Synthesis, redox properties and antibacterial activity of hindered phenols linked to heterocycles

Article abstract of DOI:10.3390/molecules25102370

A series of benzotriazole, cyclic amides and pyrimidine derivatives, containing 2,6-di-tert-butyl-phenol fragments, were synthesized. The redox properties of obtained compounds were studied using the cyclic voltammetry on a platinum electrode in acetonitrile. The oxidation potentials of all substances were comparable to those of BHT. The obtained compounds were tested for their antibacterial activity, and N-(2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl)isatin (32 μg/mL) exerted good activity against Staphylococcus aureus.

Full text of DOI:10.3390/molecules25102370

molecules
Article
Synthesis, Redox Properties and Antibacterial
Activity of Hindered Phenols Linked to Heterocycles
Vladimir N. Koshelev, Olga V. Primerova *, Stepan V. Vorobyev and Ludmila V. Ivanova
Department of organic chemistry and petroleum chemistry, Gubkin Russian State University of Oil and Gas,
Leninsky av. 65, 119991 Moscow, Russia; Koshelev.V@gubkin.ru (V.N.K.); Vorstepan@yandex.ru (S.V.V.);
Ivanova.l@gubkin.ru (L.V.I.)
* Correspondence: primerova92@yandex.ru; Tel.: +7-926-329-63-27
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Paola Di Donato and Brigida Silvestri
Received: 21 April 2020; Accepted: 19 May 2020; Published: 20 May 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of benzotriazole, cyclic amides and pyrimidine derivatives, containing
2,6-di-tert-butyl-phenol fragments, were synthesized. The redox properties of obtained compounds
were studied using the cyclic voltammetry on a platinum electrode in acetonitrile. The oxidation
potentials of all substances were comparable to those of BHT. The obtained compounds were tested for
their antibacterial activity, and N-(2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl)isatin (32
exerted good activity against Staphylococcus aureus.
µg/mL)
Keywords: hindered phenols; heterocycles; antioxidants; antibacterial activity
1. Introduction
Oxidative destruction plays an important role in biochemical processes. Free radicals and reactive
oxygen species induce the oxidative damage of cell membranes, lipids, proteins, and DNA repair
system breakdown that is connected with many degenerative diseases, such as cancer, atherosclerosis,
Alzheimer’s disease [
aiming to scavenge free radicals and inhibit oxidative stress processes, because of their ability to break
the chain process of free radical oxidation [ ]. They either naturally generated in situ (endogenous
antioxidants), or are externally provided through foods (exogenous antioxidants) [ ]. Sterically
hindered phenols have been used as antioxidant agents for more than half a century [ ]. One of
the best known representatives of this class of antioxidant agents is 2,6-di-tert-butyl-4-methylphenol
(butylated hydroxytoluene, BHT), primarily used in food, cosmetics and pharmaceuticals [ 10],
and its derivatives are widely used in the oil industry [11 12]. The efficacy of 2,6-di-tert-butylphenols as
1–3]. The main defense mechanism of the body is the use of antioxidants,
4
5
6,7
5,8–
,
inhibitors of oxidative destruction of hydrocarbons is determined by the nature of ortho-alkyl groups
and the group in the para-position of the aromatic ring, which affects the stability of the phenoxyl
radicals generated during oxidation [13–15].
In recent years, a great deal of effort has been devoted to finding multipotent antioxidants,
substances which combine antioxidant activity and other pharmacological effects: anti-inflammatory,
anticoagulant, anticarcinogenesis activities [16].
Among them, a large number of pharmacologically active substances exhibiting a wide variety
of chemotherapeutic activity have been synthesized on the basis of sterically hindered phenols and
various heterocyclic compounds.
Anti-inflammatory activity is most often found in compounds of these series [17–19]. The drugs
containing BHT or 2,6-di-tert-butylphenol and heterocycles used to treat inflammatory conditions,
which include tazofelone, darbufelone, prifelone, and tebufelone, are available commercially [20].
