fig. 3). Generally, comparing the cell viability at a concentration of 10 μM indicated that compounds with a monosubstituted group (R) on the benzene ring displayed superior anti-ferroptosis activity compared to those with bisubstituted groups. However, the impact of the structure–activity relationship, specifically the locations and size of the substituents, on the activity of the compounds was not clear and requires further investigation, as supported by the subsequent determination of IC50 values shown in Table 1.

Fig. 3. The effects of compounds 7–26 on erastin-induced HUVEC ferroptosis were evaluated by the SRB assay. (A) HUVECs were treated with 10 μM erastin or 10 μM erastin and 10 μM different compounds. Cell viability was detected by the SRB method. (B) HUVECs were treated with 10 μM erastin or 10 μM erastin and different compounds at different concentrations. Cell viability was detected by the SRB method. ***P < 0.001, vs. control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. erastin, n = 3.

Fig. 3

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Subsequently, seven promising compounds, namely 8, 11, 12, and 16–19, were selected for further evaluation of their EC50 values based on their significant protective activity (p-value < 0.01 or 0.001) compared to the erastin-injured group. The results presented in Table 1 demonstrated that all of these selected compounds exhibited similar or improved anti-ferroptosis activity compared to Fer-1, with EC50 values ranging from 0.50 to 0.73 μM. Notably, compound 12, with an EC50 value of 0.50 μM, emerged as the most effective candidate for inhibiting erastin-induced HUVEC ferroptosis.

2.3. Kinetic solubility

Furthermore, the kinetic solubility of these compounds at pH 7.4 was determined, and the results are presented in Table 1. As expected, the presence of a benzyl group at N-3 led to a decrease in kinetic solubility compared with Fer-1, which is consistent with the calculated log P values. However, these novel analogs still showed slightly improved solubility compared to SRS9-92, suggesting that the benzohydrazide moiety is slightly more effective than the ester moiety fragment in enhancing solubility.

2.4. Antioxidant activity

Concurrently with the cell-based assays mentioned earlier, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction assay was conducted to evaluate the intrinsic reducing capacity and antioxidant activity of these newly synthesized analogs,28 with Fer-1 and SRS9-92 as positive controls. The reduction of the DPPH radical served as an indicator of the ability of a compound to quench radicals. At a concentration of 50 μM, these compounds demonstrated a radical scavenging capacity of approximately 66–52% within 30 minutes. The IC50 values of these compounds were subsequently determined and are summarized in Table 2. As we can see, Fer-1 analogs containing a benzohydrazide moiety retained antioxidant activity in the DPPH assay, and the presence of different substitutions on the benzohydrazide fragment influenced the antioxidant activity. Among these compounds, 12 (4-OCH2Ph) and 17 (3-CN) exhibited the highest potency in scavenging the DPPH radical, with IC50 values of 38.08 ± 0.55 and 38.64 ± 0.69, surpassing the activities of both Fer-1 and SRS9-92.

In vitro reducing activity of the DPPH radicala.

Compd % DPPH inhibition at 50 μM IC50 (μM)
8 53.38 ± 0.46 46.83 ± 1.36
11 53.31 ± 0.82 56.77 ± 2.14
12 65.88 ± 1.97 38.08 ± 0.55
16 52.20 ± 1.30 59.20 ± 1.02
17 59.65 ± 2.49 38.64 ± 0.69
18 53.28 ± 2.10 45.87 ± 1.11
19 57.36 ± 1.73 54.49 ± 2.01
Fer-1 52.51 ± 1.85 54.28 ± 0.87
SRS9-92 56.42 ± 0.77 46.81 ± 1.07
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a

Mean ± SD value of three separate experiments is presented.

2.5. Inhibitory activity of compounds against HG-induced ferroptosis in HUVECs

There is a wealth of evidence demonstrating that the inhibition of ferroptosis can alleviate VEC dysfunction and subsequently delay the onset of related diseases, such as diabetic cardiovascular complications.14,29–34 Therefore, these ferroptosis inhibitors 8, 11, 12, and 16–19 were examined to determine their effectiveness in inhibiting HG-induced VEC ferroptosis. In this study, HUVECs were stimulated with 30 mM HG for 24 h to mimic the in vivo vascular environment of diabetes. The results of the SRB experiment, as shown in Fig. 4, confirmed that all of these compounds significantly inhibited the decline in cell survival induced by HG. Notably, compound 12 still exhibited the most pronounced effect, which is consistent with its anti-ferroptosis activity observed in the erastin-induced VEC ferroptosis model (Fig. 3 and Table 1).

Fig. 4. Analysis of the effect of compounds on the survival of HUVECs stimulated with HG by the SRB assay. HUVECs were treated with 30 mM HG or 30 mM HG and 10 μM different compounds. ****p < 0.001 vs. control, ##p < 0.01, ###p < 0.001 vs. HG, n = 3.

Fig. 4

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2.6. Mechanism of action study on compound 12

When ferroptosis occurs, mitochondria undergo obvious damage and experience a decrease in membrane potential, accompanied by an increase in intracellular reactive oxygen species (ROS) levels.12,13,35–37 Therefore, the effect of the most potent inhibitor, compound 12, on mitochondrial membrane potential using a JC-1 probe was initially investigated. The fluorescence results revealed a significant increase in the number of JC-1 monomers in HUVECs stimulated with HG, indicating damage and a decrease in membrane potential. Following incubation with compound 12, the number of JC-1 monomers in HUVECs decreased significantly (Fig. 5A and B), suggesting that compound 12 inhibited HG-induced mitochondrial damage. Subsequently, the effect of compound 12 on ROS accumulation in HUVECs was further examined using the DCFH-DA probe. In the presence of 30 mM HG, the fluorescence intensity of DCF in cells increased, but this intensity decreased after the addition of 12, indicating that compound 12 inhibited the HG-induced increase in ROS levels (Fig. 5C and D).

