Synthesis of Novel Sesamol-Based Hybrids—In Silico Analysis and In Vitro Evaluation of Radical Scavenging Activity

Abstract

New benzazole–sesamol derivatives 6a–8c were synthesized via an easily accessible reaction based on the coupling of Sesamol with in situ generated electrophilic N-alkoxycarbonylbenzazolium ions. This strategy successfully integrated benzothiazole, benzimidazole, and 5,6-dimethylbenzimidazole fragments with the biologically active natural lignan Sesamol. The structural integrity and the specific position of the newly formed C–C bond was confirmed by ¹H-, ¹³C{¹H}-, HSQC-NMR, FTIR, and HRMS analyses. The obtained compounds with yields in the range of 71–95% were evaluated for their in vitro radical scavenging activity and subjected to in silico predictions of mutagenicity and toxicity. Radical scavenging activity studies demonstrate that the introduction of a benzothiazoline ring (compounds 6a and 6b) enhances radical scavenging activity compared to Sesamol in the DPPH assay, outperforming the benzimidazole analogues. In silico analyses identified compounds 7b, 7c, 8a, 8b, and 8c as promising molecules due to the absence of mutagenic and irritant effects and their low toxicity profiles. In particular, compounds 7a, 7b, and 8a were found to be significantly safer than Sesamol. Compound 7a exhibited the highest safety profile, characterized by an LD₅₀ value of 3046.92 mg/kg.

1. Introduction

Within pharmaceutical research [1,2], there is growing interest in the discovery of new and effective anticancer agents derived from natural sources due to their considerable therapeutic potential [3]. The natural lignan Sesamol (Figure 1) is a compound of significant biological importance, isolated from the seeds of Sesamum indicum [4,5]. Sesamol has been shown to possess significant antioxidant [4,6,7], anti-inflammatory [5,8], antimicrobial [9,10], antiemetic activities [11], as well as photoprotective [12], anticancer [13], and antiproliferative properties [4,14]. In addition, it has demonstrated the ability to induce apoptosis in tumour cancer cells [15] and exhibits notable anti-ageing potential [16].

In addition to the biological activities described above, Sesamol has been investigated as a tyrosinase inhibitor, suppressing melanin synthesis in human melanoma cells (SK-MEL-2). This property classifies Sesamol as a potential anti-melanogenic agent and encourages further research into its safe application in cosmetics or for therapeutic purposes [17,18]. Accordingly, Khan et al. reported in silico predictions indicating low toxicity of Sesamol in rodents, with an LD₅₀ value of 580 mg/kg [19]. Other authors, like Koru et al., experimentally demonstrated that at a concentration of 10 µM of Sesamol is safe for human umbilical vein endothelial cells (HUVEC, ATCC^® CRL-1730™) [8].

Sesamol exhibits potent antioxidant activity (IC₅₀ < 14.48 µM in the DPPH assay; FRAP = 189.88 ± 17.56 µM) [6,7]. Its antioxidant potential has been extensively investigated and compared with other natural sources, such as carotenoids and polyphenols. Using a series of assays (DPPH, ABTS, FRAP and ORAC), the authors demonstrated a high capacity of Sesamol to neutralize free radicals, ranking its efficacy immediately after ellagic acid. The increase in antioxidant activity was established in the following order: ellagic acid > Sesamol > olive leaf extract > lutein [7].

As a consequence of its antioxidant activity, Sesamol has also been demonstrated to exert neuroprotective, cardioprotective, and hepatoprotective effects, along with improvements in cognitive functions [16,20,21]. Recent studies confirm its high lipophilicity and excellent ability to cross the blood–brain barrier, making it a promising candidate for the treatment of neuroinflammatory disorders [8]. Sesamol is also regarded as a promising candidate for anticancer therapy or for the development of drugs containing a natural structural fragment, as it modulates the key signalling pathways involved in carcinogenesis [13,15,22,23].

In recent years, the development of novel hybrid molecules containing two or more pharmacophoric bioactive fragments has emerged as an important synthetic strategy, enabling the generation of new compounds with enhanced biological potential. Several synthetic approaches reported in the literature successfully combine Sesamol with other pharmacophoric units [24,25,26,27]. In our previous work, N-acyliminium reagents, generated in situ from benzothiazole and alkyl chloroformates engaged in a Friedel–Crafts reaction with hydroxyarenes, yielding 2-(hydroxyaryl)benzothiazolines with antibacterial properties [28]. A related Friedel–Crafts-type approach has also been employed for Sesamol by other authors, who utilized in situ generated N-Boc imines to achieve regioselective C-6 alkylation under mild aqueous conditions, affording derivatives with potential antioxidant activity [24].

In this context, the focus of our research group has been the synthesis of new hybrid molecules that combine natural phenolic compounds with a benzoazole fragment through α-amidoalkylation reaction, followed by structural elucidation and evaluation of their radical scavenging, antimicrobial, and UV-B filtering activities [28,29,30,31]. The present study expands the scope of the α-amidoalkylation reaction by modifying a lignan framework and provides an opportunity to investigate the biological potential of the newly obtained hybrid molecules.