Additionally, many heterocycles, containing 2,6-di-tert-butylphenol fragment, that are active
against various types of cancer, have been found. Experimental data report on the cytotoxicity against
Molecules 2020, 25, 2370; doi:10.3390/molecules25102370
www.mdpi.com/journal/molecules

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tumor lines of epithelioid carcinoma of the cervix uteri (M-Hela) and breast adenocarcinoma (MCF-7)
of 2,6-diaminopyridine derivatives [21]. Symmetric S-BHT derivatives containing 1,3,4-thiadiazole
fragments showed antioxidant potency and potential to be useful and promising selective agents
against breast and colon cancer [22].
It is known that hindered phenols, which are part of natural extracts and synthetic particles, exhibit
antibacterial properties. [23–27]. However, the number of antibacterial agents based on heterocycles
with hindered phenol fragments is not so wide. In recent years, metal complexes of phthalocyanines and
azomethines containing fragments of 2,6-di-tert-butylphenol have been found to exhibit anti-bacterial
activity against Gram-negative bacteria E. coli [28–30].
Moreover, 2,3-dihydropyrrolo [1,2-a]imidazole I showed high antimicrobial activity when tested
for biocidal activity in jet fuels [31]. Compound II was found to have high protistocidal activity,
10–15 times greater than that of Baycox, which is used for the treatment of coccidiosis in poultry [32].
1,3,4-Thiadiazole III (Figure 1) [33] exhibited a high degree of protection in extending the lifespan of
nematodes following S. aureus infection.
Figure 1. Examples of substances, exhibiting antibacterial activity.
To sum up, multipotent antioxidants with antibacterial properties are of great interest; in particular,
for the food industry, because both effects are highly desirable to keep foods as fresh as possible.
Promising results showed above prompted us to investigate the chemical diversity of heterocyclic
substituents of hindered phenol, which have not yet been explored. The most up-to-date rational design
approach for multipotent antioxidants is to connect an antioxidant group with other pharmacophores [16].
Heterocycles, which we incorporated into the structure of antioxidant: benzotriazole, phthalimide, isatin,
succinimide, 2,4-dihydroxypyrimidine, 3,4-dihydropirimidin-2(1H)-one, are the basis of antibacterial
agents [34–39] and, in addition, have an NH group available for modification in the cycle. The antioxidant
properties can both improve and decrease quite significantly with the introduction of a substituent
in the phenol structure [40]; therefore, it is necessary to study the antioxidant properties of the
obtained compounds.
The aim of this study was to synthesize new multipotent antioxidants, coupling 2,6-di-tert-
butylphenol and a series of heterocycles. The compounds synthesized were assayed for antioxidant
and antibacterial activity.
2. Results and Discussions
2.1. Synthesis
The preparation of the target compounds is outlined in the following; Schemes 1–3.
Initially, 2-bromo-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone
1 was conveniently obtained from
2,6-di-tert-buthylphenol through acylation with acetyl chloride [41] and subsequent bromination [42].

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Scheme 1. Scheme of preparation of bromo-acetophenon 1.
Scheme 2. Scheme of preparation of compounds 2–7.
Scheme 3. Scheme of preparation of 3,4-dihydropirimidin-2(1H)-one 9a and thiones 9b–c.
The further synthesis entailed an alkylation heterocycles: benzotriazole, phtalimide, isatin,
succinimide, uracil and 5-methyluracil (thymine) by bromo-acetophenon in DMF with K2CO3.
The reaction was carried out at 100 ◦C for an hour, with the exception of interaction
with uracil and
1
1
methyluracil: in these cases, the reaction gave black resin, and optimal conditions to obtain compounds
6 and 7 were found to be 70 ◦C and 4 h.
1
In the H NMR spectra of compounds
2
–5
, all peaks, corresponding to phenol fragment are
observed: the singlet peak near 5.83 ppm is attributed to the O–H of the hindered phenol, peaks at
7.82–7.88 ppm with the integration of two protons was assigned to the two symmetrical aromatic
ring protons, singlet peaks at 4.93–5.17, corresponding to CH2-N protons, and there are no peaks of
NH-protons of starting cyclic imides and benzotriazole.

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During the alkylation of uracil and its derivatives, the attack of the alkylating reagent is possible in
two directions: N-1 atom and N-3 atom [43]. The structure of compounds and was also confirmed
with HMBC and HSQC spectra (Figure 2 for and Supplementary Material for ). The spectrum
6
7
7
6
contains correlation peaks between C-3 and H-6, C-2 and H-6, C-6 and H-2. This indicates that
bromo-acetophenone 1 attacked the N-1 atom of 5-methyluracil and uracil.
