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. 2025 Nov 6. Online ahead of print. doi: 10.1039/d5md00753d

Synthesis and evaluation of lupeol-derived triterpenic azines as potential neuroprotective agents

Florencia A Musso a,b, Natalia P Alza c,d,✉, Gabriela A Salvador c,d, María Belén Faraoni a,b,✉
PMCID: PMC12679490  PMID: 41355863

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the accumulation of α-synuclein aggregates. Current treatments are primarily symptomatic, highlighting the need for new neuroprotective strategies. Natural triterpenes have shown promise in neurodegenerative diseases, and structural modifications can enhance their bioactivity. In this study, we obtained a series of new triterpenic azines (4a–4p) from lupeol, optimizing reaction conditions through microwave-assisted synthesis. The neuroprotective potential of these derivatives was evaluated in human neuroblastoma IMR-32 cells exposed to 6-hydroxydopamine (6-OHDA), a widely used in vitro model of PD. Compounds 4c, 4m, and 4n significantly prevented 6-OHDA-induced cytotoxicity, restoring cell viability at 10 and 50 μM to control levels. Since ferroptosis is a cell death mechanism implicated in PD, we further examined the effects of these compounds in N27 dopaminergic neurons exposed to the ferroptosis inducers RSL3 and erastin. Among the tested derivatives, 4c exhibited a remarkable protective effect against RSL3-induced ferroptosis, which was comparable to ferrostatin-1, displaying an IC50 value of 9.1 μM. These findings support the development of triterpenic azines as neuroprotective agents and warrant further investigation in preclinical PD models.

New triterpenic azines from lupeol exhibited neuroprotective effects in vitro, with 4c notably preventing 6-OHDA- and ferroptosis-induced dopaminergic cell death, highlighting its potential for Parkinson's disease.graphic file with name d5md00753d-ga.jpg

1. Introduction

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder after Alzheimer's disease, affecting 2–3% of individuals over 65 years old. While the precise cause of PD remains unclear, alterations in mitochondrial function, iron regulation in the brain, neuroinflammation, and energy metabolism have been identified as main contributors to its underlying mechanisms.1–6 Neuropathologically, PD is primarily characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of α-synuclein protein aggregates, forming intracellular Lewy bodies.1,7 PD patients exhibit motor symptoms as a consequence of the dopamine depletion, including resting tremor, bradykinesia, and postural instability, among others.

Despite decades of intense efforts to develop specific therapeutic strategies for PD, current treatments primarily focus on symptomatic relief, aiming to restore dopamine levels. These treatments include the gold-standard drug levodopa, dopamine agonists, and surgical procedures.8 In 2024, a review stated that there are sixty disease-modifying therapies for PD under clinical trials,9 targeting a spectrum of triggering mechanisms, such as neuroinflammation, α-synuclein aggregation, oxidative stress, ferroptosis, mitochondrial dysfunction, and dysregulation of the gut–brain axis.2,4,5,8,10 A widely used cellular model for studying the mechanisms of neuronal death in PD and for screening of potential therapeutic agents is the induction with the neurotoxicant 6-hydroxydopamine (6-OHDA), which is an oxidative metabolite of dopamine.11 When dopaminergic neurons are exposed to 6-OHDA, cell death is promoted by a rise in the formation of reactive oxygen species and alterations in the mitochondrial respiratory chain.12,13

As natural products (NPs) play a crucial role in the treatment of various diseases, including neurodegenerative disorders,14–18 they are promising candidates for the development of new PD treatments. Triterpenes are among the most abundant NPs in the plant kingdom and exhibit a broad spectrum of biological activities, including neuroprotective properties.19–24 Despite their structural diversity, they are predominantly found as tetra- or pentacyclic structures in nature, with the latter gaining significant pharmacological interest in recent years.25–28 Lupeol (1) is a natural pentacyclic triterpene found in various plant species, including those of the Asteraceae family.29–34 In previous studies, we successfully isolated lupeol in substantial quantities as one of the major pentacyclic triterpenes from the ethanolic extract of the aerial parts of Chuquiraga erinacea (Asteraceae), an endemic species of Argentina.35 This triterpene exhibits significant biological properties, including anti-diabetic, anti-inflammatory, anti-arthritic, anti-oxidant, anti-infective, and anti-angiogenic activities.36–39 However, its low bioavailability limits its therapeutic application.38 Chemical modification of its structure is a widely used strategy to enhance this property and improve its pharmacological parameters.37,40,41 Although numerous semisynthetic analogues of lupeol have been reported,26,41–49 the development of an α,β-unsaturated carbonyl system is particularly attractive not only due to its biological relevance but also for its significant reactivity, making it a valuable building block for synthesizing novel chemical structures.42,43 Recently, Pokorny et al. synthesized a series of novel triterpenic azines through condensation reactions between an α,β-unsaturated carbonyl group of a pentacyclic triterpene and aromatic hydrazones with high yields using conventional synthesis methods.50 Azines are a class of organic compounds that have garnered significant attention for their chemical versatility and bioactive potential. These molecules, derived from the condensation of aldehydes or ketones with hydrazines, are 2,3-diaza analogues of 1,3-butadiene, also known as N–N linked diimines (C Created by potrace 1.16, written by Peter Selinger 2001-2019 N–N Created by potrace 1.16, written by Peter Selinger 2001-2019 C). Recent research on azines has demonstrated a remarkable range of biological activities, including antimicrobial, antitumor, antioxidant, and, particularly, neuroprotective properties.51–54 Based on this background, this work aims to identify the most promising compounds to advance toward preclinical trials for PD treatment. For this purpose, we synthesized the α,β-unsaturated aldehyde, 3β-hydroxy-lup-20(29)-en-30-al (2), from lupeol isolated from the natural source C. erinacea.46 This building block was employed in the synthesis of a series of triterpenic azines (4a–p), optimizing the reaction proposed by Pokorny et al. through microwave (MW)-assisted irradiation. Subsequently, the neuroprotective activity of the synthesized azines was evaluated using an in vitro PD model induced by the neurotoxin 6-OHDA. Furthermore, the effect of the most active compounds was explored in a ferroptosis model in dopaminergic neurons, showing the most promising results for compound 4c.

2. Results and discussion

Synthesis of triterpene azines 4a–p

The synthesis of new azine derivatives of the natural triterpene lupeol (1), isolated from C. erinacea, followed the reactions depicted in Schemes 1–3. The reaction between 3β-hydroxy-lup-20(29)-en-30-al (2), our building block, and diverse aromatic hydrazones (3a–p) yielded sixteen triterpene azines (4a–p). As shown in Scheme 1, compound 2 was obtained through the allylic oxidation of 1 with SeO2 (2.5 eq.) in ethanol under reflux, with 70% yield. Previous studies have reported allylic oxidation as a method to obtain α,β-unsaturated aldehydes from 1 (ref. 42, 43, 45, 55 and 56) and other triterpenes.50,57–59

Scheme 1. Synthesis of compound 2.

Scheme 1

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Scheme 2. Synthesis of aromatic hydrazones 3a–p.

Scheme 2

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Scheme 3. Synthesis of triterpenic azines 4a–p.

Scheme 3

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The aromatic hydrazones 3a–p were synthesized through the condensation of aromatic aldehydes (a–p) and hydrazine hydrate at room temperature for 30–60 minutes (Scheme 2). The reaction was monitored by thin layer chromatography (TLC), observing the disappearance of the aldehyde and the appearance of a new spot with a lower Rf. The final product was confirmed by 1H-NMR, and the hydrazones were used without further purification.

A first approach to obtain azine derivatives of 1 was explored using the protocol reported by Pokorny et al.50 To a solution of compound 2 in ethanol, the corresponding aromatic hydrazones (3a and b) were added, and the mixture was heated under reflux for 5–6 h, yielding compounds 4a and b (32% and 39%, respectively) (see method A in the Experimental section). Notably, the reaction exhibited a higher yield compared to the synthesis of azine derivatives of betulinic acid reported by Pokorny et al.50

To optimize the synthesis of lupeol azines, a MW-assisted reaction was adapted (Scheme 3). After the addition of 3a and b to a solution of 2 in ethanol, the mixture was irradiated at 70 °C with a power of 280 W for 30 min in a MW oven. Under these conditions, the yield of 4a was comparable to the reaction described above; however, 4b was obtained with a higher yield. A major advantage of the MW-assisted method is the shorter reaction times (Table 1). Therefore, derivatives 4c–p were also synthesized using MW irradiation, with reaction times ranging from 5 to 30 min (see method B in the Experimental section). The reaction yield after purification was variable (6.6–59.5%), considering that in most experiments, 20% of compound 2 remained unreacted. Additionally, a dimeric azine was obtained as a side product, which is consistent with previous reports on this type of reaction.50

Table 1. Comparison of reaction time and yields of synthesis of 4a and b.

–Ar Compound Time (h) Yield (%)
graphic file with name d5md00753d-u1.jpg 4a 0.5a 31.4a
5b 32.0b
graphic file with name d5md00753d-u2.jpg 4b 0.5a 59.4a
6b 39.0b
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a

MW-assisted reaction.

b

Heated under reflux.

