Apiol from Parsley Seeds as a Source for the Synthesis of Coenzyme Q₀ Propanoic Acid Conjugates with Amines Featuring Antioxidant Activity

Abstract

Quinone-containing compounds are known for their free-radical scavenging activity and pronounced antioxidant properties. They could be promising for the alleviation of progressive neuronal damage in neurodegenerative diseases, caused in particular by oxidative stress. In this study, coenzyme Q₀ derivatives featuring the fragment of natural quinone propanoic acid conjugated with dopamine, 5-methoxytryptamine, β-alanine, and γ-aminobutyric acid were synthesized. Apiol, the main metabolite of parsley seed extract, was used as a starting material. The aim of this study was to explore antioxidant activity of the targeted compounds in vitro using several assays, including determination of free radical scavenging capacity, inhibition of lipid peroxidation in mouse brain homogenate, and evaluation of toxicity and membrane-protective effects in mouse red blood cells (RBCs). All conjugates of the quinone propanoic acid fragment demonstrated significant antioxidant activity. The free radical scavenging capacity of the compounds in DPPH and ABTS tests was associated with the presence and amount of unsubstituted OH groups in the aromatic ring. The dopamine derivative was identified as the most potent inhibitor of both oxidative mouse RBC hemolysis and intracellular generation of reactive oxygen species. The 5-methoxytryptamine quinone derivative considerably suppressed Fe^2+/ascorbate-initiated lipid peroxidation in the mouse brain homogenate. All compounds showed no toxicity on a sea urchin embryo model and could be considered potential neuroprotective agents due to the antioxidant properties of the quinone fragment and facile cleavage of the labile amide bond, thereby providing the targeted delivery of biogenic amines into the desired location.

Introduction

Natural antioxidants, such as quinones, polyphenols, essential fatty acids, carotenoids, phytosterols, etc., are considered promising agents capable of suppressing neurodegenerative diseases progression through the interaction with various cellular targets. , Accumulation of reactive oxygen species (ROS), or oxidative stress, is thought to be one of the factors that trigger psychomotor symptoms in Parkinson’s disease (PD). , The pathogenesis of PD is associated with the accumulation of an abnormal misfolded protein, α-synuclein, resulting in a progressive degeneration of dopaminergic neurons in the substantia nigra. Current medication is aimed at the alleviation of PD symptoms but is unable to affect neuronal damage and its underlying metabolic pathways. Therefore, the development of multifunctional agents capable of ameliorating PD clinical manifestations is of particular interest.

Various quinone derivatives can serve as a suitable scaffold for the development of broad-spectrum pharmacological drugs due to their ability to participate in oxidation–reduction processes and unique chemical properties. ,− The therapeutic application of quinones can be attributed to their ability to undergo reversible oxidation–reduction transformation, thereby implementing both oxidative stress protection and prooxidant-related cytoxicity. ,,,− A vital cell antioxidant coenzyme Q₁₀ (ubiquinone, CoQ₁₀) in its reduced ubiquinol form exhibits considerable antioxidant properties in lipid-containing structures such as cell membranes and lipoproteins. ,,− CoQ₁₀ is highly hydrophobic and poor bioavailable, resulting in its low therapeutic benefit as an antioxidant. , The active reduced form of CoQ₁₀, ubiquinol, features significant bioavailability, but is chemically unstable. Similarly, CoQ₁₀ synthetic analogs idebenone and MitoQ exhibit antioxidant activity both in vitro and in vivo. Particularly, idebenone reduces oxidative stress and improves the cognitive status of patients with amnestic mild cognitive disorder. The prooxidant effect of quinones is associated with the formation of superoxide from their intermediate semiquinone radicals. ,, This effect was reported also for coenzyme Q₀ (2,3-dimethoxy-5-methyl-1,4-benzoquinone, CoQ₀) 1 (Figure ) isolated from the Taiwan endemic fungus Antrodia camphorata (syn. Antrodia cinnamomea). Numerous studies described the ability of CoQ₀ to induce ROS-dependent apoptosis and autophagy in human cancer cells, thereby suggesting CoQ₀ as a potential anticancer agent. −

1.

Coenzyme Q₀ derivatives.

Considering this, we decided to conduct the synthesis and biological evaluation of quinones conjugated with biogenic amines and amino acids. It was proposed that, like CoQ₁₀, the quinone fragment would be able to protect cells from oxidative stress and alleviate mitochondrial dysfunction. In addition, the lipophilicity of the quinone moiety ensures passage of the molecule through the blood–brain barrier, and then the labile amide bond can be easily cleaved by cell enzymes, thereby releasing free neurotransmitters and amino acids. So, the target structures can be considered both antioxidants and effective carriers of neurotransmitters or amino acids into the brain. Similar combined structures where dopamine was covalently bound to membrane receptor-targeting lipophilic molecules were reported to cross the blood–brain barrier and transport dopamine to the brain successfully. ,

Results and Discussion

The approach to quinone analogs featuring predicted redox properties can be implemented by the introduction of electron-withdrawing or electron-donating groups. ,, Specifically, the electron-donating amino and methoxy groups in the quinone ring can reduce ROS formation and, accordingly, diminish the cytotoxicity of quinones. Our tests using the sea urchin embryo model (see below) revealed that the toxicity of CoQ₀ 1 was at least 20 times higher than that of its methoxy-substituted derivatives 2, 3 (Figure ). Therefore, this study was aimed at obtaining CoQ₀ analogs where the methyl group was replaced by an electron-donating methoxy group.