Figure 2. NMR 1H-13C HMBC spectra of 7.
To expand the range of pyrimidine derivatives, a number of 3,4-dihydropyrimidine (thiones)ones
with a hindered phenol fragment were obtained by Biginelli reaction, according to Scheme 3.
The most common catalysts reported for the Biginelli reaction are mineral acids (H2SO4 [44],
HCl [45], TFA [46]), Lewis acids (CoCl2, ZnCl2 [47]), and other catalytic systems: FeCl3 + HCl [48],
TMSCl + NaI [49]. However, in our hands, the products were not obtained when all these conditions
were employed. Previously, researchers from the Vorozhtsov Novosibirsk Institute of Organic
Chemistry have synthesized compound 9a in the presence of FeCl3 6H2O as a catalyst [50]. These
•
conditions (with some correction) turned out to be suitable for the synthesis of compounds 9a–c with
yields 55–64%.
1
In the H NMR spectra of compounds 9a
–
c, there are no peaks in the region of 10.4 ppm
corresponding to the proton of the aldehyde group. Peaks at 7.02–7.66 ppm corresponding to the
proton of the phenolic group are present in all spectra. Peaks of NH protons in the spectrum of
derivatives with C=S groups are shifted to a higher-field (7.2–8 ppm), in comparison with derivatives
with the C=O group (7.8–9 ppm).
2.2. Redox Properties
The antioxidant activity of phenolic compounds is revealed, mainly due to their redox properties,
which can play an important role in absorbing and neutralizing free radicals, quenching reactive oxygen
species, such as singlet and triplet oxygen, or decomposing peroxides [51]. Absolute values of oxidation

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potentials characterize the reducing ability of antioxidant agents and therefore the manifestation of
their antioxidant effect [ ]. Cyclic voltammetry is one of the most robust methods for studying the
6
redox properties of compounds, as well as evaluating their antioxidant ability [52–54].
Cyclic voltammogrames of synthesized compounds exhibit one irreversible two-electron peak
corresponding to the phenolic fragment oxidation in the anodic region. Table 1 shows the oxidation
potentials of the studied compounds. In the case of substances
more positive region compared to the BHT, which indicates the enhancement of the electron-windrowing
character of the substituent in the para-position to the hydroxy group. The compound , which contains
a succinimide fragment, has the highest value of Epa (oxidation potential). Epa of the other compounds
2–7 the peak potentials are shifted to a
5
are close to those of BHT. The oxidation potentials of compounds 9a–c are lower than those of BHT, for
two possible reasons.
Table 1. Oxidation potentials (Epa) of compounds 2–7, 9a–c.
№
2
3
4
5
6
7
9a
9b
9c
BHT
Epa, V
1.54
1.57
1.52
1.68
1.61
1.58
1.49
1.48
1.46
1.52
Firstly, compounds 2–7 contain the electron-withdrawing carbonyl group, which decreases the
efficacy of phenolic fragment [55]. Notably, the oxidation potential, however, is still comparable to
those of BHT. Secondly, the CH group of the pyridine ring affects the oxidation potential; it contributes
to a shift in the electron density due to the effect of hyperconjugation with the benzene ring [56].
Furthermore, the products of oxidation of compounds 9a–c can be stabilized by the heterocyclic ring,
connected directly to the phenolic one [57].
Furthermore, slight differences in the oxidation potentials of compounds 9a, 9b and 9c are
observed. Compounds 9b and 9c have exhibited lower value of anodic potential peak compared to
compound 9a, due to the strongest electron-withdrawing effect of the ester group in the pirimidin ring,
compared to the acetyl group in compounds 9b and 9c. This can be explained by the fact that the
acceptor effect of the thionic group of the pyrimidine ring is weaker than that of the carbonyl group.
At the same time, the electron-withdrawing effect of the acetyl group is weaker than of the ester group,
which explains the lowest oxidation potential of compound 9c.