Fig. 5. Effects of 12 on mitochondrial membrane potential and ROS accumulation in HG-stimulated HUVECs. HUVECs were treated with 30 mM HG and 10 μM compound 12 for 24 h. (A) JC-1 probe was used to test mitochondrial damage membrane potential; (B) quantification of mitochondrial membrane potential; (C) DCHF probe was used to detect intracellular ROS levels; (D) quantification of intracellular ROS levels, non-treated groups were normalized to 1. ***p < 0.001 vs. control, ###p < 0.001 vs. HG, n = 3.

Fig. 5

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The deficiency of oxidoreductase, particularly glutathione peroxidase 4 (GPX4), is a key characteristic of ferroptosis.38,39 It has been reported that HG reduces GPX4 expression in VECs.14 Thus, the effect of 12 on the GPX4 protein level was investigated using Western blot analysis. The results demonstrated a significant downregulation of GPX4 protein expression in HUVECs incubated with 30 mM HG. However, in the group treated with compound 12, the protein level of GPX4 was higher compared to the HG group (Fig. 6A and B).

Fig. 6. The expression level of GPX4 was detected. HUVECs were treated with 30 mM HG and 10 μM compound 12 for 24 h. (A) GPX4 protein level was detected by Western blot; (B) densitometry analysis of GPX4 protein levels in panel, non-treated groups were normalized to 1. ***p < 0.001 vs. control, ###p < 0.001 vs. HG, n = 3.

Fig. 6

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Fer-1 was shown to inhibit iron accumulation and lipid peroxidation in VECs,33 thus we further evaluated the effect of compound 12 on iron content and lipid peroxide MDA and LPO in HG-stimulated HUVECs. The results demonstrated that HG increased the ferrous ion level, LPO, and MDA contents significantly, while the amounts of ferrous ion, LPO, and MDA in the HG + 12 group were lower than those in the HG group (Fig. 7). These findings collectively suggest that compound 12 inhibits HG-induced ferroptosis in HUVECs. Considering the results obtained from EC50 determination, antioxidant assay, cell protection bioassays, and mechanism of action studies, it is evident that compound 12 holds promise as a potential lead compound for the treatment of ferroptosis-associated vascular diseases.

Fig. 7. Effects of 12 on iron levels and lipid peroxides in HG-stimulated HUVECs. HUVECs were treated with 30 mM HG and 10 μM compound 12 for 24 h. (A) FerroOrange probe was used to detect intracellular ferrous ion levels; (B) quantification of intracellular ferrous ion levels; (C) the relative levels of LPO; (D) the relative levels of MDA. Non-treated groups were normalized to 1, ****p < 0.0001, ***p < 0.001, **p < 0.01 vs. control, ##p < 0.01 vs. HG, n = 3.

Fig. 7

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2.7. Microsomal stability of compound 12

Finally, the most potential candidate 12 was compared with Fer-1 for their in vitro ADME properties. We assessed their stability in microsomes (human rat and mouse) at pH 7.4 buffer mixture, with verapamil as a positive control. Unfortunately, the results indicate that 12 does not exhibit better stability in microsomes (human rat and mouse), with a short T1/2 (less than 20 min, Table 3). However, non-NADPH dependent degradation was observed for Fer-1, but not for 12, in human and rat microsomes. Both Fer-1 and 12 showed non-NADPH dependent degradation in mouse microsome. This study, unlike previous ones,21,22 evaluated microsomal stability in the presence and absence of NADPH, allowing us to identify the existence of non-NADPH-dependent degradation. This information is valuable for the development of these compounds, as neglecting it may lead to the termination of their development. Furthermore, the remaining percentage of the parent compound was similar for Fer-1 with and without the presence of NADPH, indicating that microsomal degradation (human, rat, and mouse) was caused by non-NADPH enzymes.

In vitro microsomal stability of compound 12. The final concentration in the incubation mixture was 1 μM, and the incubation temperature was 37 °C.

Species Compd Remaining% without NADPH after 60 min Remaining% with NADPH after 60 min Microsomal stability half-life T1/2 (min)
Human 12 105% 0.6% 4.9
Fer-1 6% 7.6% 16.8
Verapamil 95% 3.0% 11.7
Rat 12 110% 1.0% 9.1
Fer-1 4% 6.1% 18.4
Verapamil 90% 2.9% 11.9
Mouse 12 38% 1.0% 9.0
Fer-1 5% 6.6% 2.7
Verapamil 89% 0.4% 7.7
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3. Conclusion

In conclusion, this study aimed to design, synthesize, and evaluate novel ferrostatin derivatives for the prevention of HG-induced VEC ferroptosis. VEC injury caused by HG is known to contribute to the development of various vascular-related diseases, and ferroptosis has been implicated in VEC dysfunction and associated diseases. In this study, a series of twenty novel ferrostatin analogs were synthesized and evaluated for their anti-ferroptosis activity using HUVEC models. The results demonstrated that several compounds, notably compound 12, exhibited enhanced protective effects against erastin- and HG-induced ferroptotic cell death compared to the reference compound Fer-1. Mechanistic investigations revealed that compound 12 preserved mitochondrial membrane potential, reduced ROS accumulation, upregulated the expression of GPX4, and decreased ferrous ion levels and LPO and MDA contents. Collectively, ferroptosis inhibitor 12 emerged as a promising lead compound with potential therapeutic applications for ferroptosis-associated vascular diseases, but further optimization is needed to overcome its undesirable metabolic stability.

4. Experiment

Medium M199 and bovine serum were obtained from Gibco Co. (Carlsbad, CA, USA). Protein was extracted from cells using radio immunoprecipitation assay (RIPA) lysis buffer (Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). The primary antibodies anti-GPX4 and anti-β-actin were acquired from Proteintech Group, Inc. (Chicago, IL, USA). The peroxidase conjugated secondary antibodies were obtained from Proteintech Group, Inc. (Chicago, IL, USA). Commercially available reagents are used without further purification. Organic solvents were evaporated with reduced pressure using a Büchi R100 evaporator. TLC monitored reactions using Yantai Jingyou (China) GF254 silica gel plates. Silica gel column chromatography was performed on silica gel (200–300 mesh) from Qingdao Hailang (China). NMR spectra were measured on Bruker Avance III 600 MHz spectrometers. Chemical shifts (δ) and coupling constant (J) were valued in ppm and Hz, respectively, and solvent signals as internal standards (CDCl3, δH 7.26 ppm and δC 77.2 ppm; DMSO-d6, δH 2.50 ppm and δC 39.5 ppm). ESI-MS spectra were recorded on a Waters LC-MS ZQ2000 mass spectrometer, and elemental analysis data were acquired on a Vario EL cube (Elementar, Germany).