Recently, a methoxy-substituted quinone propanoic acid 4 (Figure ) was isolated from the bark of the tree of heaven, Ailanthus altissima. The acid was reported to exhibit significant anti-inflammatory and antioxidant effects both in vivo and in vitro without cytotoxicity toward RAW264.7 mouse macrophages at ≤ 30 μM concentration. Besides, the introduction of the hydroxyl group affords more potent antioxidant molecules (compare nothoapiol and hydroxyapiol; hydroxyquinone 2 and the respective trimethoxyquinone 3see data below). So, we synthesized derivatives of 4 where the 4-OMe in the quinone ring was replaced with 4-OH. The amide derivatives of related quinone propanoic acid would combine both the antioxidant effect of the quinone fragment and the ability to transfer biogenic amines to the desired location. The target quinone conjugates with biogenic amines and amino acids 6 (Figure ) were synthesized starting from apiol 5 (Figure ), an easily available component of parsley seeds essential oil. Apiol 5 was extracted by liquid CO₂ from parsley seeds and purified by high-efficiency distillation on a 10 kg scale.

The preparation of a structure suitable to obtain adducts with amines and amino acids under mild conditions without reagents involved in peptide synthesis was the crucial stage in the synthesis of CoQ₀ analogs. In addition, this structure should be susceptible to oxidizing agents, providing its facile conversion into quinone. With this in mind, a hydroxy-methoxy-substituted cyclic lactone 7 (Scheme ) as an intramolecularly activated ester was synthesized according to the published protocol. , Compound 7 reacted easily with different nucleophiles, yielding various hybrid molecules featuring a hydroquinone moiety. Both the highly oxygenated scaffold and the 2,4,5-trihydroxy-substituted benzene core of the opened lactone facilitated the spontaneous oxidation of the intermediate hydroquinone to quinone by atmospheric oxygen. To elucidate the mechanism of the reaction and find optimal conditions, the model procedure was executed with methylamine.

1. Oxidative Transformation of Lactone (7) with Methylamine.

Methylamine was added to lactone 7 in an argon atmosphere, followed by oxidation of the resulting hydroquinone to quinone 6a in air (Scheme ). During the reaction, the colorless lactone solution quickly acquired a black-purple hue, followed by a red color upon the complete oxidation to the quinone 6a. Notably, an excess of the amine may cause the substitution of methoxy groups with amino groups in the resulting quinone. Therefore, the lactone ring opening should be conducted under argon without an excess of the amine.

Next, we synthesized quinone propionic acid derivatives conjugated with selected biogenic amines and amino acids, namely, dopamine 8, 5-methoxytryptamine (mexamine) 11, β-alanine 12, and γ-aminobutyric acid 13 (Scheme ). Interaction of lactone 7 with unprotected dopamine 8 led to significant formation of resinous byproducts, affording 6b in low yield (28%). Therefore, protected dopamine hydrochloride 9 was used. The resulting mixture of quinone 6b and the respective hydroquinone was used in the next deprotection hydrogenation stage without separation. Then the hydroquinone was completely oxidized by atmospheric oxygen within 24 h to afford amide 6b in 50% yield in three steps. Mexamine 11 was obtained by the hydrolysis of melatonin 10. It readily reacted with 7, followed by the oxidation of the intermediate hydroquinone with atmospheric oxygen, affording amide 6c in 54% yield in two steps (Scheme ). For the synthesis of quinone propionic acid conjugates with β-alanine 12 and γ-aminobutyric acid 13, the carboxyl groups of the amino acids were converted to their benzyl esters. The respective benzyl esters 14 and 15 as hydrochlorides were obtained by the benzyl alcohol esterification of 12 and 13 in the presence of TMSCl (Scheme A). Interaction of esters 14 and 15 with 7 in the presence of Et₃N yielded mixtures of the corresponding quinones and hydroquinones that were subsequently deprotected by hydrogenation without separation. Finally, the quinones containing β-alanine 6d and γ-aminobutyric acid 6e moieties were obtained in 64% and 47% yields, respectively (Scheme B). Dihydrocoumarin 7 was converted into the quinone propanoic acid 16 by acid hydrolysis and oxidation with atmospheric oxygen. This compound is a derivative of natural quinone acid 4, where 4-OMe in the quinone ring was replaced with 4–OH.

2. Synthesis of CoQ₀ Propanoic Acid Derivatives Conjugated with Biogenic Amines and Amino Acids .

Prior to evaluation of antioxidant activity, the toxicity of quinones 1–3, 6a–e, and 16 was estimated using a phenotypic sea urchin embryo assay. Only CoQ₀ 1 exhibited toxic effect toward the sea urchin embryos, causing developmental abnormalities and embryonic death at 0.4 μM and 2 μM concentration, respectively. Other compounds were inactive up to an 8 μM concentration. The observed embryotoxicity could be attributed to the methyl substituent in the quinone ring, whereas the replacement of the methyl group with a methoxy fragment resulted in a loss of toxicity. Considering this, CoQ₀ 1 was excluded from the further assessment of antioxidant activity.