The purity of the synthesized compounds was evaluated by TLC, which showed a single spot under UV light after staining with p-anisaldehyde and heating. In addition, 1H and 13C NMR spectroscopy, melting point measurements, and HPLC analysis were performed. The identity of all compounds was confirmed by high-resolution mass spectrometry (HRMS). Detailed spectral data and images are provided in the SI.

The isolated azines 4a–p were identified as E isomers at both newly formed double bonds, as confirmed by a two-dimensional NOESY NMR experiment on the selected derivative 4a (see SI file). Symmetrical azines can exist in three configurational isomers: (E,E), (E,Z), and (Z,Z), with the (E,E) configuration being the most thermodynamically stable, as reported by Safari et al.51 This stereochemical preference has also been observed in structurally related compounds by Pokorny et al.50 The absence of key spatial correlations in the NOESY spectrum of 4a further supports that the synthesized azines predominantly adopt the (E,E) configuration.

Evaluation of the protective effect of triterpene derivatives 4a–p against 6-OHDA toxicity and ferroptosis

Our first biological evaluation was focused on the screening of azines 4a–p based on the well-established in vitro 6-OHDA model,12 which was selected because it closely reproduces the oxidative stress and mitochondrial dysfunction characteristic of PD, providing a relevant system for evaluating neuroprotective activity. For this purpose, we used human neuroblastoma IMR-32 cells, which express neuronal markers such as neurofilament subunits and dopamine transporters and provide a human-derived system for the 6-OHDA model.12

Neuroprotection was assessed by cell viability measurement when IMR-32 cells were exposed to the neurotoxin 6-OHDA in the presence of the compounds at two concentrations (10 and 50 μM) for 24 h. First, we confirmed that the working concentrations of the compounds were not cytotoxic. Based on preliminary dose–response experiments, a concentration of 25 μM 6-OHDA was established as a submaximal, marked, and moderate toxicity (45%).12 As the natural triterpene 1, the derivatives 4d, 4e, 4g, 4i–l, 4o and 4p at 10 μM did not revert the cytotoxicity of 6-OHDA. Interestingly, when IMR-32 cells were pretreated with compounds 4c (Ar = p-methoxyphenyl), 4m (Ar = 4-hydroxy-3-methoxyphenyl), and 4n (Ar = furanyl), the cytotoxic effect of 6-OHDA was prevented (Fig. 1), restoring cell viability to the control conditions even at a concentration of 10 μM. A lower efficacy was observed for compounds 4a, 4b, 4f and 4h (Fig. 1).

Fig. 1. Protective effect of compounds 4a–p against 6-OHDA cytotoxicity in IMR-32 cells. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay after cells were pre-treated with compounds 4a–p (10 and 50 μM) for 30 min, and then with 6-OHDA at 25 μM for 24 h. Results are expressed as percentage of the control. The data shown represent the mean ± SD of three independent experiments. ***p < 0.001 compared to 6-OHDA using one-way ANOVA with Tukey's post hoc tests.

Fig. 1

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Taking into account the protective activity against 6-OHDA exerted by compounds 4c, 4m and 4n, and considering that ferroptosis is a well-described mechanisms of cell death induced by this neurotoxin,2 we decided to study how the derivatives affect neurons challenged with ferroptosis inducers. Subsequent experiments addressing ferroptosis inhibition were conducted at 10 μM of derivatives 4c, 4m and 4n in N27 dopaminergic neurons exposed to RSL3 (GPX4 inhibitor) or erastin (which inhibits cystine–glutamate antiporter system XC−). The concentrations of ferroptosis inducers were fixed in preliminary dose–response assays based on their ability to elicit a reproducible decrease in neuronal viability, thereby establishing conditions suitable for assessing the neuroprotective potential of the tested compounds. At 1 μM, erastin reduced cell viability by 50%, and none of the compounds neither enhanced neuronal damage nor exerted neuroprotection. Differentially, when RSL3 was used (reducing cell viability by 80%), compound 4c efficiently rescued cells from ferroptosis, resulting in a 344% increase in viability (Fig. 2A). The effect was comparable to that of the reference ferroptosis inhibitor ferrostatin-1 (Fer-1). This finding was corroborated by microscopic analysis, as shown in Fig. 2B. While cells exposed to 4m and 4n exhibited morphological changes consistent with ferroptotic death after RSL3 treatment, neurons pretreated with 4c preserved normal morphology, resembling control conditions. To further characterize its anti-ferroptotic potential, compound 4c was tested at increasing concentrations in the RSL3-induced ferroptosis model (Fig. 2C). The compound displayed a dose-dependent inhibition, with an IC50 value of 9.1 μM, indicating moderate potency. Although less potent than Cu(ii)(atsm), a clinically explored ferroptosis modulator with IC50 values below 1 μM,60,614c effectively prevented RSL3-induced neuronal death, suggesting that 4c could serve as a promising scaffold for the development of novel ferroptosis inhibitors targeting dopaminergic neurons.

Fig. 2. Compound 4c protects neurons from ferroptosis. (A) The MTT reduction assay was used to evaluate cell viability after exposure to compounds 4c, 4m and 4n at 10 μM, followed by the induction of ferroptosis with RSL3 or erastin. Fer-1 (10 μM) was used as the positive control. (B) Cell morphology was analyzed by light field microscopy. (C) Dose–response experiments of 4c in RSL3-induced ferroptosis in N27 cells. The IC50 was determined by non-linear regression (95% CI in parentheses).

Fig. 2

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3. Conclusions

In this study, we successfully synthesized and characterized a series of novel triterpenic azines derived from lupeol (4a–4p), optimizing the reaction conditions through MW-assisted synthesis. The neuroprotective potential of these compounds was evaluated in a previously described in vitro model of PD using 6-OHDA-induced cytotoxicity in IMR-32 cells. Among the synthesized derivatives, compounds 4c, 4m, and 4n exhibited significant protective effects against 6-OHDA-induced cell death, suggesting their potential as neuroprotective agents.

Further evaluation using N27 dopaminergic neurons exposed to ferroptosis inducers revealed that compound 4c effectively prevented ferroptotic cell death induced by the GPX4 inhibitor RSL3, while 4m and 4n failed to exert such an effect. Compound 4c efficiently preserved cell viability and morphology at micromolar concentrations, displaying an IC50 of 9.1 μM, which indicates a moderate but biologically relevant anti-ferroptotic potency. These findings suggest that compound 4c may display its neuroprotective activity, at least in part, by counteracting ferroptosis, a regulated cell death mechanism increasingly recognized as a key contributor to neurodegeneration in PD.62 Although less potent than Cu(ii)(atsm), a well-characterized ferroptosis inhibitor with submicromolar activity,60,61 compound 4c achieved comparable protection to Fer-1. These results highlight 4c as a promising scaffold for the development of novel neuroprotective agents targeting ferroptosis-related pathways, potentially relevant for the treatment of neurodegenerative disorders such as PD. Overall, our results highlight the promising pharmacological potential of triterpenic azines as neuroprotective agents and provide a basis for further studies aimed at elucidating their mechanisms of action and evaluating their therapeutic potential in preclinical models of PD. Future investigations will focus on in vivo validation and structure–activity relationship studies to optimize their efficacy and bioavailability.

4. Experimental section

Materials and methods

All reagents used for chemical synthesis were of analytical grade. Masses were measured using an analytical balance (model: AUW220D, manufacturer: Shimadzu) with a readability of 0.00001 g. The balance was calibrated internally and verified with certified external weights (class E2) prior to use. Yields refer to purified products. Microwave reactions were performed with a CEM Discover Synthesis Unit (CEM Corp., Matthews, NC) with a continuous focused microwave power delivery system in glass vessels (10 mL) sealed with a septum, under magnetic stirring. The temperature of the reaction mixture inside the vessel was monitored using a calibrated infrared temperature control under the reaction vessel. Silica gel 60 (200–425 mesh, Aldrich) was used for column chromatography. The separation was monitored by TLC on silica gel 60 F254 sheets (Merck), visualized under UV light and/or using p-anisaldehyde–acetic acid in ethanol spray reagent. Nuclear magnetic resonance (NMR) spectra were recorded at 300 MHz for 1H-NMR and 75 MHz for 13C-NMR using deuterated chloroform (CDCl3, 99.8% D, Aldrich) as the solvent and tetramethyl silane (TMS) as an internal reference. Chemical shifts (δ) are reported in parts per million (ppm) from TMS using the residual solvent resonance (CDCl3: 7.26 ppm for 1H-NMR, 77.16 ppm for 13C-NMR). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br s = broad signal. Coupling constants (J) were reported in hertz (Hz). The identity of all compounds was confirmed by high-resolution mass spectrometry (HRMS) using a Bruker micrOTOF-QII mass spectrometer. Purity was assessed by HPLC analysis using a Thermo Scientific Dionex Ultimate 3000 equipped with a diode-array detector. Samples were detected at a wavelength of 254 nm following separation on a Phenomenex Luna C18 column (250 × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and methanol in a 70 : 30 ratio, with a flow rate of 1 mL min−1. Melting points were determined using a Büchi 510 apparatus and are not corrected.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, #M2128), 6-OHDA hydrochloride (#162957) and Fer-1 (#SML0583) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RSL3 (#19288) and erastin (#17754) were purchased from Cayman (Vienna, Austria). Dulbecco's modified Eagle medium (DMEM) high glucose medium (#52100047), RPMI 1640 medium (#31800022), and antibiotic–antimycotic (#15240062) were purchased from Gibco, CABA, Argentina. Fetal bovine serum (FBS) was obtained from Internegocios, Mercedes, Argentina.