2. Materials and Methods

2.1. Chemistry

2.1.1. General Information

All reagents, such as benzothiazole (BT), benzimidazole (BI), 5,6-dimethylbenzimidazole (5,6-DMBI), Sesamol, alkyl chloroformates, etc., and organic solvents were purchased from commercial suppliers Aldrich (Merck, Sofia, Bulgaria, EAD) and were used without further purification. Melting points were determined on a Boëtius PHMKO5 (Carl Zeiss, Jena, Germany) melting point metr and are uncorrected. IR spectra were measured on VERTEX 70 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany). High-resolution mass spectral measurements were performed on a Bruker MS spectrometer (Bruker, Billerica, MA, USA). NMR spectra were measured on Bruker Avance AV600 spectrometers (Bruker, Billerica, MA, USA) in DMSO-d₆ as solvent at 80 °C. All NMR chemical shifts (δ) are given in parts per million (ppm) with tetramethylsilane (TMS) serving as the reference standard. Coupling constants (J) are presented in hertz (Hz). Thin-layer chromatography (TLC) was conducted on 0.2 mm precoated silica gel 60 plates supplied by Merck. For chromatographic separation and purification, high-grade silica gel 60 (100–200 mesh) from Merck was utilized in column chromatography.

2.1.2. General Procedure for the Synthesis of Benzothiazole–Sesamol Hybrids 6a–c

A solution of benzothiazole (2 mmol) in dichloromethane (8 mL) was magnetically stirred at room temperature while the corresponding chloroformate (ethyl-, methyl chloroformate or 2,2,2-trichloroethyl chloroformate (Troc-Cl), 2.4 mmol) was added, followed by the Sesamol. The reaction was maintained under continuous stirring for the time specified in

Table 1. Optimized reaction conditions and yields of the obtained novel Sesamol hybrids 6a–c, 7a–c and 8a–c.

Product	X	R	R1	Ratio	Time, h	Yield, %
6a	S	H	CH3	2:1	2	90
6b	S	H	CH2CH3	2:1	5	78
6c	S	H	CH2CCl3	1:1	3	71 (95 *)
7a	N-COOR1	H	CH3	1:1	24	84
7b	N-COOR1	H	CH2CH3	1:1	24	86 (93 *)
7c	N-COOR1	H	CH2CCl3	1:1	24	87
8a	N-COOR1	Me	CH3	1:1	24	77
8b	N-COOR1	Me	CH2CH3	1:1	24	94
8c	N-COOR1	Me	CH2CCl3	1:1	24	92

* ratio of N-alkoxycarbonylabenzazolium ion:Sesamol 2:1.

, and its progress was monitored by TLC. After completion, the mixture was subjected to purification by column chromatography on silica gel, using petroleum ether/diethyl ether mixtures as the eluent. The desired compounds were isolated as white crystalline products.

2.1.3. General Procedure for the Synthesis of Sesamol Hybrids 7a–c or 8a–c Using Benzimidazole or 5,6-Dimethylbenzimidazole

To a solution of benzimidazole (1 mmol) in dichloromethane (10 mL), triethylamine (Et₃N, 1 mmol) was added as a hydrogen chloride scavenger. Subsequently, the corresponding acid chloride—ethyl chloroformate (2.4 mmol) or Troc-Cl (2.4 mmol), or alternatively methyl chloroformate (2.8 mmol), was slowly added dropwise under stirring. Immediately thereafter, Sesamol was added in equimolar amounts. The reaction mixture was stirred using a magnetic stirrer for 30 min at 0 °C and then transferred to room temperature, where it was stirred for an additional 24 h. Then, the organic phase was washed with 30 mL of hydrochloric acid–water solution (1:1, v/v), followed by 50 mL of water. The resulting organic layer was dried over anhydrous Na₂SO₄, and the solvent was removed by distillation.

The final products were obtained and isolated as white solids after recrystallization from petroleum ether.

Compound 6a Methyl 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate was isolated and purified by column chromatography on silica gel with mixtures of petroleum/diethyl ether (from 2:1 to 1:1), yield of 90%, m.p. = 162–164 °C, MW = 331.34 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 3.75 (s, 3H, -COOCH₃), 5.81 (d, J = 0.9, 1H, -OCH₂O-), 5.86 (d, J = 1.0, 1H, -OCH₂O-), 6.35 (s, 1H, -CH, Sesamol), 6.48 (s, 1H, -CH, Sesamol), 6.85 (s, 1H, *CH), 7.03 (t, J = 7.0, 1H, -CH, benzothiazole), 7.15 (t, J = 7.1, 1H, -CH, benzothiazole), 7.18 (d, J = 7.6, 1H, -CH, benzothiazole), 7.79 (d, J = 8.2, 1H, -CH, benzothiazole), 9.55 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 53.7(-COOCH₃), 62.4(*CH), 98.6(-CH, Sesamol), 101.3(-OCH₂O-), 103.7(-CH, Sesamol), 117.4(-C-Ar), 121.1(-C-Ar), 123.0(-C-Ar), 124.9(-C-Ar), 125.6(-C-Ar), 129.0(-C-Ar), 138.5(-C-Ar), 140.4(-C-Ar), 147.9(-C-Ar), 148.5(-C-Ar), 153.2(-C=O);