Antioxidant efficiency of quinone derivatives 2, 3, 6a–e, and 16 was evaluated using several previously published assays, including estimation of free radical scavenging capacity, accumulation of thiobarbituric acid reactive substances (TBARS) in mouse brain homogenate, and examination of toxicity and membrane-protective effects in mouse red blood cells (RBC). ,− Trolox was used as a standard in the same concentrations as those of the tested compounds.

Free radical scavenging activity data of compounds 2, 3, 6a–e, and 16 using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis­(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) assays are presented in Table .

1. Free Radical Scavenging Activity of Compounds at 10 μM and 100 μM Concentrations (DPPH and ABTS Tests) .

compound	radical scavenging activity (%)
	DPPH		ABTS
	10 μM	100 μM	10 μM	100 μM
2	20.4 ± 0.6	71.6 ± 0.4	45.2 ± 1.3	99.6 ± 0.0
3	0.0	0.0	0.0	2.1 ± 0.4
6a	19.2 ± 0.5	74.7 ± 0.4	48.1 ± 0.6	99.6 ± 0.0
6b	43.6 ± 0.4	90.7 ± 0.0	69.5 ± 1.1	99.5 ± 0.1
6c	20.6 ± 0.3	84.2 ± 0.5	60.8 ± 0.6	99.3 ± 0.0
6d	20.7 ± 2.4	75.9 ± 0.2	53.5 ± 0.4	99.5 ± 0.1
6e	26.8 ± 0.5	78.2 ± 0.5	56.7 ± 0.7	99.3 ± 0.0
16	30.7 ± 0.7	85.5 ± 0.2	49.6 ± 1.2	99.4 ± 0.1
trolox	48.2 ± 0.7	94.3 ± 0.3	51.1 ± 0.9	99.4 ± 0.0

^aThe data are expressed as the mean ± standard errors (SE) (n = 4–16).

Dimethoxyhydroxyquinone 2 and quinone propionic acid 16 showed considerable radical scavenging activity, apparently due to the presence of a hydroxyl group in the quinone moiety, since trimethoxyquinone 3 was inactive. Introduction of propionic acid fragment into the quinone ring significantly enhanced scavenging of the stable DPPH radical (16 vs 2). Compound 6b, featuring a catechol fragment was identified as the most effective in both tests. In particular, 6b inhibited the cationic radical ABTS more effectively than trolox. Thus, the free radical scavenging activity of the tested quinone derivatives was attributed mostly to the presence and amount of hydroxyl groups in the aromatic fragments.

All tested compounds 2, 3, 6a–e, and 16 significantly inhibited Fe²⁺/ascorbate-initiated lipid peroxidation in a heterogeneous substrate (oil/water emulsion) containing mouse brain lipids (Figure ). However, a correlation between the inhibition of TBARS accumulation and the free radical scavenging activity of the compounds was not observed. Particularly, trimethoxyquinone 3 failed to inhibit free radicals up to 100 μM concentration, while effectively diminishing TBARS accumulation in brain homogenate. This phenomenon could be attributed to the presence in mammalian cells, and in the mouse brain homogenate as well, of a set of non-mitochondrial enzymes capable of reducing quinone to hydroquinone. Similarly, both trimethoxyquinone 3 and dimethoxyhydroxyquinone 2 decreased TBARS accumulation considerably higher than quinone acid 16 and amides 6a, 6d, and 6e. Quinone derivative 6c, with a 5-methoxytryptamine fragment, was identified as the most potent inhibitor of TBARS formation.

2.

Effect of compounds 2, 3, 6a–e, and 16 at 100 μM concentration on TBARS accumulation in mouse brain homogenate. TBARS concentration was measured at 1 h after the initiation of lipid peroxidation by Fe²⁺/ascorbate. Control: sample without test compounds. Intact: sample without initiation of Fe²⁺/ascorbate peroxidation. Vertical bars: mean ± SE (n = 4–20).

As mentioned above, CoQ₀ exhibited cytotoxicity toward mammalian cells. − In addition, a hemolytic effect or erythrotoxicity was reported for anthraquinone alizarin, a component of the madder plant Rubia tinctorum, and for a series of naphthoquinone derivatives. , Thus, prior to the study of the effect of quinone derivatives on RBC oxidative hemolysis, their hemolytic properties should be estimated. In the series of quinone derivatives 2, 3, 6a–e, and 16, dimethoxyhydroxyquinone 2 and trimethoxyquinone 3 showed the pronounced erythrotoxicity at 10 μM concentration (Figure ) and, therefore, they were excluded from the further experiments on RBC. The hemolytic activity of compounds 2 and 3 was found to be associated with their ability to induce formation of ROS at the initial stages of incubation (p = 0.004) and then oxidation of intracellular oxyhemoglobin (oxyHB) to met- (metHB) and ferrylhemoglobin (ferrylHB) (Figure ). Functionalization of quinone 2 at position 6 yielded molecules 6a–e and 16 devoid of hemolytic effect up to 10 μM concentration (Figure ). Likewise, aminomethylation of alizarin at the C-3 position resulted in reduction in erythrotoxicity while maintaining high antioxidant activity.