4.1. Chemistry

4.1.1. Methyl 4-chloro-3-nitrobenzoate (2)

Thionyl chloride (27.2 mmol) was added to the solution of 4-chloro-3-nitrobenzoic acid (24.8 mmol) in methanol (70 mL). The reaction mixture was heated at 60 °C for 6 hours. The solvent was removed by vacuum distillation to obtain the solid product, which was used in the next step without further purification. Compound 2 was obtained as a white solid with a 95% yield. 1H NMR (600 MHz, DMSO-d6) δ 8.53 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 8.4, 2.1 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H). 13C NMR (150 MHz, chloroform-d) δ 164.4, 148.1, 133.7, 132.3, 131.8, 130.2, 126.8, 53.1.

4.1.2. Methyl 4-(cyclohexylamino)-3-nitrobenzoate (3)

Compound 2 (4.6 mmol) was dissolved in DMSO, and then K2CO3 (13.8 mmol) and cyclohexylamine (5.1 mmol) were added. The reaction mixture was heated at 80 °C for 12 hours, then poured into water. The solution was extracted with ethyl acetate three times, and the combined organic phase was concentrated under reduced pressure. The residue was purified by flash column chromatography to yield compound 3 as a yellow solid with a yield of 90%. 1H NMR (600 MHz, DMSO-d6) δ 8.61 (dd, J = 2.4, 1.2 Hz, 1H), 8.28 (d, J = 7.8 Hz, 1H), 7.96–7.93 (m, 1H), 7.21 (d, J = 9.0 Hz, 1H), 3.82 (s, 3H), 1.97–1.92 (m, 2H), 1.74–1.68 (m, 2H), 1.62–1.57 (m, 1H), 1.48–1.36 (m, 5H), 1.29–1.21 (m, 1H). 13C NMR (150 MHz, chloroform-d) δ 165.8, 147.0, 136.3, 131.1, 130.0, 116.8, 114.0, 52.2, 51.5, 32.7, 25.6, 24.6.

4.1.3. Methyl 3-amino-4-(cyclohexylamino)benzoate (4)

To a solution of compound 3 (3.6 mmol) in a mixed solvent (THF : MeOH : H2O = 4 : 2 : 1), iron powder (21.6 mmol) and ammonium chloride (35.9 mmol) were added. After the reaction mixture was heated at 80 °C for 8 hours, the reaction solution was concentrated under reduced pressure. The resulting residues were poured into water, followed by neutralization to pH ∼ 7. The solution was extracted using ethyl acetate three times, and the combined organic phase was concentrated under reduced pressure. The residue was purified by flash column chromatography to obtain compound 4 as a yellowish-brown solid, yielding 70%. 1H NMR (600 MHz, DMSO-d6) δ 7.18 (dd, J = 8.4, 1.8 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 4.91 (d, J = 7.8 Hz, 1H), 4.75 (s, 2H), 3.71 (s, 3H), 1.95 (dd, J = 12.0, 3.0 Hz, 2H), 1.75–1.70 (m, 2H), 1.64–1.59 (m, 1H), 1.39–1.31 (m, 2H), 1.27–1.10 (m, 4H). 13C NMR (150 MHz, chloroform-d) δ 167.7, 142.4, 131.8, 124.6, 118.8, 118.1, 109.6, 51.7, 51.5, 33.4, 26.0, 25.1.

4.1.4. Methyl 3-(benzylamino)-4-(cyclohexylamino)benzoate (5)

To the solution of compound 4 (4.8 mmol) in DMF were added benzyl bromide (4.8 mmol) and K2CO3 (5.8 mmol). The mixture was stirred at 70 °C for 8 h and then poured in water. The solution was extracted with ethyl acetate three times, and the combined organic solvent was removed under reduced pressure. The residue was purified by flash column chromatography to obtain compound 5 as an orange solid, yielding 70%. 1H NMR (600 MHz, CDCl3) δ 9.20 (s, 1H), 7.36 (d, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 7.23 (t, J = 7.2 Hz, 1H), 7.08 (dd, J = 8.4, 1.8 Hz, 1H), 6.92 (d, J = 1.8 Hz, 1H), 6.45 (d, J = 8.4 Hz, 1H), 5.31 (t, J = 5.4 Hz, 1H), 4.84 (d, J = 7.2 Hz, 1H), 4.32 (d, J = 5.4 Hz, 2H), 4.24 (s, 2H), 1.98 (d, J = 11.4 Hz, 2H), 1.77–1.71 (m, 2H), 1.65–1.59 (m, 1H), 1.40–1.32 (m, 2H), 1.24–1.15 (m, 4H).13C NMR (150 MHz, chloroform-d) δ 169.4, 140.8, 139.1, 135.8, 128.8, 128.2, 127.6, 121.4, 119.5, 112.0, 110.0, 51.7, 49.3, 33.5, 26.0, 25.1.

4.1.5. 3-(Benzylamino)-4-(cyclohexylamino)benzohydrazide (6)

The solution of compound 5 (3.5 mmol) and hydrazine hydrate (7.0 mmol) in methanol was heated at 80 °C for 10 hours. After completion of the reaction, the reaction mixture was cooled down to 0 °C and poured into cool methanol for the precipitation process. The product was filtered and washed with cool methanol to yield compound 6 as a yellow solid with a yield of 68%. 1H NMR (600 MHz, CDCl3) δ 11.37 (s, 1H), 8.40 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 3H), 7.34 (t, J = 7.8 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 7.02 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 5.05 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 9.6 Hz, 2H), 1.78–1.71 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.33 (m, 2H), 1.27–1.15 (m, 4H).13C NMR (150 MHz, chloroform-d) δ 139.9, 138.6, 134.8, 134.5, 129.6, 128.8, 128.3, 127.5, 126.8, 120.0, 107.8, 50.9, 47.2, 32.6, 25.7, 24.7.