2.3. Antibacterial Activity
The antibacterial activity of the synthesized compounds was tested against five strains of
pathogenic bacteria: Staphylococcus aureus (SA), Escherichia coli (EC), Klebsiella pneumonia (Kp),
Acinetobacter baumannii (Ab), Pseudomonas aeruginosa (Pa). The investigation was carried out in
the international laboratory CO-ADD based in Institute of Molecular Biology, University of Queensland
(Brisbane, Australia). Test substances were administered at a concentration of 32 µg/mL. Substance
may be considered active if inhibition values equal to or above 80% of inhibition for either replicate
(n = 2 on different plates). The antibacterial activity for all studied compounds is given in Table 2.
The product of isatin alkylation
there is no explicit correlation between antioxidant activity and antibacterial capacity of investigated
substances, we can suppose that the activity of compound is linked with isatin moiety, which is
known to possess antibacterial activity [58 59]. This compound was selected as “hit” and tested for
4 exerted good activity against Staphylococcus aureus. Although
4
,
the minimum inhibitory concentration (MIC), cytotoxicity against human embryonic kidney cells and
haemolytic activity. It was shown that compound 4 does not inhibit the growth of bacteria in smaller
concentrations as well as the one of human cells.

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Table 2. Inhibition of bacterial growth, %.
Sample
Sa
Ec
Kp
Pa
Ab
2
3
4
5
6
7
9a
9b
17.42
16.81
96.6
−8.94
20.06
14.9
4.05
7.56
14.45
20.34
13.96
3.38
10.32
10.36
9.46
8.08
10.92
0.04
13.97
18.62
13.6
18.08
−9.39
18.75
12.56
41.59
2.47
−2.04
2.53
7.29
6.12
8.94
−2.72
−1.13
0.53
19.13
26.31
−5.78
−11.19
−3.54
3. Materials and Methods
3.1. General Information
The NMR 1H and 13C spectra of solutions in DMSO-d6 and CDCl3 were recorded on a Bruker
AM-300 spectrometer (Karlsruhe, Germany). All experiments were performed according to the
standard methods of Bruker. Chemical shifts were reported relative to Me4Si. The values of SSCCs
are given in Hz. The mass spectra were recorded on an MS-30 Kratos device (Eu, 70 eV). A peak
of the molecular ion M+ was observed for all synthesized compounds. The melting points of the
compounds obtained were determined in an open capillary. Elemental analysis was carried out using
Elemental analyzer Vario micro cube (Langenselbold, Germany). The course of reactions and purity of
the compounds obtained was monitored by TLC on silica gel plates in a 10:1 benzene-ethanol (10:1
chloroform-ethanol also can be used) solvent system.
3.2. Synthesis and Analytical Data of Preparated Compounds
3.2.1. Synthesis of Compounds 2–5
A solution of bromoacetophenone
1 (3 mmol) with benzotriazole, phthalimide, isatin or
succinimide (3 mmol) and anhydrous K2CO3 (9 mmol) was stirred for 1 h at 100 ◦C in 15 mL of DMF.
After cooling, it was poured into water and the precipitate was filtered off. The precipitate was boiled
first in hexane, then in water. The resulting crystals were recrystallized from an appropriate solvent.
2-(1H-1,2,3-benzotriazol-1-yl)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)ethanone 2. White solid. Yield 71%. m.p.
166–167 ◦C (EtOH:H2O 3:1). NMR 1H (DMSO-d6, , ppm, 3JHH, Hz): 1.43 (s, 18H, 2(CH3)3); 6.50 (s, 2H,
δ
CH2N); 7.41 (t, 1H, 5-C CH in benzotriazole, J = 8.1); 7.52 (t, 1H, 6-C CH in benzotriazole, J = 8.1);
7.79 (d, 1H, 4-C CH in benzotriazole, J = 10.3), 7.88 (s, 2H, Ar), 8.06 (d, 1H, 7-C CH in benzotriazole,
J = 8.06). NMR13C (DMSO-d6,
δ, ppm): 30.4; 35.0; 54.2; 111.4; 118.3; 119.5; 124.2; 126.0; 126.2; 127.6;
134.5; 138.8; 145.6; 160.3; 191.5. Elemental analysis found: C, 72.15; H, 7.65; N, 11.41. Calculated for
C22H27N3O2: C, 72.30; H, 7.45; N, 11.50.