3.

Hemolytic activity (erythrotoxicity) of quinone derivatives 2, 3, 6a–e, and 16 (10 μM) after 1, 3, and 5 h of incubation. Control: RBC without test compounds. Vertical bars: mean ± SE (n = 3–9).

4.

Effect of quinones 2 and 3 (20 μM) on ROS generation, metHb/oxyHb ratio, and ferrylHb/oxyHb ratio in mouse RBC after 1 h incubation. Vertical bars: mean ± SE (n = 6–12).

The majority of quinone derivatives displayed statistically significant antioxidant activity in the model of H₂O₂-induced hemolysis of mouse RBC except compound 6a (Figure A). Dopamine derivative 6b featuring three hydroxyl groups enhanced cell survival most potently. More pronounced differences in activity of the tested compounds were observed in 2,2′-azobis­(amidinopropane) dihydrochloride (AAPH)-induced RBC hemolysis model (Figure B). All quinone derivatives considerably increased cell survival under acute oxidative stress, and 6b with a catechol fragment was found to be the most effective. This compound almost completely inhibited hemolysis throughout the entire period of incubation, statistically significantly exceeding trolox activity. Alike most of the tested molecules, quinone dopamine conjugate 6b inhibited oxyHB oxidation to metHB and ferrylHB (Figure ). Noticeably, compound 6b demonstrated an excellent RBC protective effect from AAPH-induced oxidative damage in the cellular antioxidant activity assay (CAA), exceeding trolox activity (Figure ). Quinone derivatives 6a and 6c also statistically significantly inhibited the formation of AAPH-induced ROS generation in RBC. In contrast, quinone propionic acid 16 demonstrated weak prooxidant activity in the CAA-RBC assay.

5.

Effect of quinone derivatives 6a–e and 16 (10 μM) on mouse RBC hemolysis under oxidative stress induced by H₂O₂ (A) and AAPH (B) after 1–5 h incubation. Control: RBC without test compounds. Vertical bars: mean ± SE (n = 4–5).

6.

Effect of quinone derivatives 6a–e and 16 (10 μM) on metHb/oxyHb and ferrylHb/oxyHb ratio in mouse RBC under AAPH-induced hemolysis after 5 h incubation. Control: RBC without test compounds. Vertical bars: mean ± SE (n = 10–12).

7.

Effect of compounds 6a–e and 16 (20 μM) on AAPH-induced intracellular ROS generation in mouse RBC (CAA-RBC assay). Intact: RBC without compounds and ROS initiator. The results were calculated as % of control (ROS production in AAPH-treated RBC without test compounds). Vertical bars: mean ± SE (n = 12–24).

In general, evaluation of antioxidant activity identified quinone dopamine conjugate 6b as the most potent agent, displaying considerable RBC protection under oxidative stress. According to our previous study, compound 6b exhibited neuroprotective effect and reduced the ROS level under AAPH and rotenone-induced oxidative stress on SH-SY5Y human neuroblastoma cells. In vivo experiments on laboratory mice demonstrated the ability of 6b to penetrate the blood–brain barrier and to increase the content of dopamine in the striatum, thereby providing dopamine delivery to the brain.

Conclusions

The coenzyme Q₀ derivatives featuring the fragment of natural quinone propanoic acid conjugated with dopamine, 5-methoxytryptamine, β-alanine, and γ-aminobutyric acid were synthesized without reagents involved in peptide synthesis. Intermediate hydroxy-methoxy-substituted coumarin 7 was synthesized from parsley metabolite apiol and then reacted easily with various nucleophiles, yielding hybrid molecules 6a–e containing a hydroxyquinone moiety. All tested quinone derivatives demonstrated statistically significant antioxidant activity in in vitro assays. Generally, the free radical scavenging capacity of the compounds in the DPPH and ABTS tests was associated with the presence and amount of unsubstituted OH groups in the aromatic fragments. The dopamine derivative 6b was the most active. In addition, 6b was identified as the most potent inhibitor of both oxidative RBC hemolysis initiated by H₂O₂ or AAH and intracellular AAPH-induced generation of ROS in the CAA-RBC assay. Trimethoxyquinone 3 and 6c, containing the 5-methoxytryptamine fragment, considerably suppressed Fe²⁺/ascorbate-initiated lipid peroxidation in mouse brain homogenate. Quinones 2 and 3 displayed a high hemolytic effect, apparently due to increased ROS generation, confirmed by the substantial oxidation of oxyhemoglobin with the formation of unstable ferryl forms. Introduction of substituents in the quinone ring allows to minimize erythrotoxicity while maintaining high antioxidant activity. The synthesized amide derivatives of quinone propanoic acid 6 could be considered promising agents for the targeted delivery of biogenic amines and amino acids to the brain.