FTIR (KBr, cm⁻¹): 3313, 3081, 2955, 2877, 1677, 1584, 1522, 1474, 1439, 1375, 1263, 1168, 1036, 935, 753;

HRMS m/z (ESI): calcd. for C₁₆H₁₃NNaO₅S⁺ [M+Na]⁺ 354.0407; found 354.0409;

Compound 6b Ethyl 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate was isolated and purified by column chromatography on silica gel with mixtures of petroleum/diethyl ether (from 2:1 to 1:1), yield of 78%, m.p. = 179–181 °C, MW = 345.37 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 1.19 (t, J = 7.0, 3H, -COOCH₂CH₃), 4.15–4.25 (m, 2H, -COOCH₂CH₃), 5.81 (d, J = 1.0, 1H, -OCH₂O-), 5.86 (d, J = 1.1, 1H, -OCH₂O-), 6.35 (s, 1H, -CH, Sesamol), 6.48 (s, 1H, -CH, Sesamol), 6.87 (s, 1H, *CH), 7.02 (t, J = 7.0, 1H, -CH, benzothiazole), 7.15 (t, J = 8.2, 1H, -CH, benzothiazole), 7.18 (d, J = 7.6, 1H, -CH, benzothiazole), 7.80 (d, J = 8.2, 1H, -CH, benzothiazole), 9.52 (s, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 14.6(-COOCH₂CH₃), 62.3(*CH), 62.6(-COOCH₂CH₃), 98.6(-CH, Sesamol), 101.3(-OCH₂O-), 103.7(-CH, Sesamol), 117.4(-C-Ar), 121.2(-C-Ar), 122.9(-C-Ar), 124.7(-C-Ar), 125.6(-C-Ar), 129.0(-C-Ar), 138.6(-C-Ar), 140.4(-C-Ar), 147.9(-C-Ar), 148.5(-C-Ar), 152.7(-C=O);

FTIR (KBr, cm⁻¹): 3278, 2987, 2904, 1666, 1581, 1481, 1447, 1329, 1263, 1169, 1036, 933, 752;

HRMS m/z (ESI): calcd. for C₁₇H₁₅NNaO₅S⁺ [M+Na]⁺ 368.0563; found 368.0567;

Compound 6c 2,2,2-trichloroethyl 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)benzo[d]thiazole-3(2H)-carboxylate was isolated and purified by column chromatography on silica gel with mixtures of petroleum/diethyl ether (from 2:1 to 1:1), yield of 95%, m.p. = 121–123 °C, MW = 448.70 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 4.96 (d, ²J = 12.2, 1H, -COOCH₂CCl₃), 4.99 (d, ²J = 12.2, 1H, -COOCH₂CCl₃), 5.81 (d, J = 1.0, 1H, -OCH₂O-), 5.85 (d, J = 1.0, 1H, -OCH₂O-), 6.37 (s, 1H, -CH, Sesamol), 6.47 (s, 1H, -CH, Sesamol), 6.96 (s, 1H, *CH), 7.08 (t, J = 7.0, 1H, -CH, benzothiazole), 7.19 (t, J = 8.2, 1H, -CH, benzothiazole), 7.24 (d, J = 7.8 Hz, 1H, -CH, benzothiazole), 7.87 (d, J = 8.2 Hz, 1H, -CH, benzothiazole), 9.56 (s, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 62.5(*CH), 75.3(-COOCH₂CCl₃), 95.7(-COOCH₂CCl₃), 98.5(-CH, Sesamol), 101.3(-OCH₂O-), 103.7(-CH, Sesamol), 117.5(-C-Ar), 120.5(-C-Ar), 123.2(-C-Ar), 125.8(-C-Ar), 126.8(-C-Ar), 129.2(-C-Ar), 137.8(-C-Ar), 140.4(-C-Ar), 148.0(-C-Ar), 148.7(-C-Ar), 151.0(-C=O);

FTIR (KBr, cm⁻¹): 3317, 3065, 2926, 1649, 1581, 1469, 1445, 1393, 1231, 1172, 1035, 920, 743;

HRMS m/z (ESI): calcd. for C₁₇H₁₂Cl₃NNaO₅S⁺ [M+Na]⁺ 469.9394; 471.9366; found 469.9399; 471.9367;

Compound 7a Dimethyl 2-(6-hydroxybenzo-[d][1,3]dioxol-5-yl)-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 84%, m.p. = 203–205 °C, MW = 372.33 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 3.69 (s, 6H, 2x-COOCH₃), 5.88 (s, 2H, -OCH₂O-), 6.36 (s, 1H, -CH, Sesamol), 6.52 (s, 1H, -CH, Sesamol), 6.96 (s, 1H, -NCHN-), 7.01 (dd, J = 3.3, 5.8 Hz, 2H, 2x-CH, benzimidazole), 7.56 (dd, J = 3.4, 5.7 Hz, 2H, 2x-CH, benzimidazole), 9.04 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 53.1(2x-COOCH₃), 74.4(-NCHN-), 98.5(-CH, Sesamol), 101.4(-OCH₂O-), 107.4(-CH, Sesamol), 113.8(2x-CH, benzimidazole), 117.2(-C-Ar), 123.3(2x-CH, benzimidazole), 133.0(-C-Ar), 140.2(-C-Ar), 148.5(-C-Ar), 151.2(-C-Ar), 151.8(2x-C=O);