4.1.6. General procedure for the synthesis of compounds 7–26

Compound 6 (0.24 mmol) was reacted with different aromatic aldehydes (0.26 mmol) in a mixed solution of acetic acid (0.01 mL) and methanol under microwave conditions (100 W, 80 °C) for 0.5 hours. After completion of the reaction, the solvent was distilled using a roto-evaporator, and the crude product was extracted with ethyl acetate/water. The organic phase was concentrated with anhydrous sodium sulfate to afford a crude residue, which was purified by a silica gel column (dichloromethane/methanol = 20 : 1) to produce corresponding pure compounds 7–26.

4.1.6.1. (E)-3-(Benzylamino)-N′-benzylidene-4-(cyclohexylamino)benzohydrazide (7)

Yellow solid; yield 57%; 1H NMR (600 MHz, CDCl3) δ 11.37 (s, 1H), 8.40 (s, 1H), 7.67 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.2 Hz, 2H), 7.40 (t, J = 7.2 Hz, 3H), 7.34 (t, J = 7.8 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 7.02 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 5.05 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 9.6 Hz, 2H), 1.78–1.71 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.33 (m, 2H), 1.27–1.15 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 139.9, 138.6, 134.8, 134.5, 129.6, 128.8, 128.3, 127.5, 126.8, 120.0, 107.8, 50.9, 47.2, 32.6, 25.7, 24.7. ESI-MS m/z: 426.9 [M + H]+. Anal. calcd. For C27H30N4O: C, 76.03; H, 7.09; N, 13.13. Found: C, 76.06; H, 7.10; N, 13.11.

4.1.6.2. (E)-3-(Benzylamino)-N′-(4-chlorobenzylidene)-4-(cyclohexylamino)benzohydrazide (8)

Yellow solid; yield 58%; 1H NMR (600 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.39 (s, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H), 7.00 (d, J = 1.8 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 5.06 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 9.6 Hz, 2H), 1.77–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.26–1.16 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 139.9, 138.7, 134.5, 134.0, 133.8, 128.9, 128.4, 128.3, 127.5, 126.8, 119.9, 107.7, 50.9, 47.2, 32.6, 25.6, 24.7. ESI-MS m/z: 460.9 [M + H]+. Anal. calcd. For C27H29ClN4O: C, 70.35; H, 6.34; N, 12.15. Found: C, 70.31; H, 6.33; N, 12.17.

4.1.6.3. (E)-3-(Benzylamino)-N′-(4-bromobenzylidene)-4-(cyclohexylamino)benzohydrazide (9)

Yellow solid; yield 50%; 1H NMR (600 MHz, DMSO-d6) δ 11.43 (s, 1H), 8.37 (s, 1H), 7.65–7.61 (m, 4H), 7.39 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.24 (t, J = 7.8 Hz, 2H), 7.00 (d, J = 1.8 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 5.06 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 12.0 Hz, 2H), 1.77–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.27–1.17 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 139.9, 138.7, 134.1, 131.8, 128.6, 128.3, 127.5, 126.8, 122.7, 119.9, 107.7, 50.9, 47.2, 32.6, 25.6, 24.7. ESI-MS m/z: 506.8 [M + H]+. Anal. calcd. For C27H29BrN4O: C, 64.16; H, 5.78; N, 11.08. Found: C, 64.13; H, 5.77; N, 11.09.

4.1.6.4. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(4-nitrobenzylidene)benzohydrazide (10)

Orange solid; yield 47%; 1H NMR (600 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.49 (s, 1H), 8.28 (d, J = 9.0 Hz, 2H), 7.93 (d, J = 9.0 Hz, 2H), 7.39 (d, J = 6.6 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.28–7.23 (m, 2H), 7.02 (d, J = 1.8 Hz, 1H), 6.54 (d, J = 8.4 Hz, 1H), 5.45 (t, J = 5.4 Hz, 1H), 5.12 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 9.6 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.35 (m, 2H), 1.30–1.12 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 148.0, 141.7, 140.3, 139.4, 135.0, 128.8, 128.0, 127.9, 127.2, 124.5, 120.0, 108.1, 51.3, 47.6, 33.0, 26.1, 25.2. ESI-MS m/z: 471.8 [M + H]+. Anal. calcd. For C27H29N5O3: C, 68.77; H, 6.20; N, 14.85. Found: C, 68.83; H, 6.21; N, 14.84.

4.1.6.5. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(4-hydroxybenzylidene)benzohydrazide (11)

Yellow solid; yield 42%; 1H NMR (600 MHz, DMSO-d6) δ 11.16 (s, 1H), 9.87 (s, 1H), 8.29 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.2 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.26–7.19 (m, 2H), 6.99 (s, 1H), 6.81 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 1H), 5.39 (t, J = 5.4 Hz, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 12.0 Hz, 2H), 1.77–1.72 (m, 2H), 1.65–1.61 (m, 1H), 1.40–1.36 (m, 2H), 1.26–1.19 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 159.0, 148.8, 141.3, 139.2, 135.4, 129.4, 128.5, 128.2, 127.3, 125.4, 120.8, 116.0, 113.11, 109.6, 51.4, 49.2, 33.3, 25.9, 24.9. ESI-MS m/z: 442.9 [M + H]+. Anal. calcd. For C27H30N4O2: C, 73.28; H, 6.83; N, 12.66. Found: C, 73.26; H, 6.84; N, 12.65.

4.1.6.6. (E)-3-(Benzylamino)-N′-(4-(benzyloxy)benzylidene)-4-(cyclohexylamino)benzohydrazide (12)

Yellow solid; yield 52%; 1H NMR (400 MHz, DMSO-d6) δ 11.26 (s, 1H), 8.34 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.8 Hz, 4H), 7.34 (t, J = 7.8 Hz, 3H), 7.26–7.20 (m, 2H), 7.08 (d, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.52 (d, J = 8.4 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 5.15 (s, 2H), 5.04 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 12.6 Hz, 2H), 1.79–1.71 (m, 2H), 1.63 (d, J = 12.6 Hz, 1H), 1.42–1.33 (m, 2H), 1.27–1.17 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 160.4, 141.0, 139.1, 136.6, 129.1, 128.7, 128.6, 128.2, 128.1, 127.5, 127.5, 126.9, 115.0, 109.6, 70.1, 51.5, 49.3, 33.4, 25.9, 25.0. ESI-MS m/z: 533.0 [M + H]+. Anal. calcd. For C34H36N4O2: C, 76.66; H, 6.81; N, 10.52. Found: C, 76.72; H, 6.80; N, 10.54.

