Synthesis, Purification, Characterization, and ABTS Antioxidant Evaluation of Novel Azo Dyes

Abstract

The search for bioactive compounds with antioxidant properties is critical in combating oxidative stress-related diseases and advancing novel therapeutic agents. Azo dyes, traditionally used in textiles, food, and cosmetics, have recently attracted attention due to their emerging biological activities, including antioxidant potential. In this study, we synthesized and characterized 267 azo dyes derived from natural phenolic cores such as salicylic acid, syringol, and 5,6,7,8-tetrahydro-2-naphthol. Eighteen of these compounds are novel. Structural characterization was performed using NMR, UV-Vis, IR spectroscopy, and mass spectrometry. Antioxidant activity was assessed using in vitro assays with ABTS radical scavenging method. SAR analysis revealed that dyes derived from syringol and 5, 6, 7, 8-tetrahydro-2-naphthol showed the most consistent and potent antioxidant activity. Notably, azo dyes bearing fluoro and nitro substituents in the para position exhibited the lowest IC₅₀ values, highlighting the influence of electron-withdrawing groups and substitution patterns on antioxidant behavior. This work establishes a precedent for SAR-driven evaluation of azo dyes using ABTS and supports their further exploration as functional antioxidant agents in medicinal chemistry.

1. Introduction

Azo compounds are the oldest and largest class of synthetic organic dyes used in industry, characterized by the presence of the azo linkage (-N=N-) in their structure, conjugated with aromatic systems [1,2,3,4]. They have been widely used for dyeing materials such as textile fibers, polymer materials, leather and papers, and pH indicators, as well as in artificial colorants for food, and cosmetics (Scheme 1) [1,5,6,7,8,9,10,11,12,13,14,15]. Azo compounds exhibit a wide range of biological activities [16], including antimicrobial [17,18,19,20,21,22,23], anti-inflammatory, analgesic [24,25], antioxidants [26,27], enzymatic inhibition [22,28,29], antifungal [30], pesticidal, antiviral [31], and anticancer properties (Scheme 1) [27,32,33,34,35,36].

The pharmaceutical use of azo compounds began with the discovery of Prontosil’s antibacterial activity against streptococcal infections [31], followed by the development of Azulfidine (Sulfasalazine) for treating rheumatoid arthritis [37] (Scheme 2). Other azo drugs include Phenazopyridine, a local anesthetic and analgesic for cystitis and urethritis, and Balsalazide, for ulcerative colitis [38]. However, some azo dyes are carcinogenic due to their degradation into harmful compounds such as Benzidine, which is linked to tumor formation in humans and animals (Scheme 2) [39,40].

1.1. General Synthesis of Azo Dyes

The synthesis of an azo dye follows an electrophilic aromatic substitution mechanism involving two main steps: (step 1) the formation of the arenediazonium ion (diazotization) through the reaction of an aniline with the in situ formed nitrous acid (HNO₂), and (step 2) the subsequent substitution reaction with highly activated benzene rings (e.g., phenols and anilines) to form the azo linkage (-N=N-) (Scheme 3) [2,41,42].

The following Section 1.2, Section 1.3, Section 1.4 and Section 1.5 provide background on the phenols and the azo dyes derived from them that were selected for exploration in this manuscript, including their structures and various applications, to contextualize previous work and highlight our contribution to this area.

1.2. Azo Dyes Derived from Salicylic Acid (1)

Salicylic acid (1) (Scheme 4) is a phenolic phytochemical with diverse biological activities and beneficial effects on human health, naturally found in fruits, vegetables, and spices [43]. Some studies have reported the synthesis and structural characterization of these dyes using salicylic acid (1) and its isomers (m- and p-hydroxybenzoic acids) coupled with o- and p-nitroanilines [39,44,45]. Crystallographic analysis of p-nitro and aniline derivatives has further confirmed their structures [46]. Moreover, Ibrahim et al. evaluated their antibacterial and antifungal activities using p-aniline derivatives bearing NO₂, Br, Cl, and CO₂H substituents [47]. Harveer and Jasmeen assessed the antioxidant activity of salicylic acid-based azo dyes derived from various aniline compounds (R = o-NO₂, p-NO₂, H, o-CH₃, m-CH₃, p-CH₃, p-OCH₃, p-Cl) using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay [48].

1.3. Azo Dyes Derived from SYRINGOL (2)

Syringol (2), also known as 2,6-dimethoxyphenol (2,6-DMP) (Scheme 5), is a biologically active natural product with antibacterial, antioxidant, and antifungal activities [49,50,51,52]. 2,6-DMP (2) has also been utilized in the synthesis of azo compound derivatives (Scheme 5) from aniline [53], aminobenzenesulfonic acid, and aminobenzoic acids, particularly for biodegradation studies in textiles [54,55].

1.4. Azo Dyes Derived from Naphthol (3)

1-Naphthol (3a) and 2-naphthol (3b) (Scheme 6) are the most commonly used phenolic compounds as a coupling component in the synthesis of azo dyes [12,56,57,58,59,60]. Several studies have demonstrated that naphthol (3)-based azo compounds exhibit important biological properties, including antimicrobial and antioxidant activity. For example, Karthika and Rajasree synthesized azo dyes from the combination of 2-naphthol (3b) with sulphanilic acid and m-chloroaniline (Scheme 6), and reported significant antibacterial activity against six bacterial strains, with inhibition zones comparable or superior to ciprofloxacin [23]. Furthermore, 1-naphthol (3a) and 2-naphthol (3b) have been used in combinatorial chemistry laboratory experiments, where structural variations influence the color properties of azo dyes, demonstrating their versatility for dyeing fibers [6]. 1-Phenylazo-2-naphthol (Scheme 6) was used to develop thin films via thermal evaporation, which exhibited stable crystalline and optical properties suitable for dye-based optoelectronic and photonic applications [61]. However, azo dyes derived from 5,6,7,8-tetrahydro-2-naphthol (4) (Scheme 6) have not been reported in the literature, and this compound is one of the phenolic scaffolds explored in the present study.

1.5. Azo Dyes Derived from Ethylphenol (5)

Azo dyes derived from ethylphenol isomers (5) (Scheme 7) have proven particularly useful due to their distinctive chromatographic behavior, intense coloration, and potential photochemical reactivity [62,63,64,65]. In chromatography chemistry, p-, m- and o-nitrophenylazo dyes formed from p-ethylphenol (5) and nitroaniline isomers (Scheme 7) were synthetized to enable efficient separation and identification of alkylphenols in complex mixtures using thin-layer chromatography (TLC), as they exhibit characteristic R_F values and develop deep purple hues upon exposure to ammonia vapor—facilitating clear distinction among structural isomers [63,64]. Moreover, azo dyes incorporating p-ethylphenol (5) (Scheme 7) can act as synthetic intermediates in the photostimulated formation of benzotriazoles, a class of compounds widely used as UV absorbers for polymer stabilization [62]. These applications highlight the versatility of ethylphenol-derived azo dyes in both analytical and materials chemistry.

1.6. Antioxidant Activity of Azo Dyes

Various studies have synthesized novel azo compounds and explored their biological activities, particularly their antioxidant potential, which has gained increasing attention [22,66,67,68,69,70,71]. Oxidative stress, caused by free radicals, plays a crucial role in aging and the progression of various diseases [69]. Therefore, investigating the antioxidant properties of azo dyes can lead to the discovery of new compounds with potential therapeutic applications as preventive or chemotherapeutic agents against diseases by scavenging free radicals [34,66,68,69]. The presence of specific structural features, such as phenolic hydroxyl groups and heterocyclic rings containing nitrogen and oxygen, in azo dyes is often associated with enhanced antioxidant activity [66,67,68,69,71]. To date, the antioxidant activity of azo dyes has been scarcely studied. Some of the reported methods include the DPPH assay [48,66,67,69,70] and the β-carotene bleaching method [69], while no other antioxidant assays have been documented. By understanding the structure–activity relationships (SAR) of these compounds, researchers aim to develop more effective antioxidants with improved bioavailability and efficacy [66].

However, despite these promising findings, the antioxidant activity of azo dyes—particularly those derived from naturally occurring phenolic compounds—remains underexplored, presenting a valuable opportunity to further investigate their potential in this area and address a significant gap in the current literature. Our work focuses on the synthesis, characterization, and evaluation of the antioxidant activity of azo dyes containing phenolic compounds such as salicylic acid (1), syringol (2), p-ethylphenol (5), and naphthol (3a–b) (Scheme 8). The azo dyes were synthesized using aniline and substituted aromatic amines, including nitroanilines and haloanilines. The synthesized compounds were characterized by IR and NMR spectroscopy, and their antioxidant activity was evaluated using ABTS (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assays. In this work, we present a systematic study of the SAR of these compounds, focusing on how they are influenced by the type of phenol and the nature and position of the substituents on the aniline moiety.