Synthesis of compound 2

Compound 1 (0.5 g; 1.17 mmol) was dissolved in absolute EtOH (20 mL), and SeO2 (2.5 eq.) was added. The reaction mixture was stirred under reflux (78 °C) for 24 h. Then, the reaction mixture was cooled to room temperature and filtered, and water (80 mL) was added to the reaction mixture. The hydroalcoholic phase was separated and extracted thrice with dichloromethane (120 mL). The combined organic phases were dried over MgSO4. The organic solvent was removed under reduced pressure and the residue was purified by flash chromatography using hexane : ethyl acetate (90 : 10) as an eluent system.

3β-Hydroxy-lup-20(29)-en-30-al (2)

Pure compound 2 was obtained as a white crystalline solid (0.3615 g, 0.82 mmol, 70.0%). Rf 0.35 (hexane : EtOAc 80 : 20). Mp: 215–218 °C. 1H (CDCl3, 300 MHz): 9.50 (s, 1H, H-30), 6.28 (br s, 1H, H-29a), 5.90 (br s, 1H, H-29b), 3.17 (dd, 1H, J = 10.6, 5.3 Hz, H-3), 2.73 (ddd, 1H, J = 10.9, 10.9, 5.6 Hz, H-19), 2.24–2.06 (m, 1H, J = 11.1 Hz, H-21a), 1.76–1.57 (m, 5H); 1.50 (br s, 3H), 1.47–1.39 (m, 5H), 1.37 (br s, 4H), 1.27–1.12 (m, 5H), 1.00 (s, 3H, H-23), 0.95 (s, 3H, H-26), 0.91 (s, 3H, H-27), 0.81 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.74 (s, 3H, H-24), 0.65 (d, 1H, J = 9.0 Hz, H-5). 13C (CDCl3, 75 MHz): 195.2 (C-30), 157.3 (C-20), 133.3 (C-29), 79.1 (C-3), 55.4 (C-5), 51.3 (C-18), 50.3 (C-9), 43.4 (C-17), 42.8 (C-14), 40.9 (C-8), 40.0 (C-22), 39.0 (C-4), 38.8 (C-1), 37.8 (C-13), 37.2 (C-10, C-19), 35.5 (C-16), 34.4 (C-7), 32.7 (C-21), 28.1 (C-23), 27.7 (C-12), 27.5 (C-15), 27.4 (C-2), 21.0 (C-11), 18.4 (C-6), 17.9 (C-28), 16.2 (C-25), 16.0 (C-26), 15.5 (C-24), 14.5 (C-27).

General procedure for the synthesis of compounds 3a–p

Hydrazine hydrate (2 eq.) was added to a solution of each aromatic aldehyde a–p in absolute EtOH (1 mL). The reaction mixture was stirred at room temperature for 30–60 min until complete disappearance of the starting material. After that time, the solvent was removed under reduced pressure, affording the substituted aromatic hydrazones 3a–p quantitatively. TLC confirmed complete consumption of the aldehydes and formation of the corresponding hydrazones, which was further corroborated by NMR analysis. The products were used without further purification, in line with the methodology previously reported by Pokorny et al.50

General procedure for the synthesis of compounds 4a and b (method A)

Compound 2 (1 eq.) was dissolved in absolute EtOH (2 mL), and the corresponding aromatic hydrazones 3a and b (1 eq.) were added. The reaction mixture was stirred under reflux (78 °C) for 5–6 h. After completion the solvent was removed under reduced pressure and the crude product was purified by flash chromatography on a silica gel column with hexane : ethyl acetate as the eluent to obtain pure compounds 4a and b.

Lup-20(29)-en-30,31-(E,E)-phenylazine (4a)

Compound 4a was prepared from 2 (0.03 g, 0.068 mmol) and 3a (0.0082 g, 0.068 mmol) following method A. The reaction time was 5 h. Pure compound 4a was obtained as a pale yellow crystalline solid (0.0114 g, 0.021 mmol, 32%) after purification by flash chromatography by using hexane : ethyl acetate (90 : 10) as an eluent system. Rf 0.40 (hexane : EtOAc 70 : 30). Mp: 126–128 °C. 1H (CDCl3, 300 MHz): 8.55 (s, 1H, H-31), 8.21 (s, 1H, H-30), 7.80 (dd, 2H, J = 6.6, 3.0 Hz, H-2′, H-6′), 7.44 (dd, 3H, J = 5.1, 1.9 Hz, H-3′, H-4′, H-5′), 5.66 (s, 1H, H-29a), 5.47 (s, 1H, H-29b), 3.17 (dd, 1H, J = 10.8, 5.2 Hz, H-3), 3.06–2.89 (m, 1H, H-19), 2.36–2.15 (m, 1H, H-21), 1.87–1.64 (m, 5H), 1.61 (br s, 3H), 1.54–1.43 (m, 5H), 1.39 (br s, 4H), 1.33–1.15 (m, 5H), 1.03 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.7 Hz, H-5). 13C (CDCl3, 75 MHz): 164.9 (C-30, C-31), 161.5 (C-1′), 152.6 (C-20), 134.3 (C-3′), 131.2 (C-5′), 128.9 (C-6′), 128.6 (C-4′), 123.5 (C-29), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.4 (C-7), 32.9 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 543.4302 calcd. for (C37H55N2O), found 573.4309 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-(4′-hydroxyphenyl)azine (4b)

Compound 4b was prepared from 2 (0.03 g, 0.068 mmol) and 3b (0.0093 g, 0.068 mmol) following method A. The reaction time was 6 h. Pure compound 4b was obtained as a light yellow crystalline solid (0.0148 g, 0.0265 mmol, 39%) after purification by flash chromatography by using hexane : ethyl acetate (80 : 20) as an eluent system. Rf 0.25 (hexane : EtOAc 70 : 30). Mp: 174–176 °C. 1H (CDCl3, 300 MHz): 8.51 (s, 1H, H-31), 8.21 (s, 1H, H-30), 7.68 (d, 2H, J = 8.7 Hz, H-2′, H-6′), 6.86 (d, 2H, J = 8.7 Hz, H-3′, H-5′), 6.71 (br s, 1H, BzOH), 5.63 (s, 1H, H-29a), 5.45 (s, 1H, H-29b), 3.19 (dd, 1H, J = 10.8, 5.0 Hz, H-3), 3.03–2.86 (m, 1H, H-19), 2.34–2.13 (m, 1H, H-21), 1.85–1.62 (m, 5H), 1.60 (br s, 3H), 1.53–1.42 (m, 5H), 1.38 (br s, 4H), 1.00 (s, 5H), 0.96 (s, 3H, H-23), 0.94 (s, 3H, H-26, H-27), 0.84 (s, 3H, H-25), 0.79 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.5 Hz, H-5). 13C (CDCl3, 75 MHz): 164.5 (C-30, C-31), 161.6 (C-4′), 159.0 (C-20), 152.5 (C-1′), 130.6 (C-2′), 126.6 (C-6′), 123.3 (C-29), 116.0 (C-3′, C-5′), 79.3 (C-3), 55.4 (C-5), 50.4 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 40.9 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.0 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.4 (C-7), 32.9 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.1 (C-11), 18.4 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 559.4240 calcd. for (C37H55N2O2), found 559.4258 [M + H]+. HPLC purity: 95%.

General procedure for the synthesis of compounds 4a–p (method B)

Compound 2 (1 eq.) was dissolved in absolute EtOH (1 mL), and aromatic hydrazones 3a–p (1 eq.) were added. The reaction was irradiated in a microwave oven at 280 W for 5–30 min at 70 °C. The reaction mixture was treated as mentioned in method A to obtain compounds 4a–p.

Lup-20(29)-en-30,31-(E,E)-phenylazine (4a)

Compound 4a was prepared from 2 (0.005 g, 0.011 mmol) and 3a (0.00135 g, 0.011 mmol) following method B. The reaction time was 30 min. Pure compound 4a was obtained as a pale yellow crystalline solid (0.00595 g, 0.01096 mmol, 31.4%) after purification under the same conditions described for method A. The Rf value, melting point, and spectroscopic data were identical to those previously reported.

Lup-20(29)-en-30,31-(E,E)-(4′-hydroxyphenyl)azine (4b)

Compound 4b was prepared from 2 (0.005 g, 0.011 mmol) and 3b (0.0015 g, 0.011 mmol) following method B. The reaction time was 30 min. Pure compound 4b was obtained as a light yellow crystalline solid (0.00375 g, 0.00671 mmol, 59.4%) after purification under the same conditions described for method A. The Rf value, melting point, and spectroscopic data were identical to those previously reported.