FTIR (KBr, cm⁻¹): 3369, 2954, 2883, 1722, 1695, 1502, 1443, 1382, 1310, 1278, 1081, 1028, 932, 752, 598; 546;

HRMS m/z (ESI): calcd. for C₁₈H₁₆N₂NaO₇⁺ [M+Na]⁺ 395.0850; found 395.0855;

Compound 7b Diethyl 2-(6-hydroxybenzo-[d][1,3]dioxol-5-yl)-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 93%, m.p. = 172–174 °C, MW = 400.39 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 1.16 (t, J = 7.1 Hz, 6H, 2x-COOCH₂CH₃), 4.12 (q, J = 7.0 Hz, 4H, 2x-COOCH₂CH₃), 5.87 (s, 2H, -OCH₂O-), 6.37 (s, 1H, -CH, Sesamol), 6.51 (s, 1H, -CH, Sesamol), 6.97 (s, 1H, -NCHN-), 7.00 (dd, J = 3.3, 5.8 Hz, 2H, 2x-CH, benzimidazole), 7.59 (dd, J = 3.4, 5.9 Hz, 2H, 2x-CH, benzimidazole), 9.05 (s, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 14.4(2x-COOCH₂CH₃), 62.0(2x-COOCH₂CH₃), 74.2(-NCHN-), 98.3(-CH, Sesamol), 101.4(-OCH₂O-), 107.4(-CH, Sesamol), 113.7(2x-CH, benzimidazole), 117.4(-C-Ar), 123.2(2x-CH, benzimidazole), 133.0(-C-Ar), 140.1(-C-Ar), 148.5(-C-Ar), 151.2(-C-Ar), 151.3(2x-C=O);

FTIR (KBr, cm⁻¹): 3383, 2990, 2874, 1694, 1685, 1506, 1421, 1414, 1280, 1228, 1172, 1035, 935, 749, 595, 545;

HRMS m/z (ESI): calcd. for C₂₀H₂₀N₂NaO₇⁺ [M+Na]⁺ 423.1163; found 423.1166;

Compound 7c Bis(2,2,2-trichloroethyl) 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 87%, m.p. = 140–142 °C, MW = 607.04 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 4.94 (d, ²J = 12.4, 2H, -COOCH₂CCl₃), 4.96 (d, ²J = 12.4, 2H, -COOCH₂CCl₃), 5.86 (s, 2H, -OCH₂O-), 6.32 (s, 1H, -CH, Sesamol), 6.71 (s, 1H, -CH, Sesamol), 7.01 (s, 1H, -NCHN-), 7.09 (dd, J = 5.9, 3.4 Hz, 2H, 2x-CH, benzimidazole), 7.68 (dd, J = 5.9, 3.3 Hz, 2H, 2x-CH, benzimidazole), 9.12 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 75.3(2x-COOCH₂CCl₃), 76.3(-NCHN-), 95.7(2x-COOCH₂CCl₃), 98.4(-CH, Sesamol), 101.4(-OCH₂O-), 110.0(-CH, Sesamol), 114.2(2x-CH, benzimidazole), 115.4(-C-Ar), 124.0(2x-CH, benzimidazole), 132.8(-C-Ar), 139.9(-C-Ar), 148.9(-C-Ar), 149.6(-C-Ar), 151.8(2x-C=O);

FTIR (KBr, cm⁻¹): 3449, 2959, 2922, 1728, 1694, 1504, 1448, 1407, 1285, 1231, 1193, 1040, 940, 861, 742, 575, 434;

HRMS m/z (ESI): calcd. for C₂₀H₁₄Cl₆N₂NaO₇⁺ [M+Na]⁺ 628.8795; 630.8765; found 628.8797; 630.8769;

Compound 8a Dimethyl 2-(6-hydroxybenzo-[d][1,3]dioxol-5-yl)-5,6-dimethyl-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 77%, m.p. = 193–195 °C, MW = 400.39 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 2.21 (s, 6H, 2x-CH₃, 5,6-dimethylbenzimidazole), 3.67 (s, 6H, 2x-COOCH₃), 5.87 (s, 2H, -OCH₂O-), 6.36 (s, 1H, -CH, Sesamol), 6.48 (s, 1H, -CH, Sesamol), 6.94 (s, 1H, -NCHN-), 7.36 (s, 2H, 2x-CH, 5,6-dimethylbenzimidazole), 9.03 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 19.8(2xCH₃, 5,6-dimethylbenzimidazole), 53.0(2x-COOCH₃), 74.1(-NCHN-), 98.5(-CH, Sesamol), 101.4(-OCH₂O-), 107.0(-CH, Sesamol), 115.2(2x-CH, 5,6-dimethylbenzimidazole), 117.4(-C-Ar), 130.6(-C-Ar), 130.8(-C-Ar), 140.3(-C-Ar), 148.5(-C-Ar), 151.0(-C-Ar), 151.7(2x-C=O);