2. Materials and Methods

2.1. General Considerations and Instrumentation

All reactions were carried out in a one neck-round bottom flask. All commercially available compounds, materials, and solvents were used as received without further purification unless otherwise noted. Reactions and column chromatography purifications were monitored by TLC using Sigma Silica Gel 60F (with or without UV indicator) plastic or aluminum sheets (0.25 mm). Unless otherwise noted, all products were purified by extraction, recrystallization, or silica gel column chromatography (depending on the compound) and fully characterized prior to use in the antioxidant assays. Proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend^TM Aeon 500 MHz NMR spectrometer using deuterochloroform relative to CDCl₃ (δ 1.50/7.26 and 77.0 for ¹H and ¹³C NMR, respectively), deuterated acetone (δ 2.05 for ¹H and 118.69 and 1.39 for ¹³C NMR), and deuterated DMSO (2.50 and 39.51 for ¹H and ¹³C NMR, respectively). Also, some compounds are analyzed in deuterated solvents (CDCl₃ and DMSO-d₆) with TMS (singlet peak in 0.00 ppm). DMSO have a peak of water in a range of 3–4 ppm in a ¹H-NMR. NMR chemical shifts (δ) are given in ppm relative to TMS and coupling constants (J) in Hz. Ultraviolet–visible (UV–Vis) spectra were collected, dissolving the compounds in DMSO, MeOH, or acetonitrile in a 96-well microplate, and the absorbance was measured in a Tecan Microplate Reader Infinite M200 Pro Infinite. All FTIR spectra for the samples was collected using a PerkinElmer Spectrum Two, UATR Two, Fourier Transform Infrared (FT-IR) Spectrophotometer. The molecular mass of selected azo dyes was determined using a Waters Xevo G2-S Q-TOF mass spectrometer. The samples were dissolved in a 10 ppm solution of HPLC-grade acetonitrile and analyzed by direct infusion into the instrument using either negative or positive electrospray ionization (ESI) mode, depending on the sample.

2.2. General Procedure for the Synthesis of Azo Dyes [72]

The aniline derivative (1.0 equiv) was diazotized by stirring with sodium nitrite (1.1 equiv), concentrated hydrochloric acid, and water at 0 °C for 30 min. In parallel, the corresponding phenol (1.1 equiv) was dissolved in 10% aqueous NaOH and stirred at 0 °C for 30 min. The freshly prepared diazonium salt solution was added dropwise to the alkaline phenol solution, maintaining the temperature at 0 °C during the addition. The resulting mixture was stirred for an additional 30 min at 0 °C to room temperature. The pH was adjusted to neutral to precipitate the crude azo product, which was collected by vacuum filtration and purified by any means: recrystallization, and extraction of column chromatography (when applied) to afford the target compound as a colored solid.

2.2.1. Synthesis of Azo Compounds (7a–c): Group 1

Synthesis of (E)-2-Hydroxy-5-((2-nitrophenyl) diazenyl) Benzoic Acid (7a)

For compound (7a), o-nitroaniline (6a) (0.7133 g, 5.1 mmol), sodium nitrite (0.3950 g, 5.6 mmol), and salicylic acid (1) (0.7501 g, 5.5 mmol) were used. The crude product was purified by recrystallization from diethyl ether (Et₂O) to afford a yellow solid (0.9838 g, 65% yield). M.P. = 206.7–211.4 °C. UV-Vis (DMSO): λ_max = 452.2 nm. ¹H NMR (500 MHz, DMSO) δ (ppm) 8.28 (s, 1H), 8.05 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.74–7.66 (m, 2H), and 7.00 (d, J = 8.8 Hz, 1H). ¹³C NMR (125 MHz, DMSO) δ (ppm) 170.80, 167.61, 146.90, 144.30, 143.48, 133.48, 130.64, 128.10, 127.39, 124.01, 118.73, 118.67, 116.17. IR (neat, cm⁻¹) 3239 (OH), 3063 (CH), 1672 (C=O), 1616 (C=C), 1522 and 1345 (NO₂), and 1454 (N=N).

The synthesis of compounds (7b) and (7c) was carried out following the same procedure and scale for (7a).

Synthesis of (E)-2-Hydroxy-5-((3-nitrophenyl) diazenyl) Benzoic Acid (7b)

Specifically, compound (7b) was obtained as an orange solid (1.105 g, 81.5% yield). M.P. = 233.1–235.4 °C. UV-Vis (DMSO): λ_max = 473.3 nm. ¹H NMR (80 MHz, DMSO) δ (ppm) 8.47 (s, 1H), 8.36 (s, 2H), 8.15 (d, J = 7.9 Hz, 2H), 8.10 (s, 1H), and 7.15 (d, J = 7.9 Hz, 1H). ¹³C NMR (125 MHz, DMSO) δ (ppm) 171.06, 165.66, 152.31, 148.69, 143.74, 131.04, 129.54, 128.85, 126.69, 124.74, 118.56, 115.33, 114.99. IR (neat, cm⁻¹) 3265 (OH), 3082 (CH), 1673 (C=O), 1575 (C=C), 1531 and 1347 (NO₂), and 1444 (-N=N).

Synthesis of (E)-2-Hydroxy-5-((4-nitrophenyl) diazenyl) Benzoic Acid (7c)

Specifically, compound (7c) was obtained as a red solid (1.227 g, 82.3% yield). M.P. = 230.1–232.0 °C. UV-Vis (DMSO): λ_max = 518.6 nm. ¹H NMR (80 MHz, DMSO) δ (ppm) 8.46 (s, 1H), 8.37 (d, J = 8.9, 2H), 8.08 (d, J = 8.9, 2H) 7.97 (d, J = 8.7, 1H), and 7.02 (d, J = 8.9 Hz, 1H). ¹³C NMR (125 MHz, DMSO) δ (ppm) 170.76, 164.49, 154.90, 147.85, 144.15, 128.89, 126.60, 124.75, 122.97, 118.39, and 113.89. IR (neat, cm⁻¹) 3206 (OH), 3096 (CH), 1668 (C=O), 1608 (C=C), 1530 and 1342 (NO₂), and 1439 (-N=N).

2.2.2. Synthesis of Azo Compounds (8a–d): Group 2

Following the general procedure (Section 2.2), the synthesis of compounds (8b) was carried out following the same scale and procedure as for (8a).

Synthesis of (E)-2,6-Dimethoxy-4-((3-nitrophenyl) diazenyl) Phenol (8a)

For compound (8a), a mixture of m-nitroaniline (6b) (0.2078 g, 1.505 mmol), sodium nitrite (0.1142 g, 1.655 mmol), concentrated HCl (12 M, 6.0 mmol), and water (0.5 mL) was added to a solution of (2,6-DMP) (2) (0.2506 g, 1.625 mmol) previously dissolved in 20% aq. NaOH (10 mL, 50 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using ethyl acetate (EtOAc) and HCl to afford an orange solid (0.3557 g, 78% yield). M.P. = 133.00–135.00 °C; UV-Vis (MeOH): λ_max = 476.0 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.70 (s, 1H), 8.29 (d, J = 7.0 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.35 (s, 2H), and 4.02 (s, 6H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 152.91, 148.91, 147.21, 145.05, 138.66, 129.77, 128.87, 124.15, 116.52, 100.96, and 56.34. IR (neat, cm⁻¹) 3433.45 (OH), 3106.00 (CH sp²), 2939.1 (CH sp³), 1610.00 (C=C), 1525.90 and 1343.48 (NO₂), and 1475.58 (N=N). MS (negative ion mode): [C₁₄H₁₃N₃O₅-H+] − calculated: 303.27, found: 302.08.

Synthesis of (E)-2,6-Dimethoxy-4-((4-nitrophenyl) diazenyl) Phenol (8b)

Specifically, compound (8b) was purified by recrystallization with ethanol (EtOH) to obtain a red solid (0.3956 g, 86%). M.P. = 144.00–145.00 °C. UV-Vis (MeCN): λ_max = 514 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 8.37 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H), 7.35 (s, 2H), and 4.02 (s, 6H). ¹³C NMR (125 MHz, Acetone) δ (ppm) 156.91, 149.18, 146.04, 142.07, 130.79, 125.70, 123.93, 102.57, and 56.74. IR (neat, cm⁻¹) 3438.65 (OH), 3062.60 (CH sp²), 2841.23 (CH sp³), 1619.40 (C=C), 1536.51 and 1314.19 (NO₂), and 1480.21 (N=N).

Synthesis of (E)-2,6-Dimethoxy-4-(phenyldiazenyl) Phenol (8c)

For compound (8c), a mixture aniline (6d) (0.1532 g, 1.645 mmol), sodium nitrite (0.1146 g, 1.661 mmol), concentrated HCl (12M, 6.0 mmol), and water (0.5 mL) was added to a solution of 2,6-DMP (2) (0.2509 g, 1.627 mmol) previously dissolved in 20% aq. NaOH (10 mL, 50 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by column chromatography using hexane and EtOAc (3:2) to afford a black liquid (0.3398 g, 80% yield). UV-Vis (MeCN): λ_max = 491.0 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.88 (d, J = 8.3 Hz, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.45 (t, J = 7.2 Hz, 1H), 7.31 (s, 2H), and 4.01 (s, 6H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 152.46, 147.13, 145.47, 137.82, 130.35, 128.97, 122.43, 100.45, and 56.31. IR (neat, cm⁻¹) 3288.83 (OH), 3013.00 (CH sp²), 2939.00 (CH sp³), 1613.38 (C=C), and 1506.95 (N=N).

Synthesis of (E)-4-((4-Chlorophenyl) diazenyl)-2,6-dimethoxyphenol (8d)

For compound (8d), a mixture of p-chloroaniline (6e) (0.1925 g, 1.509 mmol), sodium nitrite (0.1149 g, 1.655 mmol), concentrated HCl (12 M, 6.0 mmol), and water (0.5 mL) was added to a solution of 2,6-DMP (2) (0.2514 g, 1.631 mmol) previously dissolved in 20% aq. NaOH (10 mL, 50mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using EtOAc and HCl to afford a burgundy solid (0.3533 g, 80% yield). M.P. = 182.2–185.8 °C. UV-Vis (MeOH): λ_max = 494.0 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 7.83 (d, J = 8.7 Hz, 2H), 7.47 (d, J = 8.7 Hz, 2H), 7.29 (s, 2H), and 3.99 (s, 6H). ¹³C NMR (125 MHz, Acetone-d₆)) δ (ppm) 165.72, 163.74, 150.19, 150.17, 149.03, 145.75, 140.64, 125.32, 125.25, 116.93, 116.75, 101.82, and 56.67. IR (neat, cm⁻¹) 3458.81 (OH), 2,937.31 (CH sp²), 2836.20 (CH sp³), 1693.38 (C=C), 1505.03 (N=N), and (C-Cl) 757.45.