Lup-20(29)-en-30,31-(E,E)-(4′-metoxyphenyl)azine (4c)

Compound 4c was prepared from 2 (0.03 g, 0.068 mmol) and 3c (0.001015 g, 0.068 mmol) following method B. The reaction time was 20 min. Pure compound 4c was obtained as a pale yellow amorphous solid (0.0125 g, 0.0218 mmol, 32.1%) after purification by flash chromatography by using hexane : ethyl acetate (90 : 10) as an eluent system. Rf 0.56 (hexane : EtOAc 70 : 30). Mp: 122–125 °C. 1H (CDCl3, 300 MHz): 8.52 (s, 1H, H-31), 8.21 (s, 1H, H-30), 7.75 (d, 2H, J = 8.7 Hz, H-2′, H-6′), 6.95 (d, 2H, J = 8.6 Hz, H-3′, H-5′), 5.63 (s, 1H, H-29a), 5.45 (s, 1H, H-29b), 3.86 (s, 3H, Bz-OCH3), 3.17 (dd, 1H, J = 10.9, 5.1 Hz, H-3), 3.06–2.86 (m, 1H, H-19), 2.37–2.16 (m, 1H, H-21), 1.90–1.63 (m, 5H), 1.61 (br s, 3H), 1.54–1.42 (m, 5H), 1.39 (br s, 4H), 1.13–1.32 (m, 5H), 1.00 (s, 3H, H-23), 0.95 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.66 (d, 1H, J = 9.7 Hz, H-5). 13C (CDCl3, 75 MHz): 164.4 (C-30, C-31), 162.1 (C-4′), 161.3 (C-20), 130.2 (C-2′, C-6′), 127.0 (C-1′), 123.0 (C-29), 114.4 (C-3′, C-5′), 79.1 (C-3), 55.5 (C-5), 55.4 (C-18), 50.5 (C-9), 43.3 (C-17), 42.9 (C-14), 40.9 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 29.8 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.1 (C-11), 18.4 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 573.4435 calcd. for (C38H57N2O2), found 573.4415 [M + H]+. HPLC purity: 95%.

Lup-20(29)-en-30,31-(E,E)-(4′-methylphenyl)azine (4d)

Compound 4d was prepared from 2 (0.03 g, 0.068 mmol) and 3d (0.00915 g, 0.068 mmol) following method B. The reaction time was 30 min. Pure compound 4d was obtained as a pale yellow amorphous solid (0.0172 g, 0.031 mmol, 45.4%) after purification by flash chromatography by using hexane : ethyl acetate (94 : 6) as an eluent system. Rf 0.75 (hexane : EtOAc 70 : 30). Mp: 110–113 °C. 1H (CDCl3, 300 MHz): 8.53 (s, 1H, H-31), 8.21 (s, 1H, H-30), 7.69 (d, 2H, J = 7.8 Hz, H-2′, H-6′), 7.24 (d, 2H, J = 7.9 Hz, H-3′, H-5′), 5.64 (s, 1H, H-29a), 5.46 (s, 1H, H-29b), 3.24–3.09 (m, 1H, H-3), 3.06–2.88 (m, 1H, H-19), 2.41 (s, 3H, Bz-CH3), 2.32–2.15 (m, 1H, H-21), 1.87–1.60 (m, 5H), 1.57 (br s, 3H), 1.54–1.42 (m, 5H), 1.39 (br s, 4H), 1.14–1.30 (m, 5H), 1.03 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.6 Hz, H-5). 13C (CDCl3, 75 MHz): 164.6 (C-30, C-31), 161.6 (C-20), 141.6 (C-1′), 131.6 (C-4′), 129.6 (C-3′, C-5′), 128.6 (C-2′, C-6′), 123.2 (C-29), 79.2 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.2 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10), 35.7 (C-16), 34.5 (C-7), 34.2 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.8 (C-19), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 557.4462 calcd. for (C38H57N2O), found 557.4465 [M + H]+. HPLC purity: 98%.

Lup-20(29)-en-30,31-(E,E)-(4′-chlorophenyl)azine (4e)

Compound 4e was prepared from 2 (0.03 g, 0.068 mmol) and 3e (0.001052 g, 0.068 mmol) following method B. The reaction time was 8 min. Pure compound 4e was obtained as a white crystalline solid (0.00523 g, 0.00906 mmol, 13.3%) after purification by flash chromatography by using hexane : ethyl acetate (95 : 5) as an eluent system. Rf 0.53 (hexane : EtOAc 70 : 30). Mp: 113–115 °C. 1H (CDCl3, 300 MHz): 8.51 (s, 1H, H-31), 8.20 (s, 1H, H-30), 7.74 (d, 2H, J = 8.3, H-2′, H-6′), 7.41 (d, 2H, J = 8.5 Hz, H-3′, H-5′), 5.67 (s, 1H, H-29a), 5.48 (s, 1H, H-29b), 3.26–3.10 (m, 1H, H-3), 3.06–2.88 (m, 1H, H-19), 2.36–2.15 (m, 1H, H-21), 1.86–1.63 (m, 5H), 1.58 (br s, 3H), 1.54–1.41 (m, 5H), 1.39 (br s, 4H), 1.11–1.32 (m, 5H), 1.02 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.7 Hz, H-5). 13C (CDCl3, 75 MHz): 165.3 (C-30, C-31), 160.2 (C-20), 137.1 (C-4′), 132.8 (C-1′), 129.7 (C-3′, C-5′), 129.2 (C-2′, C-6′), 123.9 (C-29), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10), 36.7 (C-19), 35.7 (C-16), 34.5 (C-7), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 24.8 (C-21), 21.2 (C-11), 18.4 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 577.3919 calcd. for (C37H54ClN2O), found 577.3919 [M + H]+. HPLC purity: 95%.

Lup-20(29)-en-30,31-(E,E)-(4′-nitrophenyl)azine (4f)

Compound 4f was prepared from 2 (0.03 g, 0.068 mmol) and 3f (0.001125 g, 0.068 mmol) following method B. The reaction time was 5 min. Pure compound 4f was obtained as a pale yellow amorphous solid (0.0034 g, 0.00578 mmol, 8.5%) after purification by flash chromatography by using hexane : ethyl acetate (92 : 8) as an eluent system. Rf 0.47 (hexane : EtOAc 70 : 30). Mp: 123–125 °C. 1H (CDCl3, 300 MHz): 8.59 (s, 1H, H-31), 8.22 (s, 1H, H-30), 8.29 (d, 2H, J = 8.6 Hz, H-3′, H-5′), 7.97 (d, 2H, J = 8.5 Hz, H-2′, H-6′), 5.74 (s, 1H, H-29a), 5.54 (s, 1H, H-29b), 3.17 (dd, 1H, J = 10.9, 5.2 Hz, H-3), 3.05–2.87 (m, 1H, H-19), 2.39–2.16 (m, 1H, H-21); 1.90–1.60 (m, 5H), 1.56 (br s, 3H), 1.50–1.42 (m, 5H), 1.40 (br s, 4H), 1.12–1.32 (m, 5H), 1.03 (s, 3H, H-23); 0.96 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.3 Hz, H-5). 13C (CDCl3, 75 MHz): 166.3 (C-30, C-31), 158.7 (C-20), 149.2 (C-1′), 140.2 (C-4′), 129.1 (C-2′, C6′), 125.0 (C-29), 124.2 (C-3′, C-5′), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 29.8 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.4 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 588.4160 calcd. for (C37H54N3O3), found 588.4160 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-(2′-hydroxyphenyl)azine (4g)

Compound 4g was prepared from 2 (0.03 g, 0.068 mmol) and 3g (0.00925 g, 0.068 mmol) following method B. The reaction time was 20 min. Pure compound 4g was obtained as a white crystalline solid (0.021 g, 0.0375 mmol, 55.2%) after purification by flash chromatography by using hexane : ethyl acetate (95 : 5) as an eluent system. Rf 0.67 (hexane : EtOAc 70 : 30). Mp: 125–127 °C. 1H (CDCl3, 300 MHz): 11.76 (s, 1H, Bz-OH), 8.69 (s, 1H, H-31), 8.20 (s, 1H, H-30), 7.39–7.29 (m, 2H, H-4′, H-6′), 7.00 (d, 1H, J = 8.2 Hz, H-3′), 6.93 (t, 1H, J = 8.2 Hz, H-5′), 5.71 (s, 1H, H-29a), 5.51 (s, 1H, H-29b), 3.24–3.08 (m, 1H, H-3), 3.05–2.87 (m, 1H, H-19), 2.35–2.15 (m, 1H, H-21), 2.03–1.64 (m, 5H), 1.59 (br s, 3H), 1.54–1.42 (m, 5H), 1.39 (br s, 4H), 1.31–1.19 (m, 5H), 1.03 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.8 Hz, H-5). 13C (CDCl3, 75 MHz): 165.6 (C-30, C-31), 164.9 (C-20), 159.9 (C-2′), 152.3 (C-1′), 132.9 (C-20), 132.3 (C-6′), 124.7 (C-29), 119.5 (C-5′), 117.9 (C-4′), 117.1 (C-3′), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 32.8 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 559.4258 calcd. for (C37H55N2O2), found 559.4258 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-(2′-methoxyphenyl)azine (4h)

