Antioxidant activity of novel phenolic compounds bearing basic substituents—synthesis and insights from molecular modeling

Compounds containing phenolic groups are found in many different areas of life. The inclusion of phenolic groups within pharmaceutical compounds endows them with reactive functionality of an acidic nature. One such group of bioactive compounds are drugs in which the phenolic group forms part of the structure of beta-2-agonists, which are used in the acute treatment of obstructive airway diseases (Čižmáriková et al., 2020; Yang et al., 2021). Furthermore, it is found in the structure of the main opium alkaloid morphine (Lugo et al., 2002; Mercadante, 2010) used as an analgesic, mainly in palliative care, as well as in the tetrahydroisoquinoline alkaloid ecteinascidin used to treat patients with advanced soft tissue sarcoma where previous drugs have been unsuccessful (Zewail-Foote & Hurley, 1999; Held-Warmkessel, 2003; Lau et al., 2005). The phenol group is also present in the structure of the anthelmintic niclosamide (Barini et al., 2018), as well as in tetracycline (Agwuh & MacGowan, 2006; Nelson et al., 2011) and ansamycin (Wrona et al., 2008; Skrzypczak et al., 2022) antibiotics. Propofol, a non-barbiturate intravenous anesthetic of phenolic nature, is extensively utilized in surgical settings for the induction and maintenance of sedation (Condello et al., 2021; Ferrier et al., 2022; Čižmáriková et al., 2023). The phenol group is found in the structure of the hormonal contraceptive estrogen (Stuenkel et al., 2015), the thyroid hormone thyroxine (Mondal et al., 2016; Dutta et al., 2021) and its synthetic form levothyroxine (Sue & Leung, 2020; Jonklaas, 2022), the stress hormone adrenaline (Gough & Nolan, 2018; Gil-Jardine et al., 2022), and the neurotransmitters dopamine (Lerner at al., 2021; Mirabella et al., 2023) and serotonin (Fuller, 1992; David & Gardier, 2016; Liu et al., 2020). A large and important group of natural polyphenolic compounds are the flavonoids (Panche at al., 2016). They exhibit a broad spectrum of biological effects and are candidate compounds in the development of various medical agents in therapy as well as prevention of disease (Hasnat et al., 2024; Stachelska et al., 2025; Ullah et al., 2020). In particular, they are powerful antioxidants (Pietta, 2000; Zahra et al., 2024). Other fields of interest include infectious diseases (Liu et al., 2025; Cushnie & Lamb, 2005), cancer (Kopustinskiene et al., 2020; Mir et al., 2024), inflammation (Al-Khayri et al., 2022), and neurodegenerative diseases (Cheng et al., 2025).

The antiradical (Bendary et al., 2013; Čižmáriková et al., 2020), antimicrobial (Taguri et al., 2006; Cueva et al., 2010), antituberculosis (Mazlunat et al., 2019; Islam et al., 2021), and chelating activity of various substituted phenols were investigated (Olennikov et al., 2014; Rahim et al., 2016). Depending on the substituents present, phenols exist in so-called open and cyclic forms. Typically, this is a cyclic form that is associated with the presence of a hydrogen bond. The strength of a hydrogen bond can be studied by conformational analysis, and one of the parameters for determining its strength is the distance between the atoms forming the hydrogen bond (Rademacher et al., 2005; Lithoxoidou & Bakalbassis, 2005).

A significant number of phenolic compounds have been shown to possess substantial antiradical activity, which has been associated with their function as powerful antioxidants and anti-inflammatory agents important in the prevention of chronic diseases such as cancer, cardiovascular disease, diabetes, and neurodegenerative disorders. Both the bioactivity and toxicity of phenols are affected by the number and arrangement of their phenolic groups (Calliste et al., 2001; Amouar et al., 2009; Kadoma et al., 2010). Furthermore, phenols have the ability to form complexes with metal ions (Hider et al., 2001; Fernandez et al., 2002).

The amine moiety is also considered a potent pharmacophore, for example, in mitochondria targeting agents with anticancer activity (Gao et al., 2025; Skrzypczak et al., 2021).

The aim of this study was to prepare novel phenolic derivatives bearing substituted amino moieties (compounds I–XV, Table 1) via a two-step synthesis. The antioxidant capacity of the products was then evaluated using two different methods.

Table 1.