2.2.3. Synthesis of Azo Compounds (9a–c): Group 3

Synthesis of (E)-4-((2-Bromophenyl) diazenyl) naphthalen-1-ol (9a)

For compound (9a), a mixture of o-bromoaniline (6h) (0.2752 g, 0.174 mL, 1.60 mmol), sodium nitrite (0.1219 g, 1.76 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 1-naphthol (3a) (0.2515 g, 1.73 mmol) previously dissolved in 10% aq. NaOH (3.5 mL, 8.75 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using EtOAc and HCl to afford a brilliant solid red (0.2690 g, 51% yield). M.P. = 177.2–180.0 °C. UV-Vis (DMSO): λ_max = 416 nm. ¹H NMR (500 MHz, Acetone-d₆) δ (ppm) 10.05 (s, 1H), 9.04 (d, J = 8.5 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.75–7.71 (m, 1H), 7.64–7.60 (m, 1H), 7.57–7.52 (m, 1H), 7.45–7.40 (m, 1H), and 7.11 (d, J = 8.3 Hz, 1H). ¹³C NMR (125 MHz, Acetone-d₆) δ (ppm) 134.62, 134.06, 132.37, 129.33, 128.86, 126.48, 123.84, 123.33, 118.91, 116.27, and 109.41. IR (neat, cm⁻¹) 3329.57 (OH), 3051.58 (C-H), 1621.71 (C=C), 1515.99 (N=N), and 742.63 (C-Br). MS (negative ion mode): [C₁₆H₁₁BrN₂O-H+] − calculated: 327.18, found: 326.99.

Synthesis of (E)-1-((2-Nitrophenyl) diazenyl) naphthalen-2-ol (9b)

For compound (9b), a mixture of o-nitroaniline (6a) (0.2220 g, 1.61 mmol), sodium nitrite (0.1223 g, 1.774 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 2-naphthol (3b) (0.2509 g, 1.742 mmol) previously dissolved in 10% aq. NaOH (3.5 mL, 8.75 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using EtOAc and HCl to afford a red solid (0.2657 g, 56.4% yield). M.P. = 213.6-214.9 °C. UV-Vis (DMSO): λ_max = 484 nm. ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.04 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.46 (dd, J = 17.3, 8.8 Hz, 2H), 7.25 (d, J = 7.5 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.3 Hz, 1H), 6.94 (t, J = 7.7 Hz, 1H), and 6.24 (d, J = 9.7 Hz, 1H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm) 180.30, 143.89, 138.59, 136.40, 135.39, 132.57, 132.20, 129.91, 129.52, 128.73, 128.10, 126.59, 125.96, 124.43, 122.75, and 117.75. IR (neat, cm⁻¹) 3064.19 (C-H), 1619.92 (C=C), 1472.14 (N=N),and 1320.95 and 1568.39 (NO₂).

Following the general procedure (Section 2.2), the synthesis of compounds (9c) was carried out following the same scale and procedure as for (9b).

Synthesis of (E)-1-((4-Nitrophenyl) diazenyl) naphthalen-2-ol (9c)

Specifically, compound (9c) was obtained as a red solid (0.2765 g, 58.7% yield). M.P. = 247.2–248.9 °C. UV-Vis (DMSO): λ_max = 584 nm. ¹H NMR (80 MHz, CDCl₃) δ (ppm) 16.10 (s,1H), 8.32 (d, J = 7.5 Hz, 3H), 7.70 (t, J = 8.9 Hz, 6H), and 6.70 (d, J = 9.3 Hz, 1H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 180.40, 148.10, 144.71, 143.69, 133.20, 132.17, 129.96, 129.35, 128.81, 127.77, 126.53, 125.91, 122.70, and 116.76. IR (neat, cm⁻¹) 3117.7 (C-H), 1624.02 (C=C), 1495.80 (N=N), and 1326.88 and 1591.00 (NO₂).

2.2.4. Synthesis of Azo Compounds (10a–m): Group 4

Following the general procedure (Section 2.2), the synthesis of compounds (10b) and (10c) was carried out following the same scale and procedure as for (10a).

Synthesis of (E)-1-((2-Nitrophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10a)

For compound (10a), a mixture of o-nitroaniline (6a) (0.2155 g, 1.56 mmol), sodium nitrite (0.1193 g, 1.73 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 5,6,7,8-tetrahydro-2-naphthol (4) (0.2518 g, 1.76 mmol) previously dissolved in 10% aq. NaOH (10 mL, 25 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using ethyl acetate and HCl. ¹H NMR analysis confirmed the formation of compound 10a as a mixture of regioisomers in a 98:2 ratio. This was followed by column chromatography (5:1 hexane/EtOAc), affording pure compound 10a as a brilliant red solid (0.2374 g, 51.2% yield). M.P. = 123.0–128 °C. UV-Vis (DMSO): λ_max = 480 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.84 (s, 1H), 8.08 (d, J = 8.2 Hz, 2H), 7.98 (d, J = 8.2 Hz, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.74 (d, J = 8.7 Hz, 1H), 3.15 (m, 2H), 2.68 (m, 2H), and 1.86 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 157.33, 143.72, 143.02, 139.72, 139.02, 136.31, 134.17, 129.35, 129.07, 125.48, 118.69, 117.93, 29.06, 25.15, 22.96, and 22.76. IR (neat, cm⁻¹) 3376.31 (OH), 2926.07 (=C-H), 2857.34 (C-H), 1622.36 (C=C), 1567.43 and 1332.08 (NO₂), and 1455.95 (N=N). MS (positive ion mode): [C₁₆H₁₅N₃O₃+H] + calculated: 297.31, found: 298.12.

Synthesis of (E)-1-((3-Nitrophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10b)

Specifically, ¹H NMR analysis confirmed the formation of compound 10b as the major product in a mixture of regioisomers with an 82:15 ratio. Compound (10b) was then successfully isolated by column chromatography and was obtained in pure form as a red solid (0.2843 g, 61.3% yield). M.P. = 134–139 °C. UV-Vis (DMSO): λ_max = 510 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.27 (s, 1H), 8.62 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.14 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.13 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 3.27 (t, J = 5.5 Hz, 2H), 2.74 (t, J = 5.5 Hz, 2H), and 1.87 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.92, 151.72, 149.35, 140.42, 137.32, 135.20, 130.34, 129.41, 128.42, 124.47, 116.31, 115.99, 29.06, 25.49, 23.04, and 22.89. IR (neat, cm⁻¹) 3094.19 (OH), 2935.73 (=C-H), 2860.60 (C-H), 1594.31 (C=C), 1525.17 and 1345.15 (NO₂), and 1485.96 (N=N). MS (positive ion mode): [C₁₆H₁₅N₃O₃+H] + calculated: 297.31, found: 298.12.

Synthesis of (E)-1-((4-Nitrophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10c)

Specifically, ¹H NMR analysis confirmed the formation of compound 10c as the major product in a mixture of regioisomers with a 78:22 ratio. After column chromatography, the major isomer, compound 10c, was obtained as a red solid (0.2797 g, 60.3% yield) in an inseparable mixture of regioisomers in a 78:22 ratio. M.P. = 140–142 °C. UV-Vis (DMSO): λ_max = 515 nm. Major isomer 10c: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.61 (s, 1H), 8.35 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 10.7 Hz, 2H), 7.14 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 8.6 Hz, 1H), 3.23 (t, J = 5.8 Hz 2H), 2.73 (t, J = 5.7 Hz, 2H), and 1.93-1.78 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 154.25, 153.35, 148.17, 140.47, 138.16, 135.73, 129.53, 126.48, 125.16, 122.46, 116.43, 29.04, 25.41, 23.01, and 22.84. Minor regioisomer (E)-3-((4-nitrophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.39 (s, 1H), 8.35 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 10.7 Hz, 2H), 7.63 (s, 1H), 6.74 (s, 1H), 3.23 (t, J = 5.8 Hz 2H), 2.81 (m, 2H), and 1.93-1.78 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 154.41, 150.87, 148.38, 146.90, 136.75, 134.04, 129.92, 126.48, 125.07, 122.65, 117.89, 30.31, 28.50, 23.17, and 22.81. IR (neat, cm⁻¹) 3368.05 (OH), 2925.04 (=C-H), 2857.56 (C-H), 1604.24 (C=C), 1515.68 and 1345.15 (NO₂), and 1487.81 (N=N).

Synthesis of (E)-1-(Phenyldiazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10d)

For compound (10d), a mixture of aniline (6d) (0.37 mL, 0.3782 g, 4.06 mmol), sodium nitrite (0.2725 g, 3.95 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 5,6,7,8-tetrahydro-2-naphthol (4) (0.5958 g, 3.77 mmol) previously dissolved in 10% aq. NaOH (10 mL, 25 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified using liquid–liquid extraction using ethyl acetate and HCl. ¹H NMR analysis confirmed the formation of compound 10d as the major product in a mixture of regioisomers with a 72:27 ratio. Compound (10d) was then successfully isolated by column chromatography 3:1 (Hexane/EtOAc) to afford the pure compound 10d as a brilliant solid red (0.6698 g, 66.0% yield). M.P. = 63–65 °C. UV-Vis (DMSO): λ_max = 450 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.69 (s, 1H), 7.84 (d, J = 7.6 Hz, 2H), 7.51 (t, J = 7.7 Hz, 2H), 7.46 (t, J = 7.7 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 3.28 (t, J = 5.8 Hz 2H), 2.75 (t, J = 5.7 Hz, 2H), and 1.90–1.81 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.57, 150.98, 139.74, 135.61, 134.85, 130.73, 129.45, 128.82, 122.28, 115.77, 29.10, 25.47, 23.15, and 23.03. IR (neat, cm⁻¹) 3056.1 (OH), 2927.44 (=C-H), 2851.95 (C-H), 1595.9 (C=C), and 1452.23 (N=N). MS (positive ion mode): [C₁₆H₁₆N₂O+H] + calculated: 252.32, found: 253.13.