FTIR (KBr, cm⁻¹): 3384, 2956, 2883, 1709, 1696, 1513, 1439, 1384, 1300, 1234, 1177, 1126, 1040, 979, 866, 752, 595, 538;

HRMS m/z (ESI): calcd. for C₂₀H₂₀N₂NaO₇⁺ [M+Na]⁺ 423.1163; found 423.1164;

Compound 8b Diethyl 2-(6-hydroxybenzo-[d][1,3]dioxol-5-yl)-5,6-dimethyl-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 94%, m.p. = 195–197 °C, MW = 428.44 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 1.15 (t, J = 7.0 Hz, 6H, 2x-COOCH₂CH₃), 2.21 (s, 6H, 2x-CH₃, 5,6-dimethylbenzimidazole), 4.10 (q, J = 7.0 Hz, 4H, 2x-COOCH₂CH₃), 5.86 (s, 2H, -OCH₂O-), 6.37 (s, 1H, -CH, Sesamol), 6.46 (s, 1H, -CH, Sesamol), 6.94 (s, 1H, -NCHN-), 7.39 (s, 2H, 2x-CH, 5,6-dimethylbenzimidazole), 8.85 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 14.4(2x-COOCH₂CH₃), 19.8(2xCH₃, 5,6-dimethylbenzimidazole), 61.8(2x-COOCH₂CH₃), 73.9(-NCHN-), 98.2(-CH, Sesamol), 101.4(-OCH₂O-), 106.9(-CH, Sesamol), 115.1(2x-CH, 5,6-dimethylbenzimidazole), 117.6(-C-Ar), 130.5(-C-Ar), 130.9(-C-Ar), 140.2(-C-Ar), 148.4(-C-Ar), 151.1(-C-Ar), 151.2(2x-C=O);

FTIR (KBr, cm⁻¹): 3422, 2982, 2915, 1720, 1673, 1514, 1445, 1411, 1294, 1230, 1175, 1040, 937, 757, 597, 432;

HRMS m/z (ESI): calcd. for C₂₂H₂₄N₂NaO₇⁺ [M+Na]⁺ 451.1476; found 451.1479;

Compound 8c bis(2,2,2-trichloroethyl) 2-(6-hydroxybenzo[d][1,3]dioxol-5-yl)-5,6-dimethyl-1H-benzo[d]imidazole-1,3(2H)-dicarboxylate was isolated by recrystallization with petroleum ether, yield of 92%, m.p. = 176–177 °C, MW = 635.09 g/mol;

¹H-NMR (600 MHz, 80 °C, DMSO-d₆, δ ppm, J Hz): 2.23 (s, 6H, 2x-CH₃, 5,6-dimethylbenzimidazole), 4.92 (d, ²J = 12.4, 2H, -COOCH₂CCl₃), 4.95 (d, ²J = 12.4, 2H, -COOCH₂CCl₃), 5.86 (s, 2H, -OCH₂O-), 6.30 (s, 1H, -CH, Sesamol), 6.66 (s, 1H, -CH, Sesamol), 6.99 (s, 1H, -NCHN-), 7.50 (s, 2H, 2x-CH, 5,6-dimethylbenzimidazole), 9.10 (brs, 1H, -OH);

¹³C{¹H}-NMR (150 MHz, 80 °C, DMSO-d₆, δ ppm): 19.8(2xCH₃, 5,6-dimethylbenzimidazole), 75.2(2x-COOCH₂CCl₃), 76.0(-NCHN-), 95.8(2x-COOCH₂CCl₃), 98.4(-CH, Sesamol), 101.4(-OCH₂O-), 109.4(-CH, Sesamol), 115.5(2x-CH, 5,6-dimethylbenzimidazole), 115.7(-C-Ar), 130.8(-C-Ar), 131.4(-C-Ar), 140.0(-C-Ar), 148.8(-C-Ar), 149.5(-C-Ar), 151.7(2x-C=O);

FTIR (KBr, cm⁻¹): 3436, 2956, 2921, 2856, 1720, 1686, 1512, 1415, 1303, 1134, 1043, 942, 751, 717, 575;

HRMS m/z (ESI): calcd. for C₂₂H₁₈Cl₆N₂NaO₇⁺ [M+Na]⁺ 656.9108; 658.9078; found 656.9111; 658.9083;

2.1.4. Radical Scavenging Activity Assays

The stock solution of all compounds was prepared in a concentration of 1 mg/mL. The working solutions were prepared by dissolving aliquot parts of the stock solution with methanol. The final concentration of IC₅₀ was calculated according to the dilution factor [30]. The radical scavenging activity of the tested compounds was evaluated over a concentration range of 1–250 ppm using a series of two-fold dilutions. The linear range of each compound differed but, in all cases, it fell within the tested concentration interval.

Mehod for DPPH Free Radical Scavenging Evaluation

The DPPH free radical scavenging capabilities were assessed as previously described by Docheva et al. [32], with 0.12 mM DPPH dissolved in methanol. The absorbance variation was quantified at 515 nm using a UV-Vis spectrophotometer. The total DPPH radical scavenging activity was assessed in triplicate during 30 min in darkness. The IC₅₀ was calculated indicating the concentration at which 50% of DPPH radicals are neutralized [30].