4.1.6.7. (E)-3-(Benzylamino)-N′-(4-cyanobenzylidene)-4-(cyclohexylamino)benzohydrazide (13)

Yellow solid; yield 55%; 1H NMR (600 MHz, DMSO-d6) δ 11.62 (s, 1H), 8.45 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.27–7.22 (m, 2H), 7.01 (d, J = 1.8 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.45 (t, J = 5.4 Hz, 1H), 5.10 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (dd, J = 12.6, 3.6 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.28–1.15 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 141.5, 138.9, 138.5, 132.3, 128.7, 128.1, 127.7, 127.5, 118.6, 112.9, 109.4, 51.5, 49.2, 33.3, 25.8, 25.0. ESI-MS m/z: 451.9 [M + H]+. Anal. calcd. For C28H29N5O: C, 74.47; H, 6.47; N, 15.51. Found: C, 74.43; H, 6.48; N, 15.50.

4.1.6.8. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(4-(pyrrolidin-1-yl)benzylidene)benzohydrazide (14)

Yellow solid; yield 38%; 1H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H), 8.24 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 1H), 7.20 (t, J = 9.0 Hz, 1H), 6.98 (s, 1H), 6.57 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 1H), 5.40 (t, J = 5.4 Hz, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.27 (t, J = 9.0 Hz, 4H), 3.17 (d, J = 7.8 Hz, 1H), 2.03–1.93 (m, 6H), 1.78–1.71 (m, 2H), 1.67–1.60 (m, 1H), 1.43–1.32 (m, 2H), 1.27–1.16 (m, 3H). 13C NMR (150 MHz, CDCl3) δ 149.2, 140.8, 139.2, 129.3, 128.7, 128.2, 127.4, 120.8, 111.4, 109.8, 51.5, 49.3, 47.5, 33.4, 25.9, 25.5, 25.0. ESI-MS m/z: 495.9 [M + H]+. Anal. calcd. For C31H37N5O: C, 75.12; H, 7.52; N, 14.13. Found: C, 75.09; H, 7.53; N, 14.15.

4.1.6.9. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(4-(dimethylamino)benzylidene)benzohydrazide (15)

Yellow solid; yield 63%; 1H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.25 (s, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.27–7.18 (m, 2H), 6.99 (s, 1H), 6.74 (d, J = 8.4 Hz, 2H), 6.51 (d, J = 8.4 Hz, 1H), 5.40 (t, J = 5.4 Hz, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.96 (s, 6H), 2.00 (d, J = 12.0 Hz, 2H), 1.75 (d, J = 12.6 Hz, 2H), 1.63 (d, J = 12.6 Hz, 1H), 1.43–1.32 (m, 2H), 1.29–1.14 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 151.7, 140.8, 139.2, 129.1, 128.7, 128.2, 127.4, 121.6, 111.7, 109.7, 51.5, 49.3, 40.2, 33.4, 25.9, 25.0. ESI-MS m/z: 470.0 [M + H]+. Anal. calcd. For C29H35N5O: C, 74.17; H, 7.51; N, 14.91. Found: C, 74.20; H, 7.52; N, 14.89.

4.1.6.10. (E)-3-(Benzylamino)-N′-(3-(benzyloxy)benzylidene)-4-(cyclohexylamino)benzohydrazide (16)

Yellow solid; yield 71%; 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s, 1H), 8.37 (s, 1H), 7.47 (d, J = 7.2 Hz, 2H), 7.42–7.31 (m, 9H), 7.24 (t, J = 8.4 Hz, 3H), 7.05 (dd, J = 12.0, 2.4 Hz, 1H), 7.01 (s, 1H), 6.52 (d, J = 8.4 Hz, 1H), 5.43 (t, J = 6.0 Hz, 1H), 5.15 (s, 2H), 5.06 (d, J = 7.8 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 10.8 Hz, 2H), 1.75 (d, J = 12.6 Hz, 2H), 1.63 (d, J = 12.0 Hz, 1H), 1.43–1.33 (m, 2H), 1.28–1.16 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 159.1, 141.2, 139.1, 136.8, 135.5, 129.6, 128.7, 128.6, 128.2, 128.0, 127.6, 127.5, 121.1, 112.0, 109.5, 70.1, 51.5, 49.3, 33.3, 25.9, 25.0. ESI-MS m/z: 533.0 [M + H]+. Anal. calcd. For C34H36N4O2: C, 76.66; H, 6.81; N, 10.52. Found: C, 76.63; H, 6.82; N, 10.53.

4.1.6.11. (E)-3-(Benzylamino)-N′-(3-cyanobenzylidene)-4-(cyclohexylamino)benzohydrazide (17)

Yellow solid; yield 67%; 1H NMR (600 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.43 (s, 1H), 8.07 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.28–7.22 (m, 2H), 7.02 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.44 (d, J = 6.0 Hz, 1H), 5.09 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 11.4 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (dt, J = 13.0, 4.1 Hz, 1H), 1.42–1.34 (m, 2H), 1.28–1.14 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 141.5, 139.0, 135.6, 132.9, 131.1, 130.9, 129.5, 128.7, 128.2, 127.5, 118.3, 112.9, 109.4, 51.5, 49.3, 33.3, 25.8, 25.0. ESI-MS m/z: 451.9 [M + H]+. Anal. calcd. For C28H29N5O: C, 74.47; H, 6.47; N, 15.51. Found: C, 74.53; H, 6.48; N, 15.50.