Substance	R1	R2
I	CH2CH3	N(CH3)2
II	CH3	N(CH2CH3)2
III	CH3	NHCH(CH3)2
IV	CH2CH3	NHCH(CH3)2
V	CH3	NHCH2CH(CH3)2
VI	CH3	NHC(CH3)3
VII	CH3
VIII	CH2CH3
IX	CH3
X	CH2CH3
XI	CH3
XII	CH2CH3
XIII	CH3
XIV	CH3
XV	CH2CH3

The melting point of the synthesized substances was determined using a Kofler block (HMK, Franz Küstner, Germany) and is uncorrected. The purity of the prepared compounds was verified by thin-layer chromatography (TLC) using Silufol^® UV 254 silica gel plates (Merck) and a mixture of propan-1-ol:diethylamine solvents in a ratio of 9:1 v/v. A UV lamp was used for detection. Elemental analysis was performed using a FLASH 2000 Organic Elemental Analyzer (Thermo Scientific, Waltham, Massachusetts, USA).

Infrared spectra were measured using a reflective technique with an ATR adapter with ZnSe crystals and a Nicolet 6700 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA). Ultraviolet spectra were measured using a GENESYS 10S spectrophotometer in the wavelength range of 200–400 nm. The concentrations of the measured base and salt solutions of the prepared aryloxyaminopropanols in methanol were approximately 0.2 mol.m⁻³. ¹H-NMR spectra were measured on a Varian Gemini 2000 Spectrometer (Varian Inc., Palo Alto, USA) with an operating frequency of 300 MHz for ¹H NMR. Tetramethylsilane was used as an internal standard. Deuterated solvents were used to dissolve the samples, namely, deuterated chloroform, methanol, DMSO, and water. Chemical shifts (δ) are expressed in ppm. Signal multiplicity is expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet.

The aim of this work was to prepare 3-(aminomethyl)-4-(hydroxy)phenyl]alkylketones by means of a two-step synthesis (Scheme 1), except for [4-hydroxy-3-(piperidinomethyl)phenyl]methylketone which was synthesized by Mannich reaction. The preparation of some of the compounds has been described previously, using various synthetic procedures, that is, II (Borthakur et al., 1984; Aljohani et al., 2019), III (Rastogi et al., 1992), VII (Aljohani et al., 2019), IX (Borthakur et al., 1984; Aljohani et al., 2020), XI (Borthakur et al., 1984; Aljohani et al., 2019; Reddy et al., 2008; Dai et al., 2017), and XIV (Boesen, 2003). The products contain a phenolic group and a basic group in position 2 relative to the phenolic group. The compounds under study (I-XV) (see Table 1) were prepared from [3-(chloromethyl)-4-(hydroxy)phenyl] alkylketones as starting compounds. These were obtained from 4-hydroxyphenylalkylketones by an electrophilic substitution reaction of paraformaldehyde with hydrochloric acid (Čižmáriková et al., 2002). During the first stage, [3-(chloromethyl)-4-(hydroxy)phenyl]alkylketones were synthesized by electrophilic substitution reaction in a 55–62% yield. These are halogen derivatives with increased reactivity, which can result in diminished yields due to competing pathways, such as the formation of enamines, imines, tertiary amines, or quaternary ammonium salts. The reaction was carried out in a low-polarity environment (benzene or toluene) and in the presence of an excess of aliphatic amines (dimethylamine, diethylamine, isopropylamine, isobutylamine, tert-butylamine) and heterocyclic amines (pyrrolidine, morpholine, azepane, 4-methylpiperazine), achieving a yield of 30–60%. The products are white solids with melting points between 39 and 94 °C, soluble in organic solvents. The purity of the prepared compounds was checked using thin-layer chromatography with a propan-1-ol and diethylamine mobile phase in a 9:1 ratio, and the R_f values were calculated. Finally, the IR, UV, and ¹H-NMR spectra of the products were measured.

Scheme 1.

Synthesis of the target compounds (R¹/R² see Table 1)

In the IR spectra, valence vibration bands were identified at 2500–3300 (νOH), 1665–1685 (νC=O), 1583–1607 (νC=C), and 1251–1286 (νC-N). In UV spectra, the prepared compounds exhibited two absorption bands corresponding to transitions at λ₁ 218–220 nm (log ɛ₁ 2.52 m².mol⁻¹) and λ₂ 268–318 nm (log ɛ₂ 2.53–3.12 m².mol⁻¹). In the ¹H-NMR spectra of the prepared compounds, proton signals of the phenolic group were identified within the range of 10.50–11.51 ppm. In addition, signals of protons of the aromatic nucleus, a singlet signal of CH₂N at 3.63–4.11 ppm, signals of protons of the amine moiety, and signals of CH₂ and CH₃ groups of the acyl and of the aliphatic side chain were also identified.