Following the general procedure (Section 2.2), the synthesis of compounds (10f) and (10g) was carried out following the same scale and procedure as for (10e).

Synthesis of (E)-1-((4-Chlorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10e)

For compound (10e), a mixture of p-chloroaniline (6e) (0.1991 g, 1.561 mmol), sodium nitrite (0.1164 g, 1.687 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 5,6,7,8-tetrahydro-2-naphthol (4) (0.2552 g, 1.772 mmol) previously dissolved in 10% aq. NaOH (10 mL, 25 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified using liquid–liquid extraction using ethyl acetate and HCl. ¹H NMR analysis confirmed the formation of compound 10e as the major product in a mixture of regioisomers with an 85:15 ratio. Compound 10e was subsequently isolated as an inseparable 96:4 mixture of regioisomers and purified by column chromatography 5:3 (Heptane/Et₂O), affording a red solid (0.2251 g, 50.3% yield). M.P. = 100.0–103 °C. UV-Vis (DMSO): λ_max = 450 nm. Major isomer 10e: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.47 (s, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 3.25 (m, 2H), 2.74 (m, 2H), and 1.85 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.58, 149.44, 139.76, 136.48, 135.95, 129.65, 129.62, 129.23, 128.95, 123.37, 123.31, 115.80, 29.06, 25.43, 23.10, and 22.97. Minor isomer (E)-3-((4-chlorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.49 (s, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 6.92 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 7.8 Hz, 1H), 3.25 (t, J = 5.9 Hz, 2H), 2.79 (m, 2H), and 1.76 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 150.49, 149.27, 144.66, 136.56, 134.89, 133.30, 129.62, 129.23, 123.31, 117.63, 30.08, 28.49, 23.26, and 22.92. IR (neat, cm⁻¹) 3349.47 (OH), 2927.01 (=C-H), 2856.34 (C-H), 1579.63 (C=C), 1485.69 (N=N), and 800.0 (C-Cl).

Synthesis of (E)-1-((2-Chlorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10f)

Specifically, ¹H NMR analysis confirmed the formation of compound 10f as the major product in a mixture of regioisomers with a 77:23 ratio. After column chromatography, the major isomer, compound 10f, was obtained as an inseparable mixture of regioisomers in a 79:21 ratio and isolated as a red solid (0.1804 g, 40.3% yield). M.P. = 140–142 °C. UV-Vis (DMSO): λ_max = 455 nm. ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.75 (s, 1H), 7.87 (m, 1H), 7.55 (m, 1H), 7.37 (m, 2H), 7.11 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 3.28 (t, J = 6.1 Hz, 2H), 2.74 (t, J = 5.6 Hz, 2H), and 1.86 (m, 4H).¹³C NMR (125 MHz, CDCl₃) δ (ppm) 152.01, 146.95, 139.88, 136.54, 135.67, 133.72, 131.37, 130.66, 128.91, 127.68, 117.70, 116.20, 29.10, 25.46, 23.15, and 23.00. Minor isomer (E)-3-((2-chlorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.74 (s, 1H), 7.87 (m, 1H), 7.64 (s, 1H), 7.55 (m, 1H), 7.36 (m, 2H), 6.75 (s, 1H), 3.28 (t, J = 6.1 Hz, 2H), 2.81 (m, 2H), and 1.85 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 150.52, 145.22, 136.86, 135.65, 134.00, 133.69, 131.54, 130.64, 130.63, 129.26, 117.89, 117.34, 30.18, 28.54, 23.30, and 22.95. IR (neat, cm⁻¹) 3367.71 (OH), 2925.87 (=C-H), 2855.83 (C-H), 1585.26 (C=C), 1487.97 (N=N), and 757.87 (C-Cl).

Synthesis of (E)-1-((3-Chlorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10g)

Specifically, ¹H NMR analysis confirmed the formation of compound 10g as the major product in a mixture of regioisomers with an 81:19 ratio. After column chromatography, compound 10g was obtained as the major isomer in an inseparable 81:19 mixture of regioisomers and was isolated as a dense liquid red paste (0.1874 g, 41.86% yield). UV-Vis (DMSO): λ_max = 466 nm. Major isomer 10g: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.39 (s, 1H) 7.83 (d, J = 7.9 Hz, 1H), 7.71 (m, 1H), 7.41 (m, 2H), 7.09 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 3.26 (t, 8.1 Hz, 2H), 2.74 (m, 2H), and 1.83 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ 151.62, 148.09, 139.91, 136.52, 135.44, 134.71, 131.61, 128.31, 124.14, 118.04, 116.12, 115.86, 29.08, 25.45, 23.12, and 22.98. Minor isomer (E)-3-((3-chlorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.38 (s, 1H), 7.84 (t, J = 8.1 Hz, 1H), 7.71 (m, 1H), 7.62 (s, 1H), 7.41 (m, 2H), 6.73 (s, 1H), 3.26 (t, 8.1 Hz, 2H), 2.80 (m, 2H), and 1.83 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ 151.83, 151.70, 150.52, 136.18, 135.55, 130.46, 130.41, 130.37, 129.37, 121.66, 121.04, 117.67, 30.15, 28.51, 23.27, and 22.92. IR (neat, cm⁻¹) 3349.47 (OH), 2927.01 (=C-H), 2856.34 (C-H), 1579.63 (C=C), 1485.69 (N=N), and 800.0 (C-Cl).

Following the general procedure (Section 2.2), the synthesis of compounds (10i) and (10j) was carried out following the same scale and procedure as for (10h).

Synthesis of (E)-1-((2-Bromophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10h)

For compound (10h), a mixture of o-bromoaniline (6h) (170 μL, 0.2687 g, 1.59 mmol), sodium nitrite (0.1171 g, 1.698 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 5,6,7,8-tetrahydro-2-naphthol (4) (0.2523 g, 1.751 mmol) previously dissolved in 10% aq. NaOH (10 mL, 25 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified using liquid–liquid extraction using ethyl acetate and HCl. ¹H NMR analysis confirmed the formation of compound 10h as the major product in a mixture of regioisomers with an 85:15 ratio. Compound 10h was subsequently isolated as an inseparable 86:14 mixture of regioisomers and purified by column chromatography 5:3 (Heptane/Et₂O) to afford a dark red solid (0.2083 g, 40.8% yield). M.P. = 107.0–110 °C. UV-Vis (DMSO): λ_max = 495 nm. Major isomer 10h: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.36 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 3.28 (t, J = 6.1, 2H), 2.75 (m, 2H), and 1.85 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.01, 146.95, 139.88, 136.54, 135.67, 133.72, 131.37, 130.66, 128.91, 127.68, 117.70, 116.20, 29.10, 25.48, 23.15, and 23.00. Minor isomer (E)-3-((2-bromophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.66 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H), 7.41 (t, J = 8.1 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.55 (s, 1H), 3.28 (t, J = 6.1, 2H), 2.69 (m, 2H), and 1.76 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.91, 148.36, 140.18, 135.71, 133.96 132.05, 129.71, 129.22, 124.28, 118.13, 117.96, 116.38, 29.33, 25.71, 23.37, and 23.23. IR (neat, cm⁻¹) 3367.75 (OH), 2925.22 (=C-H), 2849.65 (C-H), 1586.60 (C=C), 1484.80 (N=N), and 659.42 (C-Br).

Synthesis of (E)-1-((3-Bromophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10i)

Specifically, ¹H NMR analysis confirmed the formation of compound 10i as the major product in a mixture of regioisomers with a 78:22 ratio. After column chromatography, compound 10i was obtained as the major isomer in an inseparable 87:13 mixture of regioisomers and was isolated as a dense liquid red paste (0.2563 g, 50.2% yield). M.P. = 70.0–75.0 °C. UV-Vis (DMSO): λ_max = 510 nm. Major isomer 10i: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.26 (s, 1H), 8.60 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.66 (m, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 3.25 (t, J = 6.2, 2H), 2.73 (t, J = 6.0, 2H), and 1.84 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.96, 145.15, 140.06, 136.38, 135.55, 134.99, 130.47, 130.38, 129.07, 121.75, 121.08, 115.86, 29.09, 25.48, 23.11, and 22.97. Minor isomer (E)-3-((3-bromophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.19 (s, 1H), 8.64 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.66 (m, 1H), 7.64 (s, 1H), 6.73 (s, 1H), 3.25 (t, J = 6.2, 2H), 2.80 (s, 2H), and 1.84 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.70, 150.52, 136.18, 135.52, 133.55, 130.46, 130.41, 129.37, 121.66, 121.04, 117.67, 30.15, 28.51, 23.27, and 22.92. IR (neat, cm⁻¹) 3374.69 (OH), 2925.50 (=C-H), 2855.77 (C-H), 1592.00 (C=C), 1488.44 (N=N), and 677.21 (C-Br).