2-[2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl]-1H-isoindole-1,3-(2H)–dione
3
. White solid. Yield
, ppm, JHH, Hz): 1.41 (s, 18H, 2(CH3)3);
5.17 (s, 2H, CH2N); 7.82 (s, 2H, Ar in phenol); 7.90 (m, 4H, Ar in phtalimide). NMR13C (DMSO-d6,
◦
1
3
67%. m.p. 188–189 C (benzene). NMR H (DMSO-d6,
δ
δ,
ppm): 30.4 (2(CH3)3); 35.1; 44.7 (CH2N); 109.6 (phtalimide); 123.8; 126.0 (phtalimide); 132.1; 135.2; 138.9
(phtalimide); 160.2 (Ar phtalimide); 168.1 (2C=O in imide); 191.3 (C=O). Elemental analysis found: C,
73.16; H, 7.08; N, 3.51. Calculated for C24H27NO4: C, 73.26; H, 6.92; N, 3.56.
1-[2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl]-1H-indole-2,3-dione
4. Yellow solid. Yield 60%. m.p.
195–196 ◦C (EtOH:H2O 2:1). NMR 1H (DMSO-d6, , ppm, 3JHH, Hz): 1.41 (s, 18H, 2(CH3)3); 5.08 (s, 2H,
δ
CH2N); 5.83 (bs, 1H, OH); 6.68 (d, 1H, 7-C CH in isatin, J = 7.3); 7.05 (t, 1H, 5-C CH in isatin, J = 8.1);
7.46 (t, 1H, 6-C CH in isatin, J = 8.1); 7.56 (d, 1H, 4-C in isatin, J = 7.3); 7.83 (s, 2H, Ar). NMR13C

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(DMSO-d6, δ, ppm): 30.1 (2(CH3)3); 34.5; 46.1 (CH2N); 109.6; 110.8; 117.7; 123.9; 125.4; 126.1; 136.5;
138.4; 151.2; 158.5 (10 Ar); 159.6 (C=O 2-C in isatin); 181.6 (C=O 3-C in isatin); 196.7 (C=O). Elemental
analysis found: C, 73.18; H, 7.05; N, 3.38. Calculated for C24H27NO4: C, 73.26; H, 6.92; N, 3.56.
1-[2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl]pyrrolidin-2,5-dione
259–260 ◦C (acetonitrile). NMR 1H (CDCl3,
, ppm, 3JHH, Hz): 1.47 (s, 18H, 2(CH3)3); 2.86 (s, 4H,
2CH2 in imide); 4.93 (s, 2H, CH2N); 5.83 (bs, 1H, OH); 7.85 (s, 2H, Ar). NMR13C (CDCl3,
, ppm): 28.4
5. White solid. Yield 74%. m.p.
δ
δ
(2CH2 in imide); 30.1 (2(CH3)3); 34.4; 44.5 (CH2N); 109.6; 125.9; 136.9; 159.3 (4 Ar); 176.8 (2C=O in
imide); 189.2 (C=O). Elemental analysis found: C, 69.37; H, 7.97; N, 3.95. Calculated for C20H27NO4:
C, 69.54; H, 7.88; N, 4.05.
3.2.2. Synthesis of Compounds 6, 7.
A solution of bromoacetophenone 1 (3 mmol) with uracil or 5-metyluracil (3 mmol) and anhydrous
K2CO3 (9 mmol) was stirred for 4 h at 70 ◦C in 15 mL of DMF. After cooling, reaction mixture was
poured into water and the precipitate was filtered off. The precipitate was refluxed in hexane, and then
dissolved in acetone. The unsolved part was filtered off, and acetone was evaporated under residue
pressure to give crystalline product, which was recrystallized from an appropriate solvent.
1-(2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl)pyrimidine-2,4(1H,3H)-dione
m.p. 192–193 ◦C (EtOH:H2O 3:1). NMR 1H (DMSO-d6, , ppm, 3JHH, Hz): 1.43 (s, 18H, 2(CH3)3); 5.26
(d, 1H, CH in uracil, J = 3.9); 5.61 (d, 1H, CH in uracil, J = 4.0); 7.53 (s, 1H, OH); 7.77 (s, 2H, Ar); 11.32 (s,
NH). NMR13C (DMSO-d6,
, ppm): 30.4 (2(CH3)3); 35.0; 53.7 (CH2N); 101.2; 125.6; 138.8; 146.9; 151.6
6. Yellow solid. Yield 52%.
δ
δ
(C=O); 160.6; 164.4 (C=O); 192.0 (C=O). Elemental analysis found: C, 67.15; H, 7.22; N, 7.68. Calculated
for C20H26N2O4: C, 67.02; H, 7.31; N, 7.82.