4.1.6.12. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(3-nitrobenzylidene)benzohydrazide (18)

Orange solid; yield 51%; 1H NMR (600 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.52 (s, 1H), 8.50 (s, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.26 (t, J = 8.4 Hz, 2H), 7.03 (s, 1H), 6.54 (d, J = 8.4 Hz, 1H), 5.45 (t, J = 5.4 Hz, 1H), 5.10 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 9.6 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.30–1.13 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 148.4, 141.5, 139.0, 136.1, 132.5, 129.6, 128.7, 128.2, 127.5, 124.2, 122.3, 109.4, 51.5, 49.3, 33.3, 25.8, 25.0. ESI-MS m/z: 471.9 [M + H]+. Anal. calcd. For C27H29N5O3: C, 68.77; H, 6.20; N, 14.85. Found: C, 68.82; H, 6.19; N, 14.82.

4.1.6.13. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(2-methoxybenzylidene)benzohydrazide (19)

Yellow solid; yield 77%; 1H NMR (600 MHz, DMSO-d6) δ 11.38 (s, 1H), 8.74 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.41–7.36 (m, 3H), 7.34 (t, J = 7.8 Hz, 2H), 7.27–7.22 (m, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.03 (s, 1H), 7.00 (t, J = 7.2 Hz, 1H), 6.52 (d, J = 8.4 Hz, 1H), 5.39 (t, J = 5.4 Hz, 1H), 5.04 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 3.85 (s, 3H), 2.00 (d, J = 12.0 Hz, 2H), 1.78–1.73 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 3H), 1.26–1.16 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 157.9, 141.1, 139.1, 131.4, 128.7, 128.2, 127.5, 127.1, 122.3, 120.9, 110.8, 109.6, 55.6, 51.5, 49.4, 33.4, 25.9, 25.0. ESI-MS m/z: 457.0 [M + H]+. ESI-MS m/z: 533.0 [M + H]+. Anal. calcd. For C28H32N4O2: C, 73.66; H, 7.06; N, 12.27. Found: C, 73.71; H, 7.07; N, 12.25.

4.1.6.14. (E)-3-(Benzylamino)-N′-(2-(benzyloxy)benzylidene)-4-(cyclohexylamino)benzohydrazide (20)

Yellow solid; yield 45%; 1H NMR (600 MHz, DMSO-d6) δ 11.45 (s, 1H), 8.76 (s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.51 (d, J = 7.8 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.36–7.31 (m, 4H), 7.24 (t, J = 7.2 Hz, 2H), 7.18 (d, J = 8.4 Hz, 1H), 7.03–6.99 (m, 2H), 6.51 (d, J = 8.4 Hz, 1H), 5.40 (t, J = 5.4 Hz, 1H), 5.20 (s, 2H), 5.03 (d, J = 7.2 Hz, 1H), 4.34 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 10.2 Hz, 2H), 1.77–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.41–1.34 (m, 2H), 1.26–1.17 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 175.3, 157.2, 157.1, 139.1, 136.6, 131.3, 129.4, 128.8, 128.7, 128.7, 128.7, 128.2, 128.2, 127.7, 127.6, 121.3, 70.5, 51.5, 33.3, 25.9, 25.0, 21.00. ESI-MS m/z: 533.0 [M + H]+. Anal. calcd. For C34H36N4O2: C, 76.66; H, 6.81; N, 10.52. Found: C, 76.60; H, 6.80; N, 10.53.

4.1.6.15. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(2,3-dihydroxybenzylidene)benzohydrazide (21)

Yellow solid; yield 45%; 1H NMR (600 MHz, DMSO-d6) δ 11.65 (s, 1H), 8.50 (s, 1H), 7.40 (d, J = 7.8 Hz, 2H), 7.35 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.3 Hz, 2H), 7.02 (s, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 6.72 (t, J = 7.8 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 5.44 (t, J = 5.4 Hz, 1H), 5.10 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 10.2 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.27–1.16 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 145.0, 141.6, 138.9, 135.5, 128.7, 128.1, 127.5, 121.5, 120.2, 119.5, 117.6, 116.5, 109.6, 51.5, 49.2, 33.3, 25.8, 25.0. ESI-MS m/z: 458.0 [M + H]+. Anal. calcd. For C27H30N4O3: C, 70.72; H, 6.59; N, 12.22. Found: C, 70.75; H, 6.60; N, 12.22.

4.1.6.16. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(3,4-dichlorobenzylidene)benzohydrazide (22)

Yellow solid; yield 37%; 1H NMR (600 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.36 (s, 1H), 7.90 (s, 1H), 7.71–7.65 (m, 2H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 7.01 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.44 (t, J = 4.8 Hz, 1H), 5.08 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 12.0 Hz, 2H), 1.77–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.41–1.34 (m, 2H), 1.27–1.15 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 141.5, 139.1, 134.4, 133.9, 133.1, 130.7, 129.1, 128.8, 128.3, 127.6, 126.5, 109.6, 51.6, 49.4, 33.5, 26.0, 25.1. ESI-MS m/z: 494.9 [M + H]+. Anal. calcd. For C27H28Cl2N4O: C, 65.45; H, 5.70; N, 11.31. Found: C, 65.41; H, 5.71; N, 11.30.

4.1.6.17. (E)-3-(Benzylamino)-N′-(3-bromo-4-fluorobenzylidene)-4-(cyclohexylamino)benzohydrazide (23)

Yellow solid; yield 42%; 1H NMR (600 MHz, DMSO-d6) δ 11.51 (s, 1H), 8.36 (s, 1H), 8.00 (dd, J = 6.6, 2.4 Hz, 1H), 7.74–7.70 (m, 1H), 7.45 (t, J = 8.4 Hz, 1H), 7.39 (d, J = 6.6 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.26–7.22 (m, 2H), 7.01 (d, J = 2.4 Hz, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.43 (t, J = 5.4 Hz, 1H), 5.07 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 2.03–1.97 (m, 2H), 1.78–1.72 (m, 2H), 1.66–1.60 (m, 1H), 1.43–1.33 (m, 2H), 1.28–1.14 (m, 4H). 13C NMR (150 MHz, DMSO-d6) 158.7 (d, J = 248.2 Hz), 140.3, 139.2, 135.0, 133.7 (d, J = 3.6 Hz), 131.6, 128.8, 128.5 (d, J = 7.9 Hz), 127.9, 127.2, 120.2, 117.7 (d, J = 22.7 Hz), 109.9, 109.1 (d, J = 21.6 Hz), 108.2, 51.3, 47.6, 33.1, 26.1, 25.2. ESI-MS m/z: 524.9 [M + H]+. Anal. calcd. For C27H28BrFN4O: C, 61.95; H, 5.39; N, 10.70. Found: C, 61.91; H, 5.40; N, 10.72.