Synthesis of (E)-1-((4-Bromophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10j)

Specifically, ¹H NMR analysis confirmed the formation of compound 10j as the major product in a mixture of regioisomers with an 84:16 ratio. After column chromatography, compound 10j was obtained as the major isomer in an inseparable 85:15 mixture of regioisomers and was isolated as a red granular solid (0.2563 g, 60.7% yield). M.P. = 96.0–99.0 °C. UV-Vis (DMSO): λ_max = 520 nm. Major isomer 10j: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.47 (s, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 7.3 Hz, 1H), 6.78 (d, J = 8.1 Hz, 1H), 3.23 (t, J = 6.0 Hz, 2H), 2.79–2.68 (m, 2H), and 1.85–1.76 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.53, 149.45, 139.76, 136.48, 135.96, 134.87, 129.65, 128.94, 123.37, 115.78, 29.06, 25.43, 23.10, and 22.97. Minor isomer (E)-3-((4-bromophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.4 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 6.72 (s, 1H), 6.59 (d, J = 7.9 Hz, 2H), 6.58 (s, 1H), 3.24 (7, J = 6.0 Hz, 2H), 2.75 (m, 2H), and 1.92–1.78 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 150.48, 149.29, 144.68, 136.58, 136.12, 133.32, 129.62, 129.23, 123.31, 117.63, 30.08, 28.50, 23.26, and 22.92. IR (neat, cm⁻¹) 3339.45 (OH), 2927.89 (=C-H), 2853.71 (C-H), 1572.89 (C=C), 1488.19 (N=N), and 522.02 (C-Br). MS (positive ion mode): [C₁₆H₁₅BrN₂O+H] + calculated: 331.21, found: 333.04.

Following the general procedure (Section 2.2), the synthesis of compounds (10l) and (10m), was carried out following the same scale and procedure as for (10k).

Synthesis of (E)-1-((2-Fluorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10k)

For compound (10k), a mixture of o-fluoroaniline (6k) (206 μL, 0.2412 g, 2.171 mmol), sodium nitrite (0.1506 g, 2.182 mmol), concentrated HCl (2 mL, 21 mmol), and water (1 mL) was added to a solution of 5,6,7,8-tetrahydro-2-naphthol (4) (0.2696 g, 2.857 mmol) previously dissolved in 10% aq. NaOH (5 mL, 12.5 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected after purification using liquid–liquid extraction using ethyl acetate and HCl. ¹H NMR analysis confirmed the formation of compound 10k as the major product in a mixture of regioisomers with an 88:12 ratio. Compound 10k was subsequently isolated as an inseparable 90:10 mixture of regioisomers and purified by column chromatography 5:3 (Heptane/Et₂O) to afford a dark red liquid (0.2137 g, 40.96% yield). UV-Vis (DMSO): λ_max = 443 nm. Major isomer 10k: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.51 (s, 1H), 7.93 (dd, J = 8.8, 5.0 Hz, 3H), 7.29 (t, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 3.35 (t, J = 6.0 Hz, 2H), 2.84 (t, J = 15.9 Hz, 2H), and 1.95 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 163.11, 151.27, 147.58, 139.61, 135.55, 134.72, 128.88, 124.09, 123.95, 116.50, 116.31, 115.59, 29.07, 25.48, 23.12, and 23.00. Minor isomer (E)-3-((2-fluorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.53 (s, 1H), 7.93 (dd, J = 8.8, 5.0 Hz, 3H), 7.69 (s, 1H), 7.29 (t, J = 8.4 Hz, 2H), 6.82 (s, 1H), 3.35 (t, J = 6.0 Hz, 2H), 2.90 (d, J = 5.2 Hz, 2H), and 1.95 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 165.11, 150.36, 147.55, 144.23, 136.00, 133.18, 129.11, 124.02, 123.95, 117.58, 116.46, 116.28, 30.04, 28.50, 23.28, and 22.94. IR (neat, cm⁻¹) 3368.23 (OH), 2930.12 (=C-H), 2856.06 (C-H), 1604.40 (C=C), 1484.35 (N=N), and 1217.14 (C-F).

Synthesis of (E)-1-((3-Fluorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10l)

Specifically, ¹H NMR analysis confirmed the formation of compound 10l as the major product in a mixture of regioisomers with an 81:19 ratio. After column chromatography, compound 10l was obtained as the major isomer in an inseparable 87:13 mixture of regioisomers and was isolated as a dark red liquid (0.2198 g, 42.14% yield). UV-Vis (DMSO): λ_max = 564 nm. Major isomer 10l: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.44 (s, 1H), 7.63 (t, J = 6.7 Hz, 1H), 7.54 (d, J = 9.8 Hz, 1H), 7.47 (dd, J = 14.7, 7.3 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 3.26 (t, J = 6.0 Hz, 2H), 2.74 (t, J = 8.5 Hz, 2H), and 1.86 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.82, 144.93, 136.20, 133.48, 132.04, 131.98, 128.85, 124.71, 117.84, 117.61, 117.14, 116.06 29.09, 25.46, 23.15, and 23.02. Minor isomer (E)-3-((3-fluorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.42 (s, 1H), 7.57 (s, 1H), 7.54 (d, J = 9.8 Hz, 1H), 7.47 (dd, J = 14.7, 7.3 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 6.73 (s, 1H), 3.26 (t, J = 6.0 Hz, 2H), 2.80 (m, 2H), and 1.86 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 150.60, 147.06, 139.82, 135.64, 132.21, 132.15, 129.18, 124. 74, 117.27, 117.11, 116.96, 116.07, 30.15, 28.55, 23.31, and 22.96. IR (neat, cm⁻¹) 3374.51 (OH), 2926.69 (=C-H), 2856.33 (C-H), 1594.86 (C=C), 1474.10 (N=N), and 1284.12 (C-F).

Synthesis of (E)-1-((4-Fluorophenyl) diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol (10m)

Specifically, ¹H NMR analysis confirmed the formation of compound 10m as the major product in a mixture of regioisomers with an 85:15 ratio. After column chromatography, compound 10m was obtained as the major isomer in an inseparable 80:20 mixture of regioisomers and was isolated as a dark red liquid (0.2650 g, 50.8% yield). UV-Vis (DMSO): λ_max = 460 nm. Major isomer 10m: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 13.44 (s, 1H), 7.75 (m, 2H), 7.45 (m, 2H), 7.07 (d, J = 8.4 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 3.23 (t, J = 6.1 Hz, 2H), 2.73 (m, 2H), and 1.84 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 151.32, 147.87, 139.70, 135.80, 128.98, 124.18, 124.11, 116.59, 116.40, 115.75, 29.10, 25.50, 23.15, and 23.03. Minor isomer (E)-3-((4-fluorophenyl)diazenyl)-5,6,7,8-tetrahydronaphthalen-2-ol: ¹H NMR (500 MHz, CDCl₃) δ (ppm) 12.45 (s, 1H), 7.75 (m, 2H), 7.58 (s, 1H), 7.45 (m, 2H), 6.71 (s, 1H), 3.23 (t, J = 6.1 Hz, 2H), 2.73 (m, 2H), and 1.84 (m, 4H). ¹³C NMR (125 MHz, CDCl₃) δ (ppm) 150.34, 148.33, 138.55, 134.23, 127.84, 122.98, 123.45, 116.67, 116.46, 115.20, 27.99, 25.86, 23.15, and 22.70. IR (neat, cm⁻¹) 3318.21 (OH), 2926.68 (=C-H), 2854.71 (C-H), 1614.62 (C=C), 1488.72 (N=N), and 1219.08 (C-F).

2.2.5. Synthesis of Azo Compounds (11a–c): Group 5

Following the general procedure (Section 2.2), the synthesis of compounds (11b) and (11c) was carried out following the same scale and procedure as for (11a).

Synthesis of (E)-4-Ethyl-2-((2-nitrophenyl) diazenyl) Phenol (11a)

For compound (11a), a mixture of o-nitroaniline (6a) (0.5221 g, 3.78 mmol), sodium nitrite (0.3160 g, 4.158 mmol), concentrated HCl (1 mL, 12 mmol), and water (1 mL) was added to a solution of 4-ethylphenol (5) (0.5041 g, 4.09 mmol) previously dissolved in 10% aq. NaOH (8 mL, 20 mmol), maintaining the temperature at 0 °C. The mixture was stirred for 30 min, gradually allowing the temperature to rise from 0 °C to room temperature. The crude product was collected by vacuum filtration and purified by liquid–liquid extraction using EtOAc and NaOH to afford a brilliant liquid red (0.4973 g, 48% yield). UV-Vis (DMSO): λ_max = 408 nm. ¹H NMR (500 MHz, Acetone) δ (ppm) 10.87 (s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.10 (t, J = 7.7 Hz, 1H), 7.02–6.98 (m, 2H), 6.59 (d, J = 10.4 Hz, 1H), 6.19 (d, J = 8.5 Hz, 1H), 1.90 (q, J = 7.6 Hz, 2H), and 0.47 (t, J = 7.6 Hz, 3H). ¹³C NMR (125 MHz, Acetone) δ (ppm) 152.17, 144.22, 138.97, 137.13, 136.17, 136.11, 134.91, 132.29, 131.13, 125.85, 119.24, 118.95, 28.20, and 15.95. IR (neat, cm⁻¹) 3337.51 (OH), 2968.66 (CH), 2938.18 (CH₃), 1621.91 (C=C), 1345.11 and 1523.50 (NO₂), and 1493.82 (N=N).

Synthesis of (E)-4-Ethyl-2-((3-nitrophenyl) diazenyl) Phenol (11b)

Specifically, compound (11b) was obtained as a red solid (0.6501 g, 51% yield). M.P. = 84.1–86.2 °C. UV-Vis (DMSO): λ_max = 400 nm. ¹H NMR (500 MHz, ¹H NMR (500 MHz, Acetone) δ (ppm) 11.58 (s, 1H), 8.73 (s, 1H), 8.38 (t, J = 9.2 Hz, 2H), 7.89 (t, J = 8.1 Hz, 1H), 7.80 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 2.70 (q, J = 7.6 Hz, 2H), and 1.27 (t, J = 7.6 Hz, 3H). ¹³C NMR (125 MHz, Acetone) δ (ppm) 152.72, 152.49, 150.22, 138.36, 137.09, 135.63, 131.70, 129.90, 129.87, 125.82, 119.05, 116.76, 28.25, and 16.01. IR (neat, cm⁻¹) 3341.08 (OH), 3090.87 (CH), 2970.03 (CH₃), 1621.05 (C=C), 1521.33 and 1345.92 (NO₂), and 1402.83 (N=N).