1-[2-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl]-5-methylpyrimidin-2,4 (1H, 3H)-dione
Yield 58%. m.p. 145–146 ◦C (EtOH:H2O 2:1). NMR 1H (DMSO-d6, , ppm, 3JHH, Hz): 1.43 (s, 18H,
2(CH3)3); 1.77 (s, 3H, CH3); 5.22 (s, 2H, CH2N); 7.42 (s, 1H, CH-N); 7.77 (s, 2H, Ar); 7.99 (s, 1H, OH);
11.31 (s, 1H, NH). NMR13C (DMSO-d6,
, ppm): 12.4 (CH3); 30.41 (2(CH3)3); 35.0; 53.6 (CH2N); 108.7;
7. Yellow solid.
δ
δ
125.6; 126.3; 138.9; 142.6; 151.6 (C=O); 160.0; 164.9 (C=O); 192.4 (C=O). Elemental analysis found: C,
67.82; H, 7.74; N, 7.50. Calculated for C21H28N2O4: C, 67.72; H, 7.58; N, 7.52.
3.2.3. Synthesis of Compounds 9a–c.
A mixture of aldehyde
compound (5.2 mmol), was dissolved in 13 mL of ethanol. Then, FeCl3
to the mixture as a catalyst. The reaction proceeded for 18 h; its progress was monitored by TLC.
After cooling the reaction mixture, a precipitate formed which was filtered under vacuum, washed
with benzene, and recrystallized from an appropriate solvent.
8
(4 mmol), urea or thiourea (8 mmol) and appropriate dicarbonyl
6H2O (4 mmol) was added
•
Ethyl-4-(3,5-di-tert-butyl-4-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate 9a
White solid. Yield 55%. m.p. 227–228 ◦C (EtOH:H2O 4:1). NMR 1H (CDCl3, , ppm, 3JHH, Hz): 1.11 (t,
3H, CH3, J = 7.1 Hz,); 1.41 (s, 18H, t-Bu); 2.27 (s, 3H, CH3); 4.02 (q, 2H, CH2, J = 7.1 Hz,); 5.12 (s, 1H,
CH); 5.80 (s, 1H, OH); 7.03 (s, 2H, Ar); 7.66 (s, 1H, NH); 9.78 (s, 1H, NH). NMR13C (CDCl3,
, ppm):
.
δ
δ
14.2; 18.6; 30.2; 34.3; 55.7; 59.9; 102.0, 123.1; 134.6; 136.0; 145.8; 153.4; 162.3, 165.9. Elemental analysis
found: C, 68.11; H, 8.48; N, 7.13. Calculated for C22H32N2O4: C, 68.01; H, 8.30; N, 7.21.
Ethyl-4-(3,5-di-tert-butyl-4-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylate 9b
White solid. Yield 61%. m.p. 235–237 C (EtOH:H2O 2:1). NMR H (CDCl3, δ, ppm, JHH, Hz):
.
◦
1
3
1.11 (t, 3H, CH3, J = 7.1 Hz,); 1.34 (s, 18H, t-Bu); 2.37 (s, 3H, CH3); 4.12 (q, 2H, CH2, J = 7.1 Hz,);
5.24 (s, 1H, CH); 5.35 (s, 1H, OH); 7.01 (s, 2H, Ar); 7.38 (s, 1H, NH); 8.13 (s, 1H, NH). NMR13C (CDCl3,

Molecules 2020, 25, 2370
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δ
, ppm): 14.2; 18.2; 30.2; 3.3; 56.2; 60.3; 103.6; 123.4; 128.4; 133.2; 136.2; 142.4; 153.8; 165.5. Elemental
analysis found: C, 65.15; H, 8.08; N, 6.77; S, 7.82. Calculated for C22H32N2O3S: C, 65.31; H, 7.97; N,
6.92; S, 7.92.