Compound 4h was prepared from 2 (0.03 g, 0.068 mmol) and 3h (0.01022 g, 0.068 mmol) following method B. The reaction time was 5 min. Pure compound 4h was obtained as a white amorphous solid (0.0232 g, 0.0405 mmol, 59.5%) after purification by flash chromatography by using hexane : ethyl acetate (92 : 8) as an eluent system. Rf 0.67 (hexane : EtOAc 70 : 30). Mp: 113–116 °C. 1H (CDCl3, 300 MHz): 8.96 (s, 1H, H-31), 8.20 (s, 1H, H-30), 8.04 (dd, 1H, J = 7.4, 1.8 Hz, H-6′), 7.41 (ddd, 1H, J = 8.7, 7.4, 1.8 Hz, H-5′), 7.00 (t, 1H, J = 7.5 Hz, H-4′), 6.93 (d, 1H, J = 8.3 Hz, H-3′), 5.63 (s, 1H, H-29a), 5.45 (s, 1H, H-29b), 3.89 (s, 3H, Bz-OCH3), 3.17 (dd, 1H, J = 10.9, 5.2 Hz, H-3), 3.07–2.90 (m, 1H, H-19), 2.36–2.18 (m, 1H, H-21), 1.98–1.63 (m, 5H), 1.59 (br s, 3H), 1.53–1.42 (m, 5H), 1.39 (br s, 4H), 1.29–1.12 (m, 5H), 1.02 (s, 3H, H-23), 0.95 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.9 Hz, H-5). 13C (CDCl3, 75 MHz): 164.3 (C-30, C-31), 159.1 (C-20), 157.4 (C-2′), 132.5 (C-1′), 127.4 (C-6′), 122.9 (C-4′), 120.9 (C-5′), 111.3 (C-29), 79.1 (C-3), 55.7 (C-18), 55.4 (C-5), 50.5 (C-9), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 32.8 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 573.4414 calcd. for (C38H57N2O2), found 573.4415 [M + H]+. HPLC purity: 97%.

Lup-20(29)-en-30,31-(E,E)-(2′-methylphenyl)azine (4i)

Compound 4i was prepared from 2 (0.03 g, 0.068 mmol) and 3i (0.00913 g, 0.068 mmol) following method B. The reaction time was 20 min. Pure compound 4i was obtained as a white crystalline solid (0.0025 g, 0.00449 mmol, 6.6%) after purification by flash chromatography by using hexane : ethyl acetate (95 : 5) as an eluent system. Rf 0.62 (hexane : EtOAc 70 : 30). Mp: 119–122 °C. 1H (CDCl3, 300 MHz): 8.84 (s, 1H, H-31), 8.23 (s, 1H, H-30), 8.00 (dd, 1H, J = 7.6, 1 Hz, H-3′), 7.38–7.16 (m, 3H, H-4′, H-5′, H-6′), 5.65 (s, 1H, H-29a), 5.48 (s, 1H, H-29b), 3.25–3.09 (m, 1H, H-3), 3.06–2.88 (m, 1H, H-19), 2.54 (s, 3H, CH3), 2.38–2.17 (m, 1H, H-21), 1.90–1.60 (m, 5H), 1.58 (br s, 3H), 1.54–1.42 (m, 5H), 1.39 (br s, 4H), 1.14–1.32 (m, 5H), 1.03 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.87 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.4 Hz, H-5). 13C (CDCl3, 75 MHz): 164.8 (C-30, C-31), 160.1 (C-20), 138.5 (C-1′), 132.4 (C-2′), 131.1 (C-3′), 130.8 (C-6′), 127.6 (C-4′), 126.4 (C-5′), 123.2 (C-29), 79.1 (C-3), 55.4 (C-5), 53.6 (C-18), 50.5 (C-9), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 33.0 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 19.8 (Bz-CH3), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 557.4442 calcd. for (C38H57N2O), found 557.4465 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-(2′-chlorophenyl)azine (4j)

Compound 4j was prepared from 2 (0.03 g, 0.068 mmol) and 3j (0.01052 g, 0.068 mmol) following method B. The reaction time was 7 min. Pure compound 4j was obtained as a white crystalline solid (0.0226 g, 0.0391 mmol, 57.5%) after purification by flash chromatography by using hexane : ethyl acetate (95 : 5) as an eluent system. Rf 0.65 (hexane : EtOAc 70 : 30). Mp: 83–86 °C. 1H (CDCl3, 300 MHz): 8.95 (s, 1H, H-31), 8.20 (s, 1H, H-30), 8.14 (dd, 1H, J = 7.4, 2.0 Hz, H-3′), 7.46–7.27 (m, 3H, H-4′, H-5′, H-6′), 5.68 (s, 1H, H-29a), 5.49 (s, 1H, H-29b), 3.24–3.10 (m, 1H, H-3), 3.07–2.91 (m, 1H, H-19), 2.38–2.17 (m, 1H, H-21), 1.97–1.64 (m, 5H), 1.57 (br s, 3H), 1.54–1.42 (m, 5H), 1.40 (br s, 4H), 1.10–1.32 (m, 5H), 1.03 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.87 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.5 Hz, H-5). 13C (CDCl3, 75 MHz): 165.2 (C-30, C-31), 157.9 (C-20), 135.7 (C-1′), 132.0 (C-2′), 131.8 (C-3′), 130.2 (C-6′), 128.2 (C-4′), 127.1 (C-5′), 123.9 (C-29), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10), 36.7 (C-19), 35.7 (C-16), 34.5 (C-7), 32.1 (C-21), 29.8 (C-23), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 577.3940 calcd. for (C37H54ClN2O), found 577.3919 [M + H]+. HPLC purity: 90%.

Lup-20(29)-en-30,31-(E,E)-(2′-nitrophenyl)azine (4k)

Compound 4k was prepared from 2 (0.03 g, 0.068 mmol) and 3k (0.01124 g, 0.068 mmol) following method B. The reaction time was 30 min. Pure compound 4k was obtained as a light orange crystalline solid (0.0165 g, 0.028 mmol, 41.2%) after purification by flash chromatography by using hexane : ethyl acetate (87 : 13) as an eluent system. Rf 0.45 (hexane : EtOAc, 70 : 30). Mp: 117–120 °C. 1H (CDCl3, 300 MHz): 8.96 (s, 1H, H-31), 8.21 (dd, 1H, J = 7.8, 1.6 Hz, H-3′), 8.16 (s, 1H, H-30), 8.04 (dd, 1H, J = 8.1, 1.4 Hz, H-6′), 7.68 (t, 1H, J = 7.3 Hz, H-5′), 7.58 (t, 1H, J = 8.2 Hz, H-4′), 5.71 (s, 1H, H-29a), 5.50 (s, 1H, H-29b), 3.17 (dd, 1H, J = 10.8, 5.3 Hz, H-3), 3.05–2.88 (m, 1H, H-19), 2.37–2.14 (m, 1H, H-21), 1.87–1.60 (m, 5H), 1.64–1.54 (m, 3H), 1.53–1.42 (m, 5H), 1.39 (br s, 4H), 1.18–1.31 (m, 5H), 1.04 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.86 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.7 Hz, H-5). 13C (CDCl3, 75 MHz): 165.5 (C-30, C-31), 156.5 (C-20), 149.1 (C-2′), 133.5 (C-1′), 131.1 (C-5′), 129.5 (C-6′), 129.3 (C-4′), 124.8 (C-3′), 124.6 (C-29), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 33.0 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 588.4132 calcd. for (C37H54N3O3), found 588.4160 [M + H]+. HPLC purity: 96%.

Lup-20(29)-en-30,31-(E,E)-(4′-N,N-dimethylphenyl)azine (4l)

Compound 4l was prepared from 2 (0.03 g, 0.068 mmol) and 3l (0.0111 g, 0.068 mmol) following method B. The reaction time was 25 min. Pure compound 4l was obtained as a yellow crystalline solid (0.0101 g, 0.0172 mmol, 25.2%) after purification by flash chromatography by using hexane : ethyl acetate (92 : 8) as an eluent system. Rf 0.55 (hexane : EtOAc 70 : 30). Mp: 123–125 °C. 1H (CDCl3, 300 MHz): 8.50 (s, 1H, H-31), 8.22 (s, 1H, H-30), 7.67 (d, 2H, J = 8.9 Hz, H-2′, H-6′), 6.70 (d, 2H, J = 8.9 Hz, H-3′, H-5′), 5.58 (s, 1H, H-29a), 5.41 (s, 1H, H-29b), 3.23–3.11 (m, 1H, H-3), 3.04 (s, 6H, Bz-N(CH3)2), 3.03–2.98 (m, 1H, H-19), 2.37–2.14 (m, 1H, H-21), 1.99–1.64 (m, 5H), 1.61 (br s, 3H), 1.58–1.46 (m, 5H), 1.39 (br s, 4H), 1.12–1.31 (m, 5H), 1.02 (s, 3H, H-23), 0.95 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.66 (d, 1H, J = 9.5 Hz, H-5). 13C (CDCl3, 75 MHz): 163.4 (C-30, C-31), 162.3 (C-20), 152.4 (C-4′), 130.2 (C-2′, C-6′), 122.1 (C-29), 122.0 (C-1′), 111.8 (C-3′, C-5′), 79.2 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.3 (Bz-N(CH3)2), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.8 (C-16), 34.5 (C-7), 33.0 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 586.4736 calcd. for (C39H60N3O), found 586.4731 [M + H]+. HPLC purity: 98%.