Experimental Section

General Experimental Procedures

Melting points were measured on a Nagema PHMK 05 polarizing microscope with a Boetius heating plate.¹H and ¹³C NMR spectra were recorded on Bruker AM300 and Bruker DRX500 instruments (working frequencies 300.13 and 500.13 MHz for ¹H and 75.47 and 125.76 MHz for ¹³CC, respectively). Chemical shifts in the ¹H NMR spectra are given relative to the residual proton signal of the solvent (CHCl₃δH 7.27 ppm, DMSO-d ₅δH 2.50 ppm), in the ¹³C NMR spectrarelative to the solvent signal (CDCl₃δC 77.0 ppm, DMSO-d ₆δC 39.5 ppm). Spin–spin coupling constants (J) were reported in hertz (Hz). Low resolution mass spectra (m/z) were recorded on a Finnigan MAT/INCOS 50 mass spectrometer at 70 eV with direct probe injection. High resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument (electrospray ionization). Reactions were monitored by TLC on silica gel 60 F₂₅₄ Merck plates, and compounds were visualized under UV-light. Products were purified by silica gel column chromatography (CC) using 0.125–0.200 mm silica gel (Acros Organics) and the indicated solvent system or recrystallization. Solvents and reagents were purified by standard procedures. Apiol with 98–99% purity was isolated by liquid CO₂ extraction of parsley seeds (var. Sakharnaya) followed by high-efficiency distillation. Coenzyme Q₀ 1 was purchased from Acros Organics. Quinones 2 and 3 were synthesized according to published procedures.

3,4-Dibenzyloxyphenethylamine Hydrochloride 9

Synthesized from commercially available dopamine hydrochloride 8 according to the published method. Yield: 87%; colorless crystals; mp 134–135 °C (Lit. 133 °C); ¹H NMR (DMSO-d ₆): δ 8.14 (3H, br s, NH₃ ⁺), 7.28–7.49 (10H, m, 2× Ph), 7.02 (1H, d, J = 1.7 Hz, 2-CH), 6.99 (1H, d, J = 8.2 Hz, 6-CH), 6.76 (1H, dd, J = 8.2 Hz, J = 1.7 Hz, 5-CH), 5.12 (2H, s, CH₂O), 5.10 (2H, s, CH₂O), 2.98 (2H, t, J = 7.9 Hz, CH₂), 2.82 (2H, t, J = 7.9 Hz, CH₂). ¹³C NMR (DMSO-d ₆): δ 148.4, 147.1, 137.4, 137.3, 130.4, 128.4 (2C), 128.3 (2C), 127.8, 127.7, 127.6 (2C), 127.4 (2C), 121.2, 115.1, 114.8, 70.2 (2C), 40.0, 32.5.

5-Methoxytryptamine Hydrochloride 11

Synthesized from commercially available melatonin 10 according to the published method. Yield 66%; colorless crystals; mp 247–250 °C (Lit. 243–245 °C); ¹H NMR (DMSO-d ₆): δ 10.84 (1H, br s, NH), 8.15 (3H, br s, NH₃ ⁺), 7.25 (1H, d, J = 8.8 Hz, 7-CH), 7.19 (1H, d, J = 1.8 Hz, 4-CH), 7.09 (1H, s, 2-CH), 6.73 (1H, dd, J = 8.8 Hz, J = 1.8 Hz, 6-CH), 3.77 (3H, s, OMe), 3.00 (4H, br s, 2× CH₂).

Benzyl Esters Hydrochlorides of β-Alanine 14 and γ-Aminobutyric Acid 15

Synthesized from commercially available β-alanine 12 and γ-aminobutyric acid 13 according to the published method.

14: Yield 63%; colorless crystals; mp 110–112 °C (Lit. 100–101 °C) ¹H NMR (DMSO-d ₆): δ 8.23 (3H, br s, NH₃ ⁺), 7.28–7.43 (5H, m, Ph), 5.12 (2H, s, CH₂O), 3.03 (2H, t, J = 7.1 Hz, CH₂), 2.79 (2H, t, J = 7.1 Hz, CH₂).

15: Yield 84%; colorless crystals, mp 110–111 °C (Lit. 109–110 °C); ¹H NMR (D₂O): δ 7.38–7.49 (5H, m, Ph), 5.16 (2H, s, CH₂O), 3.01 (2H, t, J = 7.7 Hz, 4-CH₂), 2.54 (2H, t, J = 7.3 Hz, 2-CH₂), 1.96 (2H, t, J = 7.3 Hz, 3-CH₂).

Synthesis of (3-(4-Hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)-N-methylpropanamide) 6a

To a solution of 6,7-dihydroxy-5,8-dimethoxychroman-2-one 7 (0.120 g, 0.5 mmol) in dioxane (2.5 mL) under an argon atmosphere, a 12 M solution of methylamine (0.050 mL, 0.6 mmol) was added. The resulting deep violet mixture was stirred at ambient temperature for 24 h. The solvent was then removed under reduced pressure, MeOH (2.5 mL) was added, and the mixture was stirred under ambient temperature for an additional 24 h and then concentrated to dryness. Purification of the product was accomplished by CC (15 g of SiO₂, CH₂Cl₂/CH₃OH = 95:5). Yield 0.070 g (52%); red crystals; mp 136–137 °C (CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃): δ 6.74 (1H, br s, NH), 5.82 (1H, br s, OH), 4.05 (3H, s, OMe), 4.02 (3H, s, OMe), 2.78 (3H, d, J = 4.8 Hz, NHCH ₃), 2.73 (2H, t, J = 7.7 Hz, CH₂), 2.33 (2H, t, J = 7.7 Hz, CH₂); ¹³C NMR (126 MHz CDCl₃): δ 183.6, 180.5, 172.3, 153.0, 138.5, 137.2, 129.5, 61.4, 60.4, 35.0, 26.3, 19.7; HRMS (ESI-TOF) m/z: [M + Na]⁺ calcd for C₁₂H₁₅NO₆Na, 292.0792; found, 292.0794.