Synthesis of (E)-4-Ethyl-2-((4-nitrophenyl) diazenyl) Phenol (11c)

Specifically, compound (11c) was obtained as a red solid (0.9561 g, 93% yield). M.P. = 159.7–160.9 °C. UV-Vis (DMSO): λ_max = 416 nm. ¹H NMR (500 MHz, Acetone) δ (ppm) 7.47 (d, J = 8.9 Hz, 2H), 7.23 (d, J = 8.9 Hz, 2H), 6.83 (s, 1H), 6.41 (d, J = 8.5 Hz, 1H), 6.03 (d, J = 8.5 Hz, 1H), 1.72 (dd, J = 15.2, 7.6 Hz, 2H), and 0.29 (t, J = 7.6 Hz, 3H). ¹³C NMR (125 MHz, Acetone) δ (ppm) 155.53, 152.70, 149.66, 138.74, 137.19, 136.09, 129.86, 125.80, 124.02, 119.13, 28.21, and 15.96. IR (neat, cm⁻¹) 2970.11 (CH), 2928.88 (CH₃), 1607.41 (C=C), 1520.29 and 1341.13 (NO₂), and 1493.60 (N=N).

2.3. General Antioxidant ABTS Assay

The ABTS^•+ scavenging activity was evaluated following the methods described in the literature [73,74,75], with results expressed as IC₅₀ values. This assay is based on the reduction of the ABTS radical cation (ABTS^•+) in the presence of an antioxidant, which causes a color change from light blue/green to transparent. The ABTS^•+ radical was generated combining two solutions. The first solution was obtained dissolving 38.41 mg (7 mM) of ABTS in 10 mL of distilled water. The second solution was obtained dissolving 6.62 mg (2.45 mM) of potassium persulfate (K₂S₂O₈) in 10 mL of water. Then, these solutions were combined and incubated in the dark for 24 h. Before use, the solution’s absorbance was adjusted to 0.700–1.000 at 734 nm using methanol. For the assay, 25 µL of the azo dye solution was mixed with 175 µL of ABTS^•+ solution in a 96-well microplate. The reaction mixture was incubated for 30 min at room temperature, and the absorbance was measured at 734 nm using a Tecan Microplate Reader Infinite M200 Pro Infinite (Tecan, Männedorf, Switzerland). The azo dyes were diluted in DMSO, acetonitrile, or methanol. The percentage of inhibition was calculated using the following formula to determine antioxidant capacity:

% inhibition (scavenging) = [(A_control − A_sample)/A_control] × 100

A_control: Absorbance at 30 min of the ABTS at 734 nm.

A_sample: Absorbance at 30 min of the sample with ABTS at 734 nm.

3. Results

This project focuses on the synthesis, purification, characterization, and antioxidant evaluation of azo dyes (Scheme 8), which were classified into five main groups based on the phenolic compound used: Group 1—derived from salicylic acid (1); Group 2—derived from syringol (2); Group 3—derived from naphthol (3a–b); Group 4—derived from 5,6,7,8-tetrahydro-2-naphthol (4); and Group 5—derived from p-ethylphenol (5).

3.1. Synthesis, Purification, and Characterization of Groups 7, 8, 9, 10, and 11

3.1.1. Synthesis of Salicylic Acid (1)-Derived Azo Dyes (7a–c): Group 1

Group 1 of azo dyes 7a (o-NO₂), 7b (m-NO₂), and 7c (p-NO₂), were synthesized from nitroanilines (6a–c) and salicylic acid (1) following established procedures [6,12,46,47,48] (Scheme 9). The products included known dyes such as 7a (alizarin yellow R or mordant orange R), 7b, and 7c (alizarin yellow GG or mordant yellow).

Azo dyes (7a–7c) were obtained in good yields (65–82%) and high purity after recrystallization, with melting points in narrow ranges, indicative of their purity (

Table 1. Physicochemical and antioxidant properties of azo dyes (7a–c).

Azo Dye (R)	Yield (%) 1	Melting Point (°C)	λmax (nm)	$\mathit{\upsilon }$ (N=N) (cm−1)	IC50 ABTS (mM)
7a (o-NO2)	65.1	206.7–211.4	452.2	1451	18.43
7b (m-NO2)	81.5	233.1–235.4	473.3	1444	15.26
7c (p-NO2)	82.3	230.1–232.0	518.6	1439	0.23

¹ Isolated Yield after purification by recrystallization.

). The experimental procedure was revised based on recent findings reported by T. Praneenararat et al. (2024), which indicated that the diazonium salt 2 should be added dropwise to the phenol solution to improve reaction yields [72]. Prior to implementing this modification, the yields for compounds 7a–7c averaged between 20 and 50%. After adopting this adjustment, yields increased as shown in Table 1. This revised procedure was applied to the subsequent syntheses described in the experimental section.

UV-Vis analysis revealed a clear red shift, with λ_max values ranging from 452 to 518 nm. FTIR absorption bands corresponding to the azo (-N=N-) functional group appeared between 1439 and 1450 cm⁻¹ (Table 1), consistent with the expected values for azo compounds. To the best of our knowledge, the yield, characterization, and biological properties of the (7b) (m-NO₂) azo dye had not been previously reported, in contrast to its (7a) and (7c) counterparts.

3.1.2. Synthesis of Syringol (2)-Derived Azo Dyes (8a–d): Group 2

The Group 2 azo dyes were innovative as they explored the synthesis and characterization compounds derived from syringol (2), and the coupling of nitroaniline isomers (6b–c), aniline (6d), and p-chloroaniline (6e) (Scheme 10) [53,54,55].

The synthesis of the syringol (2)-based azo dyes (8a–8d) proceeded in good yields ranging from 75 to 86% (

Table 2. Physicochemical and antioxidant properties of azo dyes (8a–8d).

Azo Dye (R)	Yield (%) 1	Melting Point (°C)	λmax (nm)	$\mathit{\upsilon }$(N=N) (cm−1)	IC50 ABTS (mM)	Purification Method
8a (m-NO2)	78	133–135	476	1450	0.30	Extraction
8b (p-NO2)	86	144–145	514	1505	0.78	Recrystallization
8c (H)	86	Liquid	491	1505	0.21	Column Chromatography
8d (p-Cl)	80	182.2–185.8	494	1505	0.41	Extraction

¹ Isolated Yield.

). The melting point analysis confirmed high purity for most of the dyes (8a–c), except for 8d (p-Cl), which, due to the presence of a chloro substituent at para position, was isolated as a liquid. The UV-Vis spectra showed λ_max between 476 and 525 nm. FTIR analysis confirmed the presence of the azo linkage with –N=N– stretching bands in the range of 1450–1505 cm⁻¹. A notable finding of this study was the variability in purification methods required for each dye derived from syringol (2). While 8b (p-NO₂) was purified by recrystallization, 8a (m-NO₂) and 8d (p-Cl) required only a liquid–liquid extraction, and (8c) was purified by silica gel column chromatography. Importantly, only three syringol (2)-based azo dyes have been previously reported in the literature [53,54,55] (Scheme 4). In this work, we report the synthesis and full characterization of three additional dyes, specifically compounds 8a (m-NO₂), 8b (p-NO₂), and 8d (p-Cl) (Scheme 6), significantly expanding the structural and functional diversity of this novel class of compounds.

3.1.3. Azo Dyes (9a–c) Derived from 1-Naphthol (3a) and 2-Naphthol(3b): Group 3

Group 3 explored the synthesis of azo dyes derived from naphthol’s (3a–b) and (3b), through the coupling of o-bromoaniline (6h) with 1-naphthol (3a), and anilines (6a) and (6c) with 2-naphthol (3b) (Scheme 11).

Compound 9a (o-Br) was obtained in 50% yield and its purity was confirmed by melting point analysis (

Table 3. Physicochemical and antioxidant properties of azo dyes (9a–c).

Azo Dye (R)	Yield (%) 1	Melting Point (°C)	λmax (nm)	$\mathit{\upsilon }$(N=N) (cm−1)	ABTS IC50 (mM)
9a (o-Br)	50	177.2–180.0	416	1515.99	0.20
9b (o-NO2)	56.4	213.6–214.9	484	1472.14	15.87
9c (p-NO2)	58.7	247.2–248.9	484	1495.80	0.47

¹ Isolated Yield.

). Dyes, 9b (o-NO₂) and 9c (p-NO₂), were synthesized in 56% and 59%, respectively. All three dyes (9a–c) were purified by liquid–liquid extraction and fully characterized by ¹H and ¹³C NMR spectroscopy. Compounds 9b (o-NO₂) and 9c (p-NO₂), both containing nitro groups, exhibited identical absorption maxima at 484 nm, suggesting enhanced π-conjugation due to the strong electron-withdrawing nature of the nitro substituents. In contrast, the bromo-substituted dye 9a (o-Br) showed a lower λ_max at 416 nm. FTIR analysis confirmed the presence of the azo bond (–N=N–) with stretching frequencies ranging from 1472 to 1516 cm⁻¹ (see Table 3). Notably, two of the synthesized dyes—9a and 9b—have not been previously reported in the literature. In contrast, dye 9c (p-NO₂) corresponds to the well-known commercially available compound Para Red or American flag red [6,60].