Method for ABTS Free Radical Scavenging Evaluation

The ABTS free radical was prepared following Re et al. [33], with several modifications. ABTS·+ was generated by dissolving 7 mM ABTS and 2.45 mM K₂S₂O₈ in deionised H₂O, with the mixture incubated in the dark at room temperature for 12–16 h before to use. A 1:1 (v/v) mixture of the reagents was diluted with methanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm for the ABTS·+ solution. The ABTS radical scavenging activity was assessed in triplicate in the absence of light at room temperature during a 10-min period. The percentage inhibition (%) of radical scavenging activity was computed in ac-accordance with the relevant equation of the approach [30].

2.1.5. In Silico Prediction of Toxicological Profiles Using QSAR Models

The application of specialized QSAR models supports the design, synthesis, and safety assessment of new synthetic compounds, including those with potential applications in the cosmetic industry [34,35,36]. This approach enables the evaluation of toxicological, ecotoxicological, and physicochemical risks associated with newly developed chemical substances. A widely used tool for such purposes is VEGA (version 1.2.3, Mario Negri Institute for Pharmacological Research, Milano (MI), Italy), a software platform for in silico analyses [34,35,36].

In the present study, the VEGA system was employed to assess potential risks such as mutagenicity, skin irritation, acute toxicity (LD₅₀), and reproductive toxicity for compounds 6a–8c using various QSAR models. Mutagenicity was evaluated using the Mutagenicity model (CAESAR, version 2.1.14) and the Mutagenicity (Ames test) model (ISS, version 1.0.3). Skin irritation was assessed using the Skin Irritation models CONCERT/Coral (version 1.0.0) and CONCERT/SarPy (version 1.0.0).

The effects on the thyroid hormone receptor alpha (TRα) were analyzed using the Thyroid Receptor Alpha Effect model (NRMEA, version 1.0.1), while the Thyroid Receptor Beta Effect model (NRMEA, version 1.0.1) was applied for the β-receptor (THRβ). Acute toxicity (LD₅₀) was predicted using the Acute Toxicity model (KNN, version 1.0.0), and reproductive toxicity was evaluated using the Developmental/Reproductive Toxicity library (PG, version 1.1.2).

2.1.6. Statistics

The data obtained were expressed as the mean ± standard error of the mean (SEM). All statistical analyses were performed using the specialized software—SPSS (version 16.0 SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Molecular Hybridization by α-Amidoalkylation of Sesamol

Molecular hybridization of Sesamol was successfully accomplished using N-acyliminium reagents generated from heterocyclic precursors, such as benzothiazole 1, benzimidazole 2, and 5,6-dimethylbenzimidazole 3, in the presence of acid chlorides—methyl chloroformate, ethyl chloroformate, and Troc-Cl (4a–c, Scheme 1).

For the α-amidoalkylation of Sesamol, the optimal reaction conditions previously established by our group for the synthesis of monoterpenoid–benzothiazole hybrids were successfully applied [30]. A reactant ratio of N-acyliminium reagent to Sesamol 2:1 was required when benzothiazole 1 was employed. Under these conditions, the reaction proceeded significantly faster, with reaction times ranging from 2 h for compound 6a to 5 h for compound (6b, Table 1). A pronounced dependence of the amidoalkylation rate leading to the formation of hybrid compounds 6a–c (see Section 2 and Section 2.1.2) on the structure of the used alkyl chloroformate was observed.

In the amidoalkylation involving N-acyliminium reagents derived from benzimidazole 2 and 5,6-dimethylbenzimidazole 3, the reactions were found to proceed successfully with an equimolar ratio of the starting reactants. However, the reaction rate was significantly slower, requiring 24 h for completion (monitored by TLC). In this case, triethylamine was used as a hydrogen chloride scavenger in the initial N-acylation step (Scheme 2), analogous to the previous reactions with resorcinol [37].

The obtained benzimidazoline-Sesamol hybrid molecules 7a–c and 8a–c (see Section 2 and Section 2.1.3) were isolated by recrystallization in yields ranging from 77% 8a to 94% 8b (Table 1). To enhance the yield of product 7b with N-acyliminium reagents generated from benzimidazole and ethyl chloroformate, the reactant ratio was adjusted to 2:1, affording 93% yield of 7b.

Synthetic modification of the natural lignan Sesamol afforded nine novel compounds by reaction of α-amidoalkylation. Spectral analyses of all newly synthesized compounds indicated substitution at the less hindered “ortho” position 6th in the Sesamol ring, clearly corresponding to already published synthetic procedures and analogues [24,27].

The structure of the synthesized novel benzazole–Sesamol hybrids 6a–8c was fully characterized spectrally using ¹H-, ¹³C{¹H}-, HSQC-NMR, FTIR, and HRMS data (original spectra are available in Supporting Information). Recording the NMR-spectra at room temperature or CDCl₃ as a solvent, previously resulted in the registration of strongly broadened or doubled signals, which made their correct interpretation impossible [28,29]. Thus, to average out the rotamers reaching adequate assignment of peaks and structure determination, the spectra were measured under heating at 80 °C. Under optimized conditions, clear signals were registered, corresponding to the chemical shifts and multiplicity for the expected structural fragments of the obtained products 6a–8c.