Synthesis of Quinone Amides 6b,d,e (General Procedure)

General procedure for the preparation of compounds 6b,d,e was carried out using the same methodology as reported in our recent article.

• Step 1. To a stirred slurry of 6,7-dihydroxy-5,8-dimethoxychroman-2-one 7 (3.753 g, 15.62 mmol) and amine hydrochloride (15.62 mmol) in dioxane (52 mL) under an argon atmosphere, Et₃N (3.2 g, 4.4 mL, 31.2 mmol) was added. The resulting deep violet mixture was stirred at ambient temperature for 72 h until complete consumption of the starting material (TLC monitoring). The solvent was then removed under reduced pressure, and water (50 mL) together with CH₂Cl₂ (30 mL) were added to the deep violet residue. The organic phase was separated, and the aqueous phase was extracted with CH₂Cl₂ (2 × 30 mL). The combined violet organic extracts were dried and concentrated in vacuo to afford a dark, ink-colored oil (10.4 g). After filtration through a SiO₂ pad (CH₂Cl₂MeOH), a mixture of quinone and hydroquinone was obtained and taken into the next step without purification.

• Step 2. In a Schlenk tube, 10% Pd/C (0.519 g, 0.488 mmol) and CH₃OH (50 mL) were added to the mixture of quinone and hydroquinone. The resulting solution was degassed 3 times and hydrogenated at ambient temperature and atmospheric pressure for 24 h until complete consumption of the starting material (TLC monitoring). The resulting discolored solution was filtered through a Celite pad and stirred in air for 72 h until oxidation of hydroquinone to quinone was complete, as indicated by the development of a red coloration. Reaction progress was monitored by ¹H NMR. The red solution was then concentrated to dryness, and the product was purified by CC (72 g SiO₂, CH₂Cl₂/CH₃OH/AcOH = 100:10:1) to obtain amides 6b,d,e as red solids.

N-(3,4-Dihydroxyphenethyl)-3-(4-hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)­propanamide 6b

Yield 50%; red solid; mp 58–60 °C; ¹H NMR (300 MHz, acetone-d ₆): δ 8.00 (1H, br s, OH), 7.14 (t, J = 4.6 Hz, 1H, NH), 6.71 (d, J = 8.1 Hz, 1H, CH_Ar), 6.70 (d, J = 1.8 Hz, 1H, CH_Ar), 6.53 (dd, J = 8.1 Hz, J = 1.8 Hz, 1H, CH_Ar), 3.95 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 3.33 (q, J = 6.5 Hz, 2H, CH₂), 2.67 (t, J = 7.9 Hz, 2H, CH₂), 2.62 (t, J = 7.4 Hz, 2H, CH₂), 2.27 (t, J = 7.9 Hz, 2H, CH₂); ¹³C NMR (75 MHz, acetone-d ₆): δ = 184.3, 181.4, 172.2, 154.2, 145.9, 144.3, 142.9, 139.0, 132.1, 130.8, 120.9, 116.6, 116.1, 61.6, 60.7, 41.9, 36.0, 35.7, 20.5; HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₉H₂₂NO₈, 392.1340; found, 392.1343.

3-(3-(4-Hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)­propanamido)­propanoic Acid 6d

Yield 64%; red crystals; mp 159–160 °C (MeOH). ¹H NMR (500 MHz, DMSO-d ₆): δ 12.09 (1H, br s, COOH), 10.35 (1H, br s, OH), 7.88 (t, J = 5.2 Hz, 1H, NH), 3.86 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.20 (q, J = 6.3 Hz, 2H, 3-CH₂), 2.50 (m, CH₂), 2.35 (t, J = 6.9 Hz, 2H, 2-CH₂), 2.12 (t, J = 7.9 Hz, 2H, CH₂); ¹³C NMR (126 MHz, DMSO-d ₆): δ 183.3, 180.2, 172.9, 171.0, 153.1, 143.2, 138.0, 129.0, 60.9, 60.0, 34.8, 34.2, 33.9, 19.2; HRMS (ESI-TOF) m/z: [M + Na]⁺ calcd for C₁₄H₁₇NO₈Na, 350.0846; found, 350.0845.