3.1.4. Azo Dyes (10a–m) Derived from 5,6,7,8-Tetrahydro-2-naphthol (4): Group 4

Group 4 azo dyes (10a–m) were synthesized from a novel naphthol, 5,6,7,8-tetrahydro-2-naphthol (4), which has not been previously used in azo dye synthesis (Scheme 12). Unlike 2-naphthol (3b), this compound contains only one aromatic ring, making it structurally distinct and worthy of biological investigation. Our laboratory has explored this phenol (4) in the development of steroid hormone-derived pharmaceuticals and chose to incorporate it in this study due to its novelty in azo dye synthesis. Azo dyes (10a–m) were synthesized by coupling this phenol (4) with various aniline derivatives (Scheme 12), including nitroaniline isomers (6a–6c), aniline (6d), chloroanilines isomers (6e–6g), bromoanilines isomers (6h–6j), and fluoroanilines isomers (6k–6m).

Interestingly, phenol 4 possesses two ortho positions at carbons C1 and C3 (highlighted in blue in Scheme 12), which, in principle, could both undergo azo coupling to afford a mixture of regioisomers. However, previous studies with structurally related 2-naphthol (3b) have shown that azo-coupling reactions tend to proceed regioselectively at the C1 position [76]. In our study, we similarly observed the formation of regioisomeric mixtures, with the C1-substituted azo compounds consistently being the major products in the series of dyes 10a–10m (Scheme 12 and Table 3). These findings align with the prior literature [76] and support the preferential electrophilic substitution at the C1 position of phenol 4. Regioselectivity ratios determined by ¹H NMR analysis ranged from 73:27 (10d) to as high as 98:2 (10a), with an overall average of approximately 83:17 favoring the C1-substituted isomer (Table 3). This trend was observed regardless of the electronic nature or position (ortho, meta, or para) of the substituents on the aniline coupling partners.

The regioselectivity ratio of ortho substitution at C1 vs. C3 was determined by ¹H NMR analysis (Table 3). Specifically, the hydroxyl proton (OH) of the phenolic ring appears at approximately 13.5 ppm for the C1-substituted isomer (major product) and at around 12.5 ppm for the C3-substituted minor isomer. Additionally, regioselectivity was further confirmed by analyzing the splitting pattern of the aromatic protons in the phenolic ring of the products. In the major C1-substituted isomer, the adjacent protons at positions three and four appear between 7.0 and 6.0 ppm, as well-defined doublets, each with a coupling constant of J = 8.7 Hz, approximately, which is consistent with expected ortho proton–proton coupling in a benzene ring (typically in the range of 7–9 Hz). In contrast, in the C3-substituted minor isomer, the aromatic ring contains two isolated protons at positions C1 and C5 that do not exhibit vicinal coupling, thus appearing as singlets. This distinct NMR pattern allowed for unambiguous identification of the regioisomers and confirmation of the regioselectivity of the azo-coupling reaction.

A summary of the experimental results for azo dyes (10a–10m) is presented in

Table 4. Physicochemical and Antioxidant Properties of Azo Dyes (10a-m).

Azo-Dyes (R) 1	Regioselectivity Ratio 2	Yield (%) 3	Melting Point (°C) 4	λmax (nm)	$\mathit{\upsilon }$ (N=N) (cm−1)	IC50 ABTS (mM)
10a (o-NO2)	98:2	51.2	123–128	480	1543.2	3.47
10b (m-NO2)	85:15	61.3	134–139	510	1550.3	12.44
10c (p-NO2)	78:22	60.3	140–142	515	1584.2	0.25
10d (H)	73:27	66.0	63–65	450	1563.6	3.20
10e (p-Cl)	85:15	50.3	100–103	450	1485.6	0.46
10f (o-Cl)	77:23	40.3	78–82	455	1448.2	0.92
10g (m-Cl)	81:19	41.86	liquid	466	1437.8	0.40
10h (o-Br)	85:15	40.8	107–110	495	1502.3	1.92
10i (m-Br)	78:22	50.2	70–75	510	1500.3	16.45
10j (p-Br)	84:16	60.7	96–99	520	1575.8	2.92
10k (o-F)	88:12	40.96	liquid	443	1484.4	0.31
10l (m-F)	81:19	42.14	liquid	464	1447.7	0.29
10m (p-F)	85:15	50.8	liquid	460	1418.4	0.16

¹ Major product corresponds to the C1-substituted azo compound; ² The regioselectivity of the products was determined by ¹H NMR spectroscopic analysis; ³ Isolated Yield after purification by silica gel column chromatography; ⁴ Melting points correspond to mixtures of regioisomers, except for compounds 10a,b and d, which correspond to the pure major isomer, and compound 10e, which corresponds to a 96:4 mixture of regioisomers.

following purification by column chromatography and characterization by spectroscopy. All compounds 10a–10m were rigorously purified by column chromatography, and in several cases, repurification was attempted in order to separate the regioisomers. However, only compounds 10a, 10b, and 10d could be successfully isolated from their corresponding isomers. Compound 10e was subsequently isolated as an inseparable 96:4 mixture of regioisomers. The remaining azo dyes, after chromatographic purification, displayed consistent regioisomeric ratios before and after purification. Therefore, the results reported in Table 4—including yields, melting points, UV–Vis λ_max, IR stretches, and IC₅₀ values—correspond, in most cases, to the indicated mixtures of regioisomers.

Yields ranged from 40.3% to 66.0%, with most dyes of group 4 exhibiting moderate isolated yields. In general, the melting points of solid azo dyes (10a–f) and (10h–j) exhibited narrow ranges, while a subset of azo dyes (10g, 10k–m) were obtained as viscous liquids. Compounds with para-substituted nitro and halogen groups (10c, 10j, 10m) exhibited the highest λ_max values, reaching up to 520 nm, which is consistent with increased conjugation. The characteristic azo stretching bands (–N=N–) in the FTIR spectra appeared between 1418.4 and 1584.2 cm⁻¹.

3.1.5. Azo Dyes (11a–c) Derived from P-Ethylphenol (5): Group 5

Group 5 involved the synthesis of dyes (11a–c) using p-nitrophenol (5) and the three nitroaniline isomers (6a–c). (Scheme 13).

The synthesis proceeded smoothly, yielding approximately 50% for dyes 11a (o-NO₂) and 11b (m-NO₂) after purification by extraction. In contrast, dye 11c (p-NO₂) was obtained with 93% purity without requiring extraction. The melting points varied from liquid (11a) to 160.9 °C (11c), indicating differences in crystallinity. UV-Vis analysis revealed λ_max values between 400 and 416 nm, with compound (11c) exhibiting the most bathochromic shift. All three dyes (11a–c) displayed similar FTIR azo stretching frequencies around 1493.5 cm⁻¹ for the azo functional group.

Although azobenzene derivatives are known to undergo reversible trans–cis photoisomerization upon light irradiation [36], no such behavior was evaluated in this study. The compounds were not exposed to controlled light sources or irradiated conditions to induce isomerization, as our focus was limited to the synthesis, purification, structural characterization, and antioxidant activity of the azo dyes.

3.2. Antioxidant Activity of Azo Dyes (7–11)

In this section, we discuss the antioxidant activity of the five groups of azo dyes based on two key structural features: the type of phenol used and the effect of the aniline substituents, considering both their nature and position. To assess the radical scavenging capacity of these azo compounds, we initially employed two widely accepted spectrophotometric methods: the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assays, both of which are frequently used to evaluate the total antioxidant capacity of natural and synthetic compounds [77,78]. Both assays are based on the principle that antioxidants react with a stable free radical (DPPH• or ABTS•+) leading to a decrease in the radical’s characteristic absorbance, which is measured spectrophotometrically [73,79].

The DPPH assay uses a deep violet methanolic solution of DPPH• with a strong absorbance at 517 nm, which turns pale yellow upon reduction by an antioxidant. However, in our study, this method proved ineffective due to significant spectral overlap: many of our synthesized azo dyes exhibited absorbance maxima within the 400–525 nm range, interfering with accurate measurements at 517 nm in methanol [73,74]. This limitation led us to adopt the ABTS assay, in which the ABTS•+ radical cation—generated by the reaction of ABTS with potassium persulfate—produces a stable bluish-green solution with an absorbance maximum at 734 nm in methanol. Upon interaction with antioxidants, this radical is reduced, and the solution gradually shifts to a lighter, less intensely colored state, correlating with a decrease in absorbance. Unlike DPPH, the ABTS assay avoided spectral interference with our azo dyes and yielded consistent, linear responses across all samples, allowing for the accurate and reproducible determination of IC₅₀ values in triplicate. Therefore, ABTS proved to be the most suitable method for evaluating the antioxidant activity of the synthesized azo dye series.

The following section presents a discussion of the antioxidant activity results, organized according to the phenolic groups used as key structural components in the synthesis of the azo dyes. The compounds were classified into five groups: Group 1 (salicylic acid (1)), Group 2 (syringol (2)), Group 3 (1- and 2-naphthol (3)), Group 4 (5,6,7,8-tetrahydro-2-naphthol (4)), and Group 5 (p-ethylphenol (5)). This classification allowed us to compare the bioactivity of the dyes based on the type of phenol incorporated. Subsequently, we analyze the influence of the type and position of the substituents on the aniline ring to identify SAR between both structural components and to establish trends that explain the observed variations in antioxidant capacity across the azo dye series.

3.2.1. ABTS-Based Antioxidant Evaluation of Azo Dyes Grouped by Phenol Type

Salicylic Acid-Derived Azo Dyes (7a–c): Group 1

In the ABTS radical scavenging assay, compound 7c (p-NO₂) exhibited significantly enhanced antioxidant activity (IC₅₀ = 0.23 mM), while 7a (o-NO₂) and 7b (m-NO₂) showed very weak activity, with IC₅₀ values exceeding 15 mM (see Table 1). These results suggest that para substitution plays a critical role in promoting electronic delocalization and improving radical scavenging efficiency of azo dyes (7a–c) derived from salicylic acid (1).