Lup-20(29)-en-30,31-(E,E)-(4′-hydroxy-3′-methoxyphenyl)azine (4m)

Compound 4m was prepared from 2 (0.03 g, 0.068 mmol) and 3m (0.0113 g, 0.068 mmol) following method B. The reaction time was 20 min. Pure compound 4m was obtained as a white crystalline solid (0.0115 g, 0.0195 mmol, 28.6%) after purification by flash chromatography by using hexane : ethyl acetate (82 : 18) as an eluent system. Rf 0.25 (hexane : EtOAc 70 : 30). Mp: 123–125 °C. 1H (CDCl3, 300 MHz): 8.48 (s, 1H, H-31), 8.21 (s, 1H, H-30), 7.50 (d, 1H, J = 1.8 Hz, H-6′), 7.18 (dd, 1H, J = 8.1, 1.8 Hz, H-2′), 6.95 (d, 1H, J = 8.1 Hz, H-5′), 5.95 (s, 1H, Bz-OH), 5.63 (s, 1H, H-29a), 5.45 (s, 1H, H-29b), 3.96 (s, 3H, Bz-OCH3), 3.23–3.11 (m, 1H, H-3), 3.03–2.89 (m, 1H, H-19), 2.35–2.15 (m, 1H, H-21), 1.84–1.63 (m, 5H), 1.58 (br s, 3H), 1.54–1.41 (m, 5H), 1.40 (br s, 4H), 1.11–1.31 (m, 5H), 1.02 (s, 3H, H-23), 0.95 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.5 Hz, H-5). 13C (CDCl3, 75 MHz): 164.3 (C-30, C-31), 161.6 (C-20), 148.8 (C-3′), 147.1 (C-4′), 126.9 (C-1′), 124.8 (C-6′), 123.0 (C-29), 114.5 (C-5′), 108.5 (C-2′), 79.1 (C-3), 56.2 (C-5), 55.4 (C-18), 50.5 (C-9), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 29.7 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 589.4376 calcd. for (C38H57N2O3), found 589.4364 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-furfurylazine (4n)

Compound 4n was prepared from 2 (0.03 g, 0.068 mmol) and 3n (0.0075 g, 0.068 mmol) following method B. The reaction time was 8 min. Pure compound 4n was obtained as a pale yellow crystalline solid (0.0158 g, 0.0296 mmol, 43.6%) after purification by flash chromatography by using hexane : ethyl acetate (90 : 10) as an eluent system. Rf 0.45 (hexane : EtOAc 70 : 30). Mp: 107–104 °C. 1H (CDCl3, 300 MHz): 8.43 (s, 1H, H-31), 8.26 (s, 1H, H-30), 7.59 (d, 1H, J = 1.7 Hz, H-5′), 6.86 (d, 1H, J = 3.5 Hz, H-3′), 6.53 (dd, 1H, J = 3.5, 1.8 Hz, H-4′), 5.64 (s, 1H, H-29a), 5.46 (s, 1H, H-29b), 3.17 (dd, 1H, J = 10.8, 5.2 Hz, H-3), 3.01–2.83 (m, 1H, H-19), 2.35–2.10 (m, 1H, H-21), 1.90–1.63 (m, 5H), 1.60 (br s, 3H), 1.57–1.42 (m, 5H), 1.39 (br s, 4H), 1.11–1.30 (m, 5H), 1.02 (s, 3H, H-23), 0.96 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.80 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.66 (d, 1H, J = 9.5 Hz, H-5). 13C (CDCl3, 75 MHz): 165.7 (C-30, C-31), 150.5 (C-20), 149.8 (C-2′), 145.7 (C-5′), 123.7 (C-29), 116.3 (C-3′), 112.3 (C-4′), 79.1 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 27.4 (C-21), 21.1 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6 (C-27). HRMS (ESI) m/z: 533.4109 calcd. for (C35H53N2O2), found 533.4102 [M + H]+. HPLC purity: >99%.

Lup-20(29)-en-30,31-(E,E)-(2′-naphthyl)azine (4o)

Compound 4o was prepared from 2 (0.03 g, 0.068 mmol) and 3o (0.0116 g, 0.068 mmol) following method B. The reaction time was 30 min. Pure compound 4o was obtained as a pale yellow crystalline solid (0.015 g, 0.0253 mmol, 37.2%) after purification by flash chromatography by using hexane : ethyl acetate (95 : 5) as an eluent system. Rf 0.45 (hexane : EtOAc 70 : 30). Mp: 128–131 °C. 1H (CDCl3, 300 MHz): 8.71 (s, 1H, H-31), 8.27 (s, 1H, H-30), 8.13–8.02 (m, 2H, H-1′, H-4′), 7.94–7.81 (m, 3H, H-3′, H-6′, H-9′), 7.59–7.47 (m, 2H, H-7′, H-8′), 5.68 (s, 1H, H-29a), 5.50 (s, 1H, H-29b), 3.23–3.10 (m, 1H, H-3), 3.08–2.91 (m, 1H, H-19), 2.36–2.19 (m, 1H, H-21), 1.92–1.64 (m, 5H), 1.58 (br s, 3H), 1.55–1.45 (m, 5H), 1.40 (br s, 4H), 1.10–1.32 (m, 5H), 1.04 (s, 3H, H-23), 0.97 (s, 6H, H-26, H-27), 0.88 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.67 (d, 1H, J = 9.7 Hz, H-5). 13C (CDCl3, 75 MHz): 165.0 (C-30, C-31), 161.6 (C-20), 135.0 (C-10′), 133.3 (C-5′), 132.1 (C-2′), 130.7 (C-1′), 128.8 (C-8′), 128.7 (C-4′), 128.1 (C-5′), 127.6 (C-3′), 126.7 (C-6′), 123.9 (C-7′), 123.6 (C-29), 79.2 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.2 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.8 (C-16), 34.5 (C-7), 32.1 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 593.4439 calcd. for (C41H57N2O), found 593.4465 [M + H]+. HPLC purity: 98%.

Lup-20(29)-en-30,31-(E,E)-(5′-piperonyl)azine (4p)

Compound 4p was prepared from 2 (0.03 g, 0.068 mmol) and 3p (0.0112 g, 0.068 mmol) following method B. The reaction time was 10 min. Pure compound 4p was obtained as a white crystalline solid (0.0162 g, 0.0276 mmol, 40.6%) after purification by flash chromatography by using hexane : ethyl acetate (93 : 7) as an eluent system. Rf 0.42 (hexane : EtOAc 70 : 30). Mp: 106–109 °C. 1H (CDCl3, 300 MHz): 8.46 (s, 1H, H-31), 8.19 (s, 1H, H-30), 7.42 (d, 1H, J = 1.6 Hz, H-4′), 7.18 (dd, 1H, J = 8.0, 1.6 Hz, H-1′), 6.85 (d, 1H, J = 7.9 Hz, H-6′), 6.02 (s, 2H, H-7′), 5.64 (s, 1H, H-29a), 5.45 (s, 1H, H-29b), 3.17 (dd, 1H, J = 10.8, 5.1 Hz, H-3), 3.04–2.86 (m, 1H, H-19), 2.35–2.14 (m, 1H, H-21), 1.91–1.61 (m, 5H), 1.58 (br s, 3H), 1.53–1.41 (m, 5H), 1.39 (br s, 4H), 1.12–1.30 (m, 5H), 1.02 (s, 3H, H-23), 0.95 (s, 6H, H-26, H-27), 0.85 (s, 3H, H-25), 0.81 (s, 3H, H-28), 0.75 (s, 3H, H-24), 0.66 (d, 1H, J = 9.6 Hz, H-5). 13C (CDCl3, 75 MHz): 164.6 (C-30, C-31), 161.1 (C-20), 150.4 (C-3′), 148.5 (C-2′), 128.9 (C-5′), 125.2 (C-6′), 123.2 (C-29), 108.5 (C-1′), 106.8 (C-4′), 101.7 (C-7′), 79.2 (C-3), 55.4 (C-5), 50.5 (C-9, C-18), 43.3 (C-17), 42.9 (C-14), 41.0 (C-8), 40.1 (C-22), 39.0 (C-4), 38.8 (C-1), 38.1 (C-13), 37.3 (C-10, C-19), 35.7 (C-16), 34.5 (C-7), 32.9 (C-21), 28.1 (C-23), 27.6 (C-2, C-12), 27.5 (C-15), 21.2 (C-11), 18.5 (C-6), 18.1 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.7 (C-27). HRMS (ESI) m/z: 587.4208 calcd. for (C38H55N2O3), found 587.4207 [M + H]+. HPLC purity: >99%.

Biological evaluation of compounds 4a–p

Cell culture

Human neuroblastoma IMR-32 cells and N27 rat dopaminergic neurons were gifted by Dr. Patricia Oteiza (UC DAVIS, USA). IMR-32 cells were cultured in DMEM high glucose medium, and N27 neurons in RPMI 1640 medium, both supplemented with 10% FBS, 100 U ml−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1 amphotericin B, at 37 °C in a humidified atmosphere containing 5% CO2. The medium was replaced by serum-free medium prior to treatments.

Preparation of stock and working solutions

Compounds 4a–p were dissolved in dimethyl sulfoxide (DMSO) to prepare 33.3 mM stock solutions. For the in vitro assays, working dilutions were freshly prepared in distilled water prior to use. Specifically, 133.2 μM and 666 μM working solutions were used to achieve final concentrations of 10 μM and 50 μM in the culture medium, respectively, by adding 7.5 μL per well. The final concentration of DMSO in all treatments did not exceed 0.15% (v/v).