4-(3-(4-Hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)­propanamido)­butanoic Acid 6e

Yield 47%; red crystals; mp 121–123 °C (MeOH); ¹H NMR (500 MHz, DMSO-d ₆): δ 12.03 (1H, br s, COOH), 10.36 (1H, br s, OH), 7.82 (t, J = 5.5 Hz, 1H, NH), 3.86 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.02 (q, J = 6.3 Hz, 2H, 4-CH₂), 2.51 (m, CH₂), 2.20 (t, J = 7.4 Hz, 2H, 2-CH₂), 2.13 (t, J = 7.9 Hz, 2H, CH₂), 1.59 (pent, J = 7.2 Hz, 2H, 3-CH₂); ¹³C NMR (126 MHz, DMSO-d ₆): δ = 183.3, 180.2, 174.2, 170.8, 153.1, 143.2, 138.0, 129.1, 60.9, 60.0, 37.9, 34.3, 31.1, 24.6, 19.3; HRMS (ESI-TOF) m/z: [M + Na]⁺ calcd for C₁₅H₁₉NO₈Na, 364.1003; found, 364.1007.

Synthesis of (3-(4-Hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)-N-(2-(5-methoxy-1H-indol-3-yl)­ethyl)­propanamide) 6c

To a stirred slurry of 6,7-dihydroxy-5,8-dimethoxychroman-2-one 7 (0.240 g, 1 mmol) and mexamine hydrochloride 11 (0.227 g, 1 mmol) in dioxane (6 mL) under an argon atmosphere, Et₃N (0.121 g, 0.167 mL, 1.2 mmol) was added. The resulting deep violet mixture was stirred at ambient temperature for 24 h until complete consumption of the starting material (TLC monitoring). The solvent was then removed under reduced pressure, MeOH (5 mL) was added, and the mixture was stirred at ambient temperature in air for 24 h. The red solution was then concentrated to dryness, and the product was purified by CC (20 g of SiO₂, CH₂Cl₂/CH₃OH = 95:5). Yield 54%; 0.230 g; red solid; mp 68–70 °C; ¹H NMR (500 MHz, DMSO-d ₆): δ 10.61 (1H, br s, NH), 7.93 (t, J = 4.6 Hz, 1H, C­(O)NH), 7.22 (d, J = 8.7 Hz, 1H, CH_Ar), 7.09 (br s, 1H, CH_Ar), 7.00 (d, J = 2.2 Hz, 1H, CH_Ar), 6.71 (dd, J = 8.7 Hz, J = 2.2 Hz, 1H, CH_Ar), 3.87 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.30 (q, J = 6.9 Hz, 2H, CH₂), 2.76 (t, J = 7.4 Hz, 2H, CH₂), 2.54 (t, J = 7.9 Hz, 2H, CH₂), 2.15 (t, J = 7.9 Hz, 2H, CH₂); ¹³C NMR (126 MHz, DMSO-d ₆): δ 183.4, 180.3, 170.8, 153.1, 153.0, 143.4, 138.1, 131.4, 129.2, 127.6, 123.3, 112.0, 111.7, 111.1, 100.2, 60.9, 60.1, 55.4, 39.5, 34.5, 25.3, 19.4; HRMS (ESI-TOF) m/z: [M + Na]⁺ calcd for C₂₂H₂₄N₂O₇Na, 451.1476; found, 451.1475.

Synthesis of 3-(4-Hydroxy-2,5-dimethoxy-3,6-dioxocyclohexa-1,4-dien-1-yl)­propanoic Acid 16

To a mixture of 7 (0.120 g, 0.5 mmol) and H₂O (5 mL), an aqueous solution of HCl (0.05 mL 1 M) was added. The mixture was heated to reflux and then stirred in air at room temperature for 24 h. The reaction product was extracted with CH₂Cl₂ (3 × 10 mL), the combined extracts were dried over MgSO₄, filtered, evaporated, and dried in vacuo to give 16 as a red crystalline solid. Yield 40%; mp 144–146 °C (CH₂Cl₂); ¹H NMR (500 MHz DMSO-d ₆): δ 2.27 (t, J = 7.9 Hz, 2H, CH₂), 2.53 (t, J = 7.9 Hz, 2H, CH₂), 3.77 (s, 3H, OCH₃), 3.88 (s, 3H, OCH₃), 10.39 (br s, 1H, OH), 12.15 (br s, 1H, CO₂H); ¹³C NMR (126 MHz DMSO-d ₆): δ 183.3, 180.2, 173.5, 153.3, 143.3, 138.0, 128.2, 60.9, 60.0, 32.7, 18.6; HRMS (ESI-TOF) m/z: 279.0477 (calcd for C₁₁H₁₂O₇Na, [M + Na]⁺, 279.0475).

Phenotypic Sea Urchin Embryo Assay

Adult sea urchins Paracentrotus lividus L. (Echinidae), were collected from the Mediterranean Sea on the Cyprus coast in March-May, 2024 and 2025, and kept in an aerated seawater tank. Experiments were conducted according to previously published procedure. Briefly, stock solutions of compounds were prepared in DMSO at a concentration of 20 mM, followed by a 10-fold dilution with 96% EtOH. This procedure improved the solubility of the test molecules in the seawater. The effect was assessed by exposing fertilized eggs (8–20 min after fertilization) and hatched blastules (8.5–9 h post fertilization) to 2-fold decreasing concentrations of the compound and estimated quantitatively as a minimum effective concentration resulting in developmental abnormalities. Embryo development was monitored at room temperature (18–23 °C) using a Biolam LOMO optical microscope (St. Petersburg, Russia) until the beginning of active feeding (four-arm middle pluteus stage, 34–36 h post fertilization).