4.1.6.18. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(4-fluoro-3-nitrobenzylidene)benzohydrazide (24)

Yellow solid; yield 38%; 1H NMR (600 MHz, DMSO-d6) δ 11.62 (s, 1H), 8.46 (s, 1H), 8.41 (d, J = 6.0 Hz, 1H), 8.08 (d, J = 6.0 Hz, 1H), 7.66 (t, J = 9.0 Hz, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.8 Hz, 2H), 7.25 (d, J = 7.2 Hz, 2H), 7.01 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 5.45 (t, J = 5.4 Hz, 1H), 5.09 (d, J = 7.2 Hz, 1H), 4.36 (d, J = 5.4 Hz, 2H), 2.00 (d, J = 12.0 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 3H), 1.27–1.17 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 155.9 (d, J = 268.7 Hz), 141.5, 138.9, 137.4 (d, J = 8.3 Hz), 135.4, 133.3 (d, J = 8.7 Hz), 131.6 (d, J = 4.1 Hz), 128.7, 128.2, 127.8, 127.5, 124.9, 120.4, 118.9 (d, J = 21.5 Hz), 109.4, 51.5, 50.9, 49.2, 33.3, 25.8, 25.0. ESI-MS m/z: 489.9 [M + H]+. Anal. calcd. For C27H28FN5O3: C, 66.24; H, 5.77; N, 14.31. Found: C, 66.28; H, 5.76; N, 14.29.

4.1.6.19. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(3,5-dimethoxybenzylidene)benzohydrazide (25)

Yellow solid; yield 36%; 1H NMR (600 MHz, DMSO-d6) δ 11.38 (s, 1H), 8.33 (s, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 7.2 Hz, 2H), 7.24 (t, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.82 (d, J = 2.4 Hz, 2H), 6.54–6.51 (m, 2H), 5.43 (t, J = 4.8 Hz, 1H), 5.05 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.78 (s, 6H), 2.00 (d, J = 13.2 Hz, 2H), 1.78–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.27–1.16 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 160.9, 141.2, 139.1, 136.0, 128.7, 128.2, 127.5, 109.5, 105.3, 103.0, 55.5, 51.5, 49.3, 33.3, 25.9, 25.0. ESI-MS m/z: 4887.0 [M + H]+. Anal. calcd. For C29H34N4O3: C, 71.58; H, 7.04; N, 11.51. Found: C, 71.64; H, 7.03; N, 11.50.

4.1.6.20. (E)-3-(Benzylamino)-4-(cyclohexylamino)-N′-(2-hydroxy-4-methoxybenzylidene)benzohydrazide (26)

Yellow solid; yield 50%; 1H NMR (600 MHz, DMSO-d6) δ 11.88 (s, 1H), 11.54 (s, 1H), 8.46 (s, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.34 (t, J = 9.0 Hz, 3H), 7.24 (dd, J = 12.6, 7.2 Hz, 2H), 7.00 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 6.50 (dd, J = 8.4, 2.4 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 5.44 (t, J = 5.4 Hz, 1H), 5.08 (d, J = 7.2 Hz, 1H), 4.35 (d, J = 5.4 Hz, 2H), 3.76 (s, 3H), 2.00 (d, J = 12.6 Hz, 2H), 1.77–1.72 (m, 2H), 1.66–1.61 (m, 1H), 1.42–1.34 (m, 2H), 1.27–1.18 (m, 4H). 13C NMR (150 MHz, CDCl3) δ 162.5, 141.2, 139.0, 135.6, 131.8, 128.7, 128.2, 127.5, 120.8, 111.3, 109.6, 106.8, 101.4, 55.4, 51.5, 49.2, 33.3, 25.9, 25.0. ESI-MS m/z: 472.9 [M + H]+. Anal. calcd. For C28H32N4O3: C, 71.16; H, 6.83; N, 11.86. Found: C, 71.20; H, 6.84; N, 11.84.

4.2. Pharmacological assay

4.2.1. Kinetic solubility sassy

A series of DMSO compound stock solutions were prepared (0.15–5 mM). An aliquot of 4 μL stock solution was added to 196 μL PBS buffer (pH 7.4). A series of concentrations were prepared (3.13–200 μM), including a blank on a microtiter plate. The microtiter plate was shaken for 10 seconds and incubated for 2 hours at 37 °C. When there was no turbidity measured at a given concentration the sample was assumed to be dissolved.

4.2.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay

The stable radical 3.94 mg of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was dissolved in 100 mL of ethanol to a concentration of 100 μM. Each tested compound was dissolved in DMSO, followed by the addition of 180 μL of DPPH solution and 20 μL of the compound solution to each well of a 96-well plate, resulting in a final concentration of 50 μM for each test compound. Incubated in the dark at room temperature for 30 minutes, the absorbance at 517 nm was measured using an enzyme marker to determine the clearance rate of DPPH. To ensure an even distribution of high and low IC50 concentrations, a concentration gradient of the test compound solution was configured and evaluated based on its ability to scavenge DPPH free radicals at 50 μM. The experimental results were plotted and analyzed using GraphPad Prism, enabling the compound's IC50 to be determined.

4.2.3. Cell culture

HUVECs were obtained from ScienCell (San Diego, CA, USA) and cultured on gelatin-coated plastic dishes in M199 medium with 20% (v/v) bovine serum and 10 IU mL−1 fibroblast growth factor 2. HUVECs were maintained at 37 °C under humidified conditions and 5% CO2. Morphologic changes of HUVECs were observed by inverted phase-contrast microscopy (ZEISS, Primovert, Germany).