The 2D HSQC-NMR technique clearly visualized the position of the distinctive signals for the obtained compounds (see all reported signals in Section 2 and Supporting Information Section). For example, in ¹H-NMR spectrum of compound 7b, the triplet at 1.16 ppm corresponds to the methyl protons of the ethoxycarbonyl fragments, while the quartet at 4.12 ppm is assigned to the methylene protons. The singlet at 5.87 ppm is attributed to the two protons of the methylenedioxy fragment in the Sesamol moiety. Singlets observed at 6.37 ppm and 6.51 ppm correspond to the aromatic protons of the Sesamol, indicating substitution at 6th position.

The characteristic singlet in the range of δ = 6.85–7.01 ppm is assigned to the proton at the asymmetric C-2 carbon of the benzazoline ring. Also, the broad singlet in the range of δ = 8.85–9.56 ppm corresponds to the proton of hydroxyl group. The signals found in of ¹³C{¹H}-NMR spectra comply with all carbon atoms from the expected structures. The FTIR and HRMS spectra match precisely to those anticipated for the synthesized compounds 6a–c, 7a–c and 8a–c (see Supplementary Information Section).

3.2. In Vitro Radical Scavenging Assay

The aim of the present study was to evaluate the radical scavenging activity of the newly synthesized Sesamol hybrids (6a–8b,

Table 2. IC₅₀ values (µM) of the investigated natural and synthetic phenolic compounds determined by the DPPH and ABTS assays.

Compound	MW	DPPH	ABTS
Quercetin [29,32]	302.24	4.60 ± 0.3	48.0 ± 4.4
Rutin [29,32]	610.52	5.02 ± 0.4	95.3 ± 4.5
Thymol [30]	150.22	506 ± 15	92.5 ± 10
Carvacrol [30]	150.22	456 ± 15	52.6 ± 5.0
Sesamol	138.12	11.60 ± 1.5	46.3 ± 5.0
6a	331.34	9.20 ± 1.0	85.7 ± 7.5
6b	345.37	11.0 ± 1.0	96.7 ± 7.5
7a	372.33	94.5 ± 7.5	151.7 ± 9.5
7b	400.39	84.8 ± 7.5	133.3 ± 9.5
8a	400.39	ND *	112.9 ± 9.5
8b	428.44	ND *	105.3 ± 9.5

* ND-not determined.

). For this purpose, previously reported data from our studies on the natural compounds Quercetin, Rutin, Thymol, Carvacrol, and Sesamol were selected. In addition to their antioxidant properties, these natural compounds exhibit a range of well-established biological activities and broad therapeutic potential [29,30,32]. Quercetin and Rutin are distinguished as potent antioxidants with pronounced anti-inflammatory properties [29,32], whereas Thymol and Carvacrol possess antimicrobial and antiseptic activities [30]. In this context, Sesamol complements the group of natural compounds with its radical scavenging capacity [4,6,7], antitumor effect [13], and other biological activities [4]. The radical scavenging activity of the synthesized Sesamol hybrids was evaluated using DPPH and ABTS assays.

Comparative analysis of the DPPH assay results indicated that the flavonoids (Quercetin and Rutin) are the most potent antioxidants [29,32]. In contrast, when evaluated by the ABTS assay, Sesamol 5 exhibited an antioxidant potential (IC₅₀ = 46.3 ± 5.0 μM) comparable to that of Quercetin (IC₅₀ = 48.0 ± 4.4 μM) [29]. The antioxidant efficiency of the selected natural compounds is directly dependent on the number and spatial arrangement of their hydroxyl groups or other substituents on the aryl fragment, which determine their ability to neutralize free radicals [38]. The calculated IC₅₀ values (µM) obtained by the two radical scavenging methods are presented in Table 2.

Comparative evaluation of the antioxidant activity of the synthetic compounds showed that, in the DPPH assay, the N-acylbenzothiazoline-Sesamol hybrids 6a (9.2 ± 1.0 µM) and 6b (11.0 ± 1.0 µM) exhibited a similar radical-scavenging activity to the natural lignan Sesamol (11.6 ± 1.5 µM). These results indicate that structural modification with a benzothiazoline moiety and an alkoxycarbonyl group do not adversely affect antioxidant efficiency comparing to Sesamol in the DPPH assay. In contrast, the benzimidazoline hybrid compounds 7a (94.5 ± 7.5 µM) and 7b (84.8 ± 7.5 µM) displayed lower activity.

When evaluated by the ABTS assay, Sesamol (46.3 ± 5.0 µM) was confirmed as the most potent antioxidant among the tested natural compounds and all synthetic molecules (Table 2). The closest activities to the reference compound were again observed for compounds 6a (85.7 ± 7.5 µM) and 6b (96.7 ± 7.5 µM), followed by analogues 8b (105.3 ± 9.5 µM) and 8a (112.9 ± 9.5 µM), whereas hybrid compounds 7a and 7b exhibited the lowest antioxidant activity (IC₅₀ = 151.7 ± 9.5 µM and 133.3 ± 9.5 µM, respectively).