The compound 4-hydroxy-3-piperidinomethylphenylmethyl ketone (IX) was synthesized in a study by Borthakur et al. (1984). However, the authors only isolated it in the form of oil. For this reason, a Mannich reaction with a 60% yield was conducted. In addition to IR, UV, and NMR spectra, the structure was confirmed by MS spectra.

To expand knowledge of the potential biological effects of synthesized substances, primary screening of their antioxidant activity was performed on selected final compounds and intermediates of their synthesis. The DPPH method was used to screen antioxidant activity, as well as the ABTS method, which is more sensitive to this type of substance than the DPPH assay. The ABTS method is based on the decolorization of the blue-green solution of the activated radical ABTS^•+ after its reaction with an antioxidant substance. The results of antioxidant activity measurements for all substances tested are shown in Table 2. The investigated compounds demonstrate significantly higher oxidation activity values than corresponding compounds with alkoxymethyl groups (Čižmáriková et al., 2020). The antioxidant activity determined by the DPPH method ranged from 2.9% to 6.2%, and that determined by the ABTS method for compounds with tertiary nitrogen ranged from 84.2% to 99.1%. The value obtained using this method for compound IV with secondary nitrogen (NH-CH(CH₃)₂) was 33.5%. The intermediate product [3-(chloromethyl)-4-(hydroxy)phenyl] methylketone (EACh) showed higher activity using both the DPPH method (4.3%) and the ABTS method (8.2%), whereas 4-hydroxyphenylmethylketone (4-OHaceto) showed low activity (0.94%) using only the ABTS method. Compounds with alkoxy groups have been shown to possess significantly lower antioxidant activity values, ranging from 0.55% to 12.58% by the DPPH method and from 0.92% to 28.14% by the ABTS method (Čižmáriková et al., 2020). Interestingly, the presence of nitrogen atoms also had a positive effect on increased antioxidant activity in beta-blocker-type substances (Čižmáriková et al., 2020, Čižmáriková et al., 2021). The distinguishing characteristic of these compounds is the formation of a hydrogen bond between the phenolic group and the nitrogen atom in the side chain, which was investigated using conformational analysis. The Sybyl program was employed to obtain 3D models of molecules and calculate the energy of a larger number of bonds around which rotation is possible and conformers that arose by rotation around simple bonds. The number of conformers for each compound was determined by the volume and spatial arrangement of substituents, as well as the number of bonds around which rotation is possible. Of the final compounds selected for analysis, the compound featuring an isobutyl substituent (V) yielded the highest number of conformers, a consequence of its flexible chain. The compound with a tert-butyl group (VI) gives the least conformers due to its conformational rigidity. When performing conformational analysis, the most suitable conformers were subjected to complete optimization using the semi-empirical AM1 method (Table 3). From the resulting conformers obtained in this way, the most optimal conformers were selected and their geometric parameters characterizing the hydrogen bond, distances between atoms, and hydrogen bond angle were measured. In compounds with branched carbon chains V, VI and a nitrogen atom, there was the possibility of forming a hydrogen bond between the hydrogen atom of the amino group and the oxygen atom of the phenol group NH⋯O or a hydrogen bond between OH⋯N. In compound (V) with an isobutyl group, given the geometric parameters and heat of formation of the given conformer, the formation of an NH⋯O intramolecular hydrogen bond is more likely than in the conformer with an intramolecular hydrogen bond between the hydrogen atom of the OH group and the amino nitrogen. The assumption is that the length (NH⋯O) is equal to 0.237 nm, the heat of formation ΔH is 332.48 kJ/mol, and the angle between the NH⋯O atoms is 120.17°. These findings are consistent with data in the literature (Nunes et al., 2007, Grabowski, 2007), where the formation of intramolecular hydrogen bonds NH⋯O is considered more likely. The energy of the hydrogen bond was approximated by the difference between the heat of formation of the conformation with the hydrogen bond and the same conformation without the hydrogen bond. When compounds with a built-in heterocycle (VII, IX, XIII, and XIV) are compared based on hydrogen bond length and bond energy, small differences in hydrogen bond energy are observed, with their energy being lower than that of compounds with an aliphatic chain (V and VI) (see Table 3).