Syringol (2)-Derived Azo Dyes (8a–8d): Group 2

Group 2 demonstrated consistently moderate to strong antioxidant activity (Table 2). Compound 8c (unsubstituted) showed one of the best IC₅₀ values in the entire dataset (0.21 mM), followed by 8a (m-NO₂, 0.30 mM). Even dyes with less favorable substitutions exhibited IC₅₀ values below 0.40 mM. The presence of methoxy groups on the syringol (2) scaffold likely enhances electron delocalization and stabilizes radical intermediates, making this phenol core one of the most favorable for antioxidant activity among the series.

Azo Dyes Derived from 1-Naphthol and 2-Naphthol (9a–c): Group 3

The compounds in Group 3 exhibited mixed antioxidant activity (Table 3). The o-Br derivative (9a) showed good activity (IC₅₀ = 0.20 mM), while the p-nitro-substituted analogs 9c (IC₅₀ = 0.47 mM) and especially 9b (o-NO₂) displayed significantly reduced IC₅₀ activity of 15.87 mM. These results indicate that o-nitro substitution strongly suppresses radical scavenging efficiency of 2-naphthol derivatives.

Azo Dyes (10a–m) Derived from 5,6,7,8-Tetrahydro-2-naphthol (4): Group 4

Group 4 includes both the best and worst performers in terms of antioxidant activity (Table 4). Compound 10m (p-F) exhibited the highest activity (IC₅₀ = 0.16 mM), followed by 10c (p-NO₂), 10l (m-F), and 10k (o-F), with IC₅₀ values of 0.25, 0.29, and 0.31 mM, respectively. In contrast, several ortho- and meta-substituted analogs (e.g., 10a, 10i, 10h) and 10d (unsubstituted) showed poor activity, with IC₅₀ values exceeding 1.9 mM. This wide activity range highlights the high tunability of the phenol 4 scaffold depending on the diazonium partner and substitution pattern. Overall, Group 4 represents the most versatile and promising group in the series of phenols.

Azo Dyes (11a–c) Derived from P-Ethylphenol (5): Group 5

Azo dyes (11a–c) derived from p-ethylphenol (5) exhibited poor antioxidant activity (IC₅₀ > 7 mM) (

Table 5. Physicochemical and antioxidant properties of azo dyes (11a–c).

Azo Dye (R)	Yield (%) 1	Melting Point (°C)	λmax (nm)	$\mathit{\upsilon }$ (N=N) (cm−1)	IC50 ABTS (mM)
11a (o-NO2)	48	liquid	408	1493.75	12.30
11b (m-NO2)	51	84.1–86.2	400	1493.43	16.39
11c (o-NO2)	93 2	159.7–160.9	416	1493.50	7.61

¹ Isolated Yield after purification by liquid–liquid extraction; ² Purification by extraction was not necessary.

), regardless of nitro group position, suggesting that this phenol core offers limited electron delocalization. These findings, combined with the weak activity of dyes bearing o- and m-nitroanilines, confirm their low radical scavenging potential compared to a more active series like Group 4.

In summary, the comparative data indicate that the most consistent and potent antioxidant activity was observed in Group 4 (tetrahydro-2-naphthol (4)) and Group 2 (syringol (2)), particularly when these phenolic scaffolds were paired with para-electron-withdrawing substituents such as fluoro, chloro, or nitro groups (F, Cl, NO₂). In contrast, Group 1 (salicylic acid (1)) and Group 3 (naphthol (3)) exhibited more variable or limited antioxidant performance, while Group 5 (p-ethylphenol (5)) appeared to be the least favorable phenol scaffold for designing azo dyes with antioxidant activity. These results highlight the importance of carefully selecting both the diazonium substitute and the phenolic core to fine-tune the redox behavior of azo dyes, thereby optimizing their potential for biomedical or industrial antioxidant applications.

3.2.2. Antioxidant Activity Analysis Based on the Type and Position of Substituents on the Aniline Derivatives

The ABTS radical scavenging assay of the synthesized azo dyes (7–11) revealed clear trends in antioxidant capacity (IC₅₀), which depended on both the type and position of the substituents on the aniline moiety used. Figure 1 illustrates the IC₅₀ values of the 15 most active azo dyes, excluding those with IC₅₀ values above 2 mM, to facilitate a clearer discussion and interpretation of the data based on the substituents of the aniline moiety.

Overall, para-substituted compounds exhibited superior antioxidant performance compared to their ortho and meta counterparts, particularly when substituted with fluoro or nitro groups. Among the most active compounds were 10m (p-F) (IC₅₀ = 0.16 mM), 9a (o-Br) (IC₅₀ = 0.20 mM), and 8c (H) (IC₅₀ = 0.21 mM) (Figure 1), suggesting that p-fluoro and unsubstituted phenols, as well as small ortho halogens like bromo, favor electron delocalization and efficient radical quenching. Notably, p-NO₂ substituted azo dyes (7c), (10c), (8b), and (9c) also exhibited strong activity in a range of IC₅₀ 0.23–0.78 mM (Figure 1), confirming that para-nitro groups—despite their electron-withdrawing nature—may enhance resonance stabilization of the resulting radical species.

In contrast, m- and o-nitro-substituted dyes such as 7a (o-NO₂) (IC₅₀ = 18.43 mM), 11a (o-NO₂) (12.30 mM), 9b (o-NO₂) (15.87 mM), and 10b (m-NO₂) (12.44 mM) showed extremely poor activity (Figure 1). This strongly suggests that substitution at the meta or ortho position with bulky and strongly electron-withdrawing groups (EWGs) disrupts conjugation with the azo linkage, limiting antioxidant efficiency.

A similar trend was observed with halogenated dyes. p-chloro and p-bromo compounds such as 8d (p-Cl) and 10e (p-Cl) exhibited IC₅₀ values of 0.41 and 0.46 mM, respectively, while ortho and meta-analogs were generally weaker (e.g., 10f (o-Cl) = 0.92 mM, 10i (m-Br) = 16.45 mM). This reinforces the idea that substitution pattern directly affects electronic delocalization and, in turn, radical scavenging. Interestingly, fluoro-substituted dyes across all positions (e.g., 10k (o-F), 10l (m-F), and 10m (p-F)) performed relatively well (IC₅₀ around 0.16–0.31 mM µg/mL) (Figure 1), likely due to fluoro’s small size and moderate electronegativity allowing extended delocalization without introducing steric congestion.

In summary, the data strongly support that EWGs at the para position, particularly F and NO₂, enhance antioxidant activity of our azo dyes, whereas their placement at the ortho or meta positions dramatically reduces efficacy. Fluorinated azo dyes show consistent and favorable radical scavenging, and sterically unhindered, para-substituted systems offer the best performance.

The findings reported in this study are pioneering, as this work presents a SAR analysis of the antioxidant activity of azo compounds using the ABTS assay. Our results reveal novel trends in the behavior of these azo dyes in relation to the type of phenol and the nature and position of the substituents on the aniline moiety. These trends do not necessarily align with those previously reported in the literature for non-azo phenolic compounds, thus establishing an important precedent for understanding the unique antioxidant properties of azo dye structures (7–11).

The literature reports consistently show that EWGs, such as nitro (-NO₂), tend to decrease antioxidant capacity, particularly when positioned ortho or para to the hydroxyl group [80]. In contrast, electron-donating groups (EDGs), especially hydroxyl (-OH) and methoxy (-OCH₃), significantly enhance antioxidant activity [81]. Regarding halogen substituents (e.g., F, Cl, Br), their influence appears to be modest and position-dependent [80,81,82,83]. Nonetheless, halogens in the meta position consistently reduce antioxidant performance due to the absence of conjugative stabilization [80].

Some of these SAR align with the trends observed in our study, where azo dyes bearing phenolic cores with EWGs in the ortho and meta positions—particularly nitro groups—showed diminished radical scavenging activity compared to those substituted with halogens. Surprisingly, the azo dye containing the most electronegative halogen, fluoro, exhibited the highest antioxidant activity, along with other azo dyes bearing para-substituents such as chloro and nitro.

4. Conclusions

This study reports the synthesis, purification, and characterization of 26 azo dyes, including 18 novel compounds—particularly those derived from syringol (2) (Group 2) and 5,6,7,8-tetrahydro-2-naphthol (4) (Group 4)—which exhibited the most consistent and potent antioxidant activity overall. Notably, this is the first time that azo dyes (10a–10m) derived from 5,6,7,8-tetrahydro-2-naphthol (4) have been reported, as well the study of those regioselective towards the azo-coupling reaction, representing a significant contribution to the field. In addition, we report for the first time the synthesis and bioactivity evaluation of syringol (2)-based azo dyes containing nitro substituents in ortho, meta, and para positions.

To our knowledge, no prior study has explored the antioxidant activity of azo dyes using the ABTS radical scavenging assay in the context of a SAR analysis. Our work systematically correlates antioxidant capacity (IC₅₀) with both the phenolic core and the type and position of substituents on the aniline moiety. One of the most remarkable findings of this study is that azo dyes bearing EWGs, such as fluoro and nitro in the para position, demonstrated the highest ABTS radical scavenging activity—an observation that contrasts with prior reports on non-azo phenols. These results establish a novel SAR profile specific to azo dye structures and suggest that the redox behavior of these compounds is influenced by electronic and positional factors in unique ways. Overall, this pioneering work sets the foundation for future investigations into the pharmacological potential of these novel dyes.