In summary, the antioxidant activity of the synthesized compounds was examined using the DPPH and ABTS assays. As the ABTS assay gives a more precise evaluation of structure–activity relationships, the antioxidant potential of hybrid compounds 8a and 8b was preferentially investigated by this method (Table 2).

3.3. In Silico Predictions of Sesamol Hybrids Toxicity

The VEGA platform [34,35,36] was utilized to generate in silico predictions of mutagenicity, acute toxicity (LD₅₀), skin irritation, interactions with thyroid hormone receptors α and β (TRα/THRβ), and reproductive toxicity for the newly synthesized compounds (6a–8c) as well as for the reference compound Sesamol. The VEGA platform was selected as the primary tool in the present study because it provides well-validated QSAR models for the prediction of a wide range of toxicological, ecotoxicological, and physicochemical properties of novel chemical substances. The use of such in silico approaches is essential for early-stage safety assessment in the development of new synthetic substances or cosmetic products. This concept is in accordance with the 3R principles formulated by Russell and Burch aimed at reducing animal experimentation [34,35,36,39,40].

It should be noted that the acute toxicity (LD₅₀) prediction models were not applicable to all compounds listed in

Table 3. Prediction results for the mutagenicity, skin irritation, thyroid receptor alpha effect (TRα) and beta effect (THRβ), and reproductive toxicity with the VEGA tool on compounds (6a–8c and Sesamol 5).

Compound	Mutagenicity Model (CAESAR) 2.1.14	Mutagenicity Model (ISS) 1.0.3	Skin Irritation	Thyroid Receptor Alpha Effect (TRα) and Beta Effect (THRβ)	Reproductive Toxicity
6a	Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
6b	Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
6c	Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
7a	Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
7b	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
7c	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
8a	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
8b	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
8c	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant
Sesamol	NON-Mutagenic	NON-Mutagenic	NON-Sensitizer	Inactive	NON-Toxicant

The predicted non mutagenic is indicated in bold.

. Thus, the discussion focuses on the predicted results obtained for selected structures (7a, 7b, and 8a), which were directly compared with the reference compound Sesamol. The predicted LD₅₀ values indicate that compounds 7a (3046.92 mg/kg), 8a (1873.06 mg/kg), and 7b (1388.57 mg/kg) exhibit low acute toxicity and are considerably less toxic than Sesamol (808.82 mg/kg).

According to mutagenicity predictions, compounds 7b, 7c, and the series 8a–8c were classified as non-mutagenic based on the CAESAR and ISS models. With respect to the remaining safety endpoints, all evaluated molecules 6a–8c were predicted to be non-irritant to the skin, inactive toward thyroid hormone receptors, and devoid of reproductive toxicity.

The presented data identify the newly synthesized structures (7a, 7b, and 8a) as promising hit molecules for future multifunctional applications.

4. Conclusions

In the present work, the synthesis of a series of novel hybrid compounds 6a–8c in high yields was successfully accomplished by combining the biologically active lignan Sesamol with benzothiazole 1, benzimidazole 2, and 5,6-dimethylbenzimidazole 3. The applied synthetic approach represents a logical continuation of our previous studies on naturally occurring phenolic compounds and monoterpenoids [28,29,30].

Incorporation of a benzothiazoline moiety into hybrid compounds 6a and 6b showed comparable radical scavenging activity in the DPPH assay relative to Sesamol, whereas the ABTS assay confirmed the high radical scavenging capacity of the natural compound. Overall, the benzothiazoline-based hybrid compounds were established as more potent antioxidant agents than their benzimidazoline and 5,6-dimethylbenzimidazoline analogues.

In silico tools implemented within the VEGA software platform [34,35,36] identified compounds 7b, 7c, and 8a–8c as non-mutagenic and non-irritant to the skin. QSAR models for acute toxicity were applicable only to three of the newly synthesized hybrid compounds 7a, 7b, and 8a. These compounds were predicted to exhibit a substantially improved safety profile compared with Sesamol (LD₅₀ = 808.82 mg/kg), Thymol (LD₅₀ = 970.11 mg/kg), and Carvacrol (LD₅₀ = 806.9 mg/kg). The predicted safety profile of compound 7a (LD₅₀ = 3046.92 mg/kg) is nearly identical to that of the previously synthesized aromatic analogues 4-(benzo[d]thiazol-2-yl)-2-isopropyl-5-methylphenol (LD₅₀ = 3192.7 mg/kg) and 4-(benzo[d]thiazol-2-yl)-5-isopropyl-2-methylphenol (LD₅₀ = 3191.84 mg/kg) [30]. Moreover, the predicted LD₅₀ values for compounds 8a (1873.06 mg/kg) and 7b (1388.57 mg/kg) significantly exceed that of Sesamol and closely approach the predicted value for the commercial UVB filter 2-phenylbenzimidazole-5-sulfonic acid (PBSA, LD₅₀ = 1465.52 mg/kg) [30].