Experimental treatments

The compounds were tested at 10 and 50 μM to ensure that they did not exert cytotoxicity. IMR-32 cells were exposed to compounds 4a–p (10 and 50 μM) for 30 min, and then treated with 6-OHDA at 25 μM for 24 h. N27 neurons were treated with 100 nM RSL3 and 1 μM erastin (ferroptosis inducers) for 24 h after pre-incubation with compounds 4c, 4m and 4n (10 μM) for 30 min. Controls received the vehicle alone. The ferroptosis inhibitor Fer-1 was used as the positive control.

Cell viability assay

Cells were seeded in a 96-well microplate at a density of 10 000 cells per well for 6-OHDA-induced cytotoxicity and 5000 cells per well for RSL3/erastin-induced ferroptosis, and after treatments, cellular viability was measured by the colorimetric MTT assay.63 The tetrazolium salt MTT is reduced by metabolically viable cells to a colored water-insoluble formazan salt. Following treatments, the MTT was incubated for 2 h at 37 °C in a 5% CO2 atmosphere (final concentration 0.5 mg ml−1). Then, the formazan crystals were dissolved with 200 μl of 20% sodium dodecyl sulfate (pH 4.7), and measured spectrophotometrically at 570 and 650 nm using a microplate reader (Multiskan GO, Thermo Scientific). Results are expressed as the mean ± SD (percentage of the control). Control wells containing each compound in the absence of cells and reagents were included in all experiments to exclude interferences with the colorimetric measurements.

Microscopic analysis

After treatments, N27 cells were analyzed by light field microscopy to evaluate cell morphology using a Nikon Eclipse E600 microscope.

Statistical analysis

Data were analyzed using one-way ANOVA accompanied by Tukey's test. p values less than 0.05 were considered statistically significant. At least eight concentrations of the test compound, and two different cell passages, each tested in three independent wells, were used. Non-linear regression analysis with a variable slope model was used to fit a four-parameter logistic curve to dose–response data to calculate IC50 with 95% CI.

Author contributions

F. A. M. conducted the synthesis, purification, and identification of the compounds, performed data analysis and evaluation, and contributed to investigation, validation, visualization, drafting of the original manuscript, and editing. N. P. A. carried out the biological experiments, interpreted and evaluated the results, and contributed to conceptualization, formal analysis, investigation, validation, visualization, drafting of the original manuscript, and editing. M. B. F. contributed to writing – review and editing, original draft preparation, visualization, validation, supervision, funding acquisition, conceptualization, investigation, project administration, and resource management. G. A. S. contributed similarly to M. B. F., providing support in writing – review and editing, original draft preparation, visualization, validation, supervision, funding acquisition, conceptualization, investigation, project administration, and resource management. All authors critically reviewed and approved the final version of the manuscript and accept responsibility for all aspects of the work.

Conflicts of interest

The authors declare no competing interests.

Supplementary Material

MD-OLF-D5MD00753D-s001

Acknowledgments

G. A. S. acknowledges financial support from the National Scientific and Technical Research Council (CONICET, grant PIP11220210100175CO), the National Agency for the Promotion of Science and Technology (ANPCyT, grant PICT 2021-0143), and Universidad Nacional del Sur (grants PGI 24B292 and PGI 24B358), Argentina. M. B. F. acknowledges financial support of ANPCyT (grant PICT 2017-1443) and Universidad Nacional del Sur (grant PGI-UNS 24/Q105), Argentina. G. A. S. and N. P. A. are Researcher Members of CONICET, and M. B. F. is a Research Member of CIC. F. A. M. gratefully acknowledges CONICET for her doctoral fellowship. The authors acknowledge the use of the image of Chuquiraga erinacea (photo by John Doe), available at Wikimedia Commons under the Creative Commons Attribution 2.0 Generic License (CC BY 2.0).

Data availability

The data supporting this article have been included in the supplementary information (SI).

Supplementary information is available. See DOI: https://doi.org/10.1039/d5md00753d.