Antioxidant Activity Evaluation. Materials

Stock solutions of test compounds were prepared in acetone at 1 and 10 mM concentration. Mouse RBCs and brain homogenate were incubated at 37 °C in a thermostated Biosan ES-20 shaker (Latvia). Absorption was measured by using a Thermo Spectronic Genesys 20 spectrophotometer (USA). Fluorescence and absorption spectra were analyzed with a CLARIOstar Plus multiplate reader (BMG LABTECH, Germany). Phosphate buffered saline (PBS, pH 7.4), 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) (Sigma-Aldrich, Germany), 2-thiobarbituric acid (TBA) (Alfa Aesar, USA), trichloroacetic acid (Alfa Aesar, USA), ascorbic acid (ICN Biomedical Inc., USA), FeCl₂ (reagent grade), (OOO Reakhim, Russia), 2,2′-azino-bis­(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (Alfa Aesar, USA), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Alfa Aesar, USA), 2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Lumiprobe, Russia), and H₂O₂ (pure grade) solution were used. Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, Sigma-Aldrich, Germany) served as a reference compound.

Determination of Free Radical Scavenging Activity

DPPH and ABTS radical scavenging activity was estimated according to previously published procedures , with minor modifications. The stock solution of the compound was added to the DPPH solution in MeOH (1% v/v). Then, the mixture was shaken vigorously and kept in the dark at room temperature for 30 min. The absorption decrease was measured at λ = 517 nm. The radical scavenging activity was calculated as a percentage of DPPH^• discoloration. ABTS cation-radical was obtained by reacting a stock solution of ABTS (7 mM) with potassium persulfate K₂S₂O₈ (2.45 mM) and keeping the mixture in the dark at room temperature for 16 h before use. The ABTS^•+ solution was diluted with ethanol to an absorption of 0.70 at λ = 734 nm. The stock solution of the compound was added to the ABTS^•+ solution (1% v/v) and was shaken vigorously and then kept in the dark at room temperature for 30 min. The decrease in absorption was measured at λ = 734 nm. The radical scavenging activity was calculated as a percentage of ABTS^•+ discoloration. ,,

Antioxidant Activity. TBARS Assay

The antioxidant activity of compounds 2, 3, 6a–e, and 16 was evaluated in vitro as inhibition of Fe²⁺/ascorbate-initiated lipid peroxidation estimated as a reduction of accumulation of secondary lipid peroxidation products (TBA-reactive species, TBARS) in mouse brain homogenates (oil-aqueous emulsion). ,− Brain extract was homogenized in physiological saline (pH 7.4) and centrifuged. The compound stock solution was added to the supernatant, and lipid peroxidation was initiated by FeCl₂/ascorbic acid. Samples were stirred carefully and shaken at 37 °C. The concentration of secondary lipid peroxidation products (TBARS) was measured by UV spectra at λ = 532 nm using an extinction coefficient of 1.56 × 10⁵ M^–1cm⁻¹. ,

Antioxidant Activity Evaluation in Mouse RBC Model

A mouse RBC suspension was prepared in phosphate-buffered saline (PBS, pH 7.4). ,− Erythrotoxicity was evaluated as the ability of the compounds to induce hemolysis of RBC after 1, 3, and 5 h incubation at 37 °C. DCFH-DA was used to assess the intracellular ROS generation (cellular antioxidant activity, CAA-RBC assay). DCFH-DA (20 μM final concentration) was added to the RBC, the samples were incubated at 37 °C for 30 min and centrifuged, and then the supernatant was removed. The compound stock solution was added to a final concentration of 20 μM, and the sample was incubated for 30 min. Then AAPH solution (3 mM) was added, and after 60 min, the fluorescein concentration was determined with a fluorescence microplate reader at 488 nm/522 nm for excitation and emission, respectively. ,− The results were calculated as % of a control (100%). Membrane-protective and antioxidant activity in RBC were determined by the inhibition of oxidative hemolysis initiated by AAPH (3 mM) or H₂O₂ (1.8 mM) and inhibition of hemoglobin oxidation. The compound stock solution (10 μM final concentration) was added to RBC 30 min prior to the initiation of hemolysis. Then the reaction mixture was shaken slowly for 5 h at 37 °C. An aliquot was taken hourly from the incubated sample and centrifuged for 5 min (1600 g). The hemolysis was estimated by the hemoglobin content in the supernatant at λ = 524 nm. In the end of incubation, an aliquot of RBC suspension was subjected to complete hemolysis and centrifuged in order to assess the accumulation of hemoglobin oxidation products. The amount of oxyhemoglobin (oxyHb), methemoglobin (metHb), and ferryl hemoglobin (ferrylHb) was calculated using relevant extinction coefficients.

Statistical analysis was conducted using the Microsoft Office Excel 2010 and Statistica 6.0 software packages. Experimental data are presented as mean values with SE. For evaluating statistical significance (p value) of measurements, the Mann–Whitney U test was employed.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c09328.

• ¹H and ¹³C NMR and HRMS spectra of compounds 6a–e (PDF)

The manuscript was written through the contributions of all authors. All authors have given their approval to the final version of the manuscript.

The authors declare no competing financial interest.