4.2.4. Cell viability assay

Cell viability was detected using the sulforhodamine B assay (SRB; Sigma-Aldrich, USA). Briefly, HUVECs were cultured in 96-well plates. After incubation overnight, the cells were treated with or without different derivatives for 24 h. The cells fixed with trichloroacetic acid and then stained with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid for 10 minutes. The cells were rinsed with 1% acetic acid. Then, 100 μL of 10 mM Tris-base (pH 10.5) was added to dissolve the conjugation. The absorbance (A) was detected at 540 nm wavelength using a microplate spectrophotometer (TECAN, USA). Data represent at least three separate experiments. Survival rate = (A value of experimental group − A value of blank group)/(A value of control group − A value of blank group) × 100%.

4.2.5. Intracellular ROS assay

The levels of intracellular ROS were detected using the fluorescent probe 2′,7′-dichlorodihydrofluorescein (DCHF, Invitrogen, Carlsbad, CA, USA). After treatment, cells were incubated with DCFH-DA (10 mM) for 30 min at 37 °C in 5% CO2 and washed with PBS three times. The cellular fluorescence was monitored under a fluorescence microscope (LEICA DMi8, Wiesbaden, Germany). The amount of ROS was quantified as the relative fluorescence intensity of DCF per cell within the sight area.

4.2.6. Mitochondrial membrane potential (MMP) measurement

The JC-1 kit instructions were used for operation (Beyotime, Beijing, China). The treated cells in 24-well plates were sucked out of the culture medium, washed once with PBS, and then 1 mL cell culture medium was added. Then, 1 ml JC-1 staining working fluid was added into each well. The cells were incubated in a cell incubator at 37 °C for 20 min. After incubation at 37 °C, the supernatant was removed and washed twice with JC-1 staining buffer (1×). 2 ml cell culture medium was added. The cellular fluorescence was monitored under a fluorescence microscope (LEICA DMi8, Wiesbaden, Germany).

4.2.7. Western blot analysis

HUVECs were harvested as scheduled after treatment under different conditions, and the total protein concentrations were determined by BCA Protein Assay kits (Beyotime, Shenzhen, Guangdong, China). Equivalent samples of cell lysates (15 μg per lane) were separated using 12% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Madison, WI, USA) for 2 h. After blocking the membrane with 5% nonfat skim milk in TBST buffer for 2 h, the membrane was incubated with the following primary antibodies at 4 °C overnight: anti-GPX4 (1 : 1000) and anti-β-actin (1 : 5000) antibodies. After washing with TBST three times, membranes were incubated with secondary antibody for 1.5 h at room temperature. Immune complexes were detected by enhanced chemiluminescence (Millipore Corporation, Billerica, MA, USA). Integrated densities of bands were quantified by Image J software.

4.2.8. Intracellular ferrous ion level assay

The ferrous ion level was detected by using an intracellular ferrous ion fluorescent probe (Dojindo, Japan). After the cells were treated, the medium was removed and washed three times with serum-free medium. The cells were cultured in 1 μM FerroOrange working solution at 37 °C, 5% CO2 incubator for 30 min. After culture, the cells were observed directly under a fluorescence microscope.

4.2.9. LPO content assay

The LPO content was detected by using a LPO assay kit (BC5240, Solarbio, China). The cells were collected into a centrifuge tube, and the supernatant was discarded after centrifugation. The extract was added, and the cells were disrupted by ultrasonic wave (ice bath, power 200 W, ultrasonic 3 s, interval 7 s, total time 3 min), centrifuged at 8000 × g 4 °C for 10 min, and the supernatant was taken to determine the LPO content.

4.2.10. MDA content assay

The MDA content was detected by using a MDA assay kit (Beyotime, Beijing, China). In brief, the cells were lysed using Western and IP cell lysate (Beyotime, Beijing, China). The lysate was collected into a centrifuge tube and centrifuged at 10 000 × g for 10 min, and the supernatant was taken for determination of the MDA level and protein content. Protein concentrations were quantified by using a protein detection kit (Sorabio, Beijing, China). MDA levels were normalized to mg protein.

4.2.11. Microsomal stability test

The disappearance of compounds Fer-1 and 12 after incubation with liver microsomes was analyzed using LC-MS/MS. The method used was sensitive, rapid, and highly selective. Incubation samples were taken at different time points (0, 5, 10, 20, 30, 60 min) and precipitated with acetonitrile containing tolbutamide as the internal standard. The samples were then vortexed and centrifuged consistently. The analytes were eluted on an Agilent Extend C18 column (2.1 × 50 mm, 5 μm) using a mobile phase consisting of acetonitrile (0.1% trifluoroacetic acid, mobile phase B) and water (0.1% trifluoroacetic acid, mobile phase A) at a flow rate of 0.50 mL min−1. The composition of the mobile phase was changed from A : B 90 : 10 (v : v) to A : B 5 : 95 (v : v) over a run time of 0.3–1.0 min before returning to A : B 90 : 10 (v : v) at 2.1 min. The retention times for Fer-1 and 12 were 1.39 min and 1.46 min, respectively, with a total run time of 3.0 min. The column temperature was set at 40 °C and the injection volume was 2 μL. Detection of Fer-1 and 12 was performed using electrospray positive ionization mass spectrometry in the multiple reaction monitoring mode, with m/z 263.2/181.0 and 471.9/307.4, respectively.

4.2.12. Statistical analysis

Data are presented as mean ± SEM. Images were processed by use of GraphPad Prism 8 (GraphPad Software, LaJolla, CA, USA) and Adobe Photoshop (Adobe, San Jose, USA). At least three independent replications were performed. One-way ANOVA combined with Bonferroni post hoc tests was used to determine the significance between different groups. p < 0.05 was considered statistically significant.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

MD-015-D4MD00038B-s001

Acknowledgments

This research work was financially supported by the National Natural Science Foundation of China (No. 82204240), Natural Science Foundation of Shandong Province (No. ZR2023MB046), and the Project of Shandong Province Higher Educational Youth Innovation Science and Technology Program (2020KJE006).

†

Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4md00038b

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MD-015-D4MD00038B-s001
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