Notes and references

  1. Lees A. J. Hardy J. Revesz T. Lancet. 2009;373:2055–2066. doi: 10.1016/S0140-6736(09)60492-X. [DOI] [PubMed] [Google Scholar]
  2. Mahoney-Sánchez L. Bouchaoui H. Ayton S. Devos D. Duce J. A. Devedjian J. C. Prog. Neurobiol. 2021;196(101890):101890. doi: 10.1016/j.pneurobio.2020.101890. [DOI] [PubMed] [Google Scholar]
  3. Lin K. J. Chen S. D. Lin K. L. Liou C. W. Lan M. Y. Chuang Y. C. et al. . Cells. 2022;11(23):3829. doi: 10.3390/cells11233829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Liu L. Cui Y. Chang Y.-Z. Yu P. Sci. Rep. 2023;13(1):15365. doi: 10.1038/s41598-023-42574-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dong-Chen X. Yong C. Yang X. Chen-Yu S. Li-Hua P. Signal Transduction Targeted Ther. 2023;8(1):73. doi: 10.1038/s41392-023-01353-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Teferi N. Challa M. Woodiwiss T. Allen B. Petronek M. Redox Experimental Medicine. 2023:1. [Google Scholar]
  7. Youdim M. B. H. Kupershmidt L. Amit T. Weinreb O. Parkinsonism Relat. Disord. 2014;20:S132–S136. doi: 10.1016/S1353-8020(13)70032-4. [DOI] [PubMed] [Google Scholar]
  8. Wang Z. L. Yuan L. Li W. Li J. Y. Trends Mol. Med. 2022;28(4):258–269. doi: 10.1016/j.molmed.2022.02.003. [DOI] [PubMed] [Google Scholar]
  9. McFarthing K. Buff S. Rafaloff G. Pitzer K. Fiske B. Navangul A. et al. . J. Parkinson's Dis. 2024;14:899–912. doi: 10.3233/JPD-240272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Alza N. P. Iglesias González P. A. Conde M. A. Uranga R. M. Salvador G. A. Front. Cell. Neurosci. 2019;13:175. doi: 10.3389/fncel.2019.00175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hernandez-Baltazar D. Zavala-Flores L. M. Villanueva-Olivo A. Neurologia. 2017;32:533–539. doi: 10.1016/j.nrl.2015.06.011. [DOI] [PubMed] [Google Scholar]
  12. Iglesias González P. A. Conde M. A. González-Pardo V. Uranga R. M. Salvador G. A. Toxicol. In Vitro. 2019;60:400–411. doi: 10.1016/j.tiv.2019.06.019. [DOI] [PubMed] [Google Scholar]
  13. Simola N. Morelli M. Carta A. R. Neurotoxic. Res. 2007;11:151–167. doi: 10.1007/BF03033565. [DOI] [PubMed] [Google Scholar]
  14. Long H. Z. Cheng Y. Zhou Z. W. Luo H. Y. Wen D. D. Gao L. C. Front. Pharmacol. 2021;12:648636. doi: 10.3389/fphar.2021.648636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rahman H. H. Bajgai J. Fadriquela A. Sharma S. Trinh T. T. Akter R. Jeong Y. J. Goh S. H. Kim C. S. Lee K. J. Molecules. 2021;26(17):5327. doi: 10.3390/molecules26175327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Andrade S. Nunes D. Dabur M. Ramalho M. J. Pereira M. C. Loureiro J. A. Pharmaceutics. 2023;15(1):212. doi: 10.3390/pharmaceutics15010212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cui D. Chen Y. Ye B. Guo W. Wang D. He J. Phytomedicine. 2023;121:155101. doi: 10.1016/j.phymed.2023.155101. [DOI] [PubMed] [Google Scholar]
  18. Goyal R. Mittal P. Gautam R. K. Kamal M. A. Perveen A. Garg V. Alexiou A. Saboor M. Haque S. Farhana A. Papadakis M. Ashraf G. M. Nutr. Metab. 2024;21:26. doi: 10.1186/s12986-024-00800-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hill R. A. Connolly J. D. Nat. Prod. Rep. 2013;30:1028–1065. doi: 10.1039/c3np70032a. [DOI] [PubMed] [Google Scholar]
  20. Bednarczyk-Cwynar B. Zaprutko L. Phytochem. Rev. 2014;14:203–231. doi: 10.1007/s11101-014-9353-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hordyjewska A. Ostapiuk A. Horecka A. J. Pre-Clin. Clin. Res. 2018;12:72–75. [Google Scholar]
  22. Wang W. Wang Y. Liu M. Zhang Y. Yang T. Li D. Huang Y. Li Q. Bai G. Shi L. J. Cell. Mol. Med. 2018;23:586–595. doi: 10.1111/jcmm.13964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. González-Cofrade L. de las Heras B. Apaza Ticona L. Palomino O. M. Planta Med. 2019;85:1304–1315. doi: 10.1055/a-0953-6738. [DOI] [PubMed] [Google Scholar]
  24. Heise N. V. Schüler J. A. Orlamünde T. E. Brandes B. Deigner H. P. Al-Harrasi A. Csuk R. Mediterr. J. Chem. 2022;12(2):188–200. [Google Scholar]
  25. Harun N. H. Septama A. W. Ahmad W. A. N. W. Suppian R. Chin. Herb. Med. 2020;12:118–124. doi: 10.1016/j.chmed.2019.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nistor G. Trandafirescu C. Prodea A. Milan A. Cristea A. Ghiulai R. Racoviceanu R. Mioc A. Mioc M. Ivan V. Şoica C. Molecules. 2022;27(19):6552. doi: 10.3390/molecules27196552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hussain H. Xiao J. Ali A. Green I. R. Westermann B. Nat. Prod. Rep. 2023;40:412–451. doi: 10.1039/d2np00033d. [DOI] [PubMed] [Google Scholar]
  28. Liu J. Yin X. Kou C. Thimmappa R. Hua X. Xue Z. Plant Commun. 2024;5(4):100845. doi: 10.1016/j.xplc.2024.100845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Duan D. D. Bu C. Y. Cheng J. Wang Y. N. Shi G. L. J. Econ. Entomol. 2011;104:375–378. doi: 10.1603/ec10129. [DOI] [PubMed] [Google Scholar]
  30. Silva L. B. Strapasson R. L. B. Riva D. Salvador M. J. Stefanello M. É. A. Rev. Bras. Farmacogn. 2011;21:556–559. [Google Scholar]
  31. Haque M. A. Hassan M. M. Das A. Begum B. Ali M. Y. Morshed H. J. Appl. Pharm. Sci. 2012;2(6):79–83. [Google Scholar]
  32. Asgarpanah J. Dakhili N. Mirzaee F. Salehi M. Janipour M. Rangriz E. Pharmacogn. J. 2015;8:42–43. [Google Scholar]
  33. Bouattour E. Fakhfakh J. Frikha Dammak D. Jabou K. Damak M. Mezghani Jarraya R. Chem. Biodiversity. 2016;13:1674–1684. doi: 10.1002/cbdv.201600118. [DOI] [PubMed] [Google Scholar]
  34. Romo-Asunción D. Ávila-Calderón M. A. Ramos-López M. A. Barranco-Florido J. E. Rodríguez-Navarro S. Romero-Gomez S. Aldeco-Pérez E. J. Pacheco-Aguilar J. R. Rico-Rodríguez M. A. Fla. Entomol. 2016;99:345–351. [Google Scholar]
  35. Gurovic M. Castro M. Richmond V. Faraoni M. Maier M. Murray A. Planta Med. 2009;76:607–610. doi: 10.1055/s-0029-1240582. [DOI] [PubMed] [Google Scholar]
  36. Laghari A. H. Memon S. Nelofar A. Khan K. M. Ind. Crops Prod. 2011;34:1141–1145. [Google Scholar]
  37. Siddique H. R. Saleem M. Life Sci. 2011;88:285–293. doi: 10.1016/j.lfs.2010.11.020. [DOI] [PubMed] [Google Scholar]
  38. Furtado N. J. C. Pirson L. Edelberg H. Miranda L. M. Loira-Pastoriza C. Preat V. Larondelle Y. André C. Molecules. 2017;22:400. doi: 10.3390/molecules22030400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Beserra F. P. Vieira A. J. Gushiken L. F. S. de Souza E. O. Hussni M. F. Hussni C. A. Nóbrega R. H. Martinez E. R. M. Jackson C. J. de Azevedo Maia G. L. Rozza A. L. Pellizzon C. H. Oxid. Med. Cell. Longevity. 2019:1–20. doi: 10.1155/2019/3182627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kumar S. Misra N. Raj K. Srivastava K. Puri S. K. Nat. Prod. Res. 2008;22:305–319. doi: 10.1080/14786410701766349. [DOI] [PubMed] [Google Scholar]
  41. Saini M. Khan M. F. Sangwan R. Khan M. A. Kumar A. Verma R. Ahamad T. Jain S. ChemistrySelect. 2019;4:1800–1805. [Google Scholar]
  42. Khan M. F. Maurya C. K. Dev K. Arha D. Rai A. K. Tamrakar A. K. Maurya R. Bioorg. Med. Chem. Lett. 2014;24:2674–2679. doi: 10.1016/j.bmcl.2014.04.059. [DOI] [PubMed] [Google Scholar]
  43. Khan M. F. Mishra D. P. Ramakrishna E. Rawat A. K. Mishra A. Srivastava A. K. Maurya R. Med. Chem. Res. 2014;23:4156–4166. [Google Scholar]
  44. Bednarczyk-Cwynar B. Wiecaszek T. Ruszkowski P. Nat. Prod. Commun. 2016;11(9):1237–1238. [PubMed] [Google Scholar]
  45. Machado V. R. Sandjo L. P. Pinheiro G. L. Moraes M. H. Steindel M. Pizzolatti M. G. Biavatti M. W. Nat. Prod. Res. 2017;32:275–281. doi: 10.1080/14786419.2017.1353982. [DOI] [PubMed] [Google Scholar]
  46. Castro M. J. Musso F. Vetere V. Faraoni M. B. J. Organomet. Chem. 2019;880:333–340. [Google Scholar]
  47. Liu K. Zhang X. Xie L. Deng M. Chen H. Song J. Long J. Li X. Luo J. Pharmacol. Res. 2021;164:105373. doi: 10.1016/j.phrs.2020.105373. [DOI] [PubMed] [Google Scholar]
  48. Phan H. V. T. Duong T. H. Pham D. D. Pham H. A. Nguyen V. K. Nguyen T. P. Sichaem J. Nat. Prod. Res. 2020;36(1):1–7. doi: 10.1080/14786419.2020.1758095. [DOI] [PubMed] [Google Scholar]
  49. Le H. T. T. Chau Q. C. Duong T. H. Tran Q. T. P. Pham N. K. T. Nguyen T. H. T. Nguyen N. H. Sichaem J. Molbank. 2021:M1306. [Google Scholar]
  50. Pokorny J. Krajcovicova S. Hajduch M. Holoubek M. Gurska S. Dzubak P. Volna T. Popa I. Urban M. Future Med. Chem. 2018;10(5):483–491. doi: 10.4155/fmc-2017-0171. [DOI] [PubMed] [Google Scholar]
  51. Safari J. Gandomi-Ravandi S. RSC Adv. 2014;4:46224–46249. [Google Scholar]
  52. Subedi L. Kwon O. W. Pak C. Lee G. Lee K. Kim H. Kim S. Y. BMC Neurosci. 2017;18:82. doi: 10.1186/s12868-017-0399-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Chourasiya S. S. Kathuria D. Wani A. A. Bharatam P. V. Org. Biomol. Chem. 2019;17:8486–8521. doi: 10.1039/c9ob01272a. [DOI] [PubMed] [Google Scholar]
  54. Muzio L. Sirtori R. Gornati D. Eleuteri S. Fossaghi A. Brancaccio D. Manzoni L. Ottoboni L. De Feo L. Quattrini A. Mastrangelo E. Sorrentino L. Scalone E. Comi G. Marinelli L. Riva N. Milani M. Seneci P. Martino G. Nat. Commun. 2020;11(1):3848. doi: 10.1038/s41467-020-17524-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wal A. Wal P. Rai A. K. Raj K. J. Pharm. Sci. Res. 2010;2(1):13–25. [Google Scholar]
  56. Castro M. J. Richmond V. Faraoni M. B. Murray A. P. Bioorg. Chem. 2018;79:301–309. doi: 10.1016/j.bioorg.2018.05.012. [DOI] [PubMed] [Google Scholar]
  57. Neukirch H. D'Ambrosio M. Sosa S. Altinier G. Della Loggia R. Guerriero A. Chem. Biodiversity. 2005;2:657–671. doi: 10.1002/cbdv.200590042. [DOI] [PubMed] [Google Scholar]
  58. Castro M. J. Richmond V. Romero C. Maier M. S. Estévez-Braun A. Ravelo Á. G. Faraoni M. B. Murray A. P. Bioorg. Med. Chem. 2014;22:3341–3350. doi: 10.1016/j.bmc.2014.04.050. [DOI] [PubMed] [Google Scholar]
  59. Sousa M. C. Varandas R. Santos R. C. Santos-Rosa M. Alves V. Salvador J. A. R. PLoS One. 2014;9:e89939. doi: 10.1371/journal.pone.0089939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Scarpellini C. Klejborowska G. Lanthier C. Hassannia B. Vanden Berghe T. Augustyns K. Trends Pharmacol. Sci. 2023;44(12):902–916. doi: 10.1016/j.tips.2023.08.012. [DOI] [PubMed] [Google Scholar]
  61. Southon A. Szostak K. Acevedo K. M. Dent K. A. Volitakis I. Belaidi A. A. Barnham K. J. Crouch P. J. Ayton S. Donnelly P. S. Bush A. I. Br. J. Pharmacol. 2020;177(3):656–667. doi: 10.1111/bph.14881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Maniscalchi A. Benzi Juncos O. N. Conde M. A. Funk M. I. Fermento M. E. Facchinetti M. M. Curino A. C. Uranga R. M. Alza N. P. Salvador G. A. Redox Biol. 2024;71:103074. doi: 10.1016/j.redox.2024.103074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Alza N. P. Murray A. P. Salvador G. A. Naunyn-Schmiedeberg's Arch. Pharmacol. 2017;390(12):1229–1238. doi: 10.1007/s00210-017-1421-0. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

MD-OLF-D5MD00753D-s001
MD-OLF-D5MD00753D-s001.pdf (5.4MB, pdf)

Data Availability Statement

The data supporting this article have been included in the supplementary information (SI).

Supplementary information is available. See DOI: https://doi.org/10.1039/d5md00753d.

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