1-(4-(3,5-Di-tert-butyl-4-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)ethan-1-one 9c
White solid. Yield 64%. m.p. 204–206 ◦C (EtOH:H2O 4:1). NMR 1H (DMSO-d6, , ppm, 3JHH, Hz):
1.41 (s, 18H, t-Bu); 1.48 (s, 3H, CH3); 5.26 (s, 1H, CH); 7.05 (s, 1H, OH); 7.36 (s, 2H, Ar); 7.73(s, 1H, NH);
8.03 (s, 1H, NH). NMR13C (DMSO-d6,
, ppm): 19.4; 30.1; 30.2; 34.4; 56.8; 111.7; 123.7; 128.4; 132.2;
.
δ
δ
136.6; 141.6; 154.1; 195.8. Elemental analysis found: C, 67.44; H, 8.25; N, 7.36; S, 8.47 Calculated for
C21H30N2O2S: C, 67.34; H, 8.07; N, 7.48; S, 8.56.
3.3. Redox Properties
Cyclic voltammetry (CV) was carried out in the argon atmosphere in a three-electrode cell using an
Ecotest-VA potentiostat (Moscow, Russia). The working electrode was a stationary platinum electrode
S = of 3 mm2; the auxiliary electrode was a platinum plate (S = 18 mm2). The reference electrode was
(Ag/AgCl/KCl), with a waterproof diaphragm. The potential sweep rate was 0.2 V
s–1. Et4NClO4 (99%,
·
Acros) for the supporting electrolyte was twice recrystallized from the aqueous ethanol and dried for
48 h in vacuum at 50 ◦C. The concentration of the studied compounds was 5 mM.
3.4. Biological Activity
Antimicrobial screening was performed by CO-ADD (The Community for Antimicrobial Drug
Discovery), funded by the Wellcome Trust (London, UK) and The University of Queensland (Brisbane,
Australia). The antimicrobial activities were evaluated against cultures of Staphylococcus aureus ATCC
43300, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii
ATCC 19606, and Pseudomonas aeruginosa ATCC 27853. Compounds were plated as a 2-fold dose
response from 32 to 0.25 µg/mL (or 20 to 0.156 uM), with a maximum of 0.5% DMSO, final in assay
concentration. The growth inhibition of all bacteria was determined measuring absorbance at 600 nm
(OD600), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition
was calculated for each well, using the negative control (media only) and positive control (bacteria
without inhibitors) on the same plate as references. The minimum inhibitory concentration (MIC) was
determined following the CLSI guidelines, identifying the lowest concentration at which full inhibition
of the bacteria has been detected. Full inhibition of growth has been defined at <20% growth (or >80%
inhibition), and concentrations have only been selected if the next highest concentration displayed full
inhibition (i.e., 80–100%) as well (eliminating ‘singlet’ active concentration).
Growth inhibition of HEK293 cells was determined measuring fluorescence at ex:530/10 nm and
em:590/10 nm (F560/590), after the addition of resazurin (25 µg/mL final concentration) and incubation
at 37 ◦C and 5% CO2, for an additional 3 h. The fluorescence was measured using a Tecan M1000
Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well,
using the negative control (media only) and positive control (cell culture without inhibitors) on the
same plate as references.
4. Conclusions
In this study, the synthesis, redox properties and anibacterial activity of nine
2,6-di-tert-buthylphenol derivatives linked to different heterocycles are described. For uracil and
thymine, it is revealed by 2D-NMR spectroscopy that substitution takes place at the N-1 atom.
Electrochemical studies of obtained compounds show, that the nature of the substituent in the
4th position of the phenolic ring strongly influences on the antioxidant activity. Compounds
2
–
5
with an electron-withdrawing linker between heterocycle and phenol showed a shift in the
oxidation potential to the positive region, but its values are still comparable to those of BHT.

Molecules 2020, 25, 2370
9 of 12
Dihydropyrimidines 9a
–
c, in which the heterocycle was connected with phenol, directly showed lower
value of oxidation potential and higher antioxidant abilities. Studies of antibacterial properties showed
that N-(2-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-oxoethyl)isatin is active against Staphylococcus
aureus in concentration 32 g/mL, with a low toxicity against human cells. Thus, compound was
4
µ
4
found to be the multipotent antioxidant.
Supplementary Materials: 1H-NMR and 13C-NMR spectra of obtained compounds.
Author Contributions: O.V.P., concept, performing chemical synthesis and purification, spectroscopy experiments,
writing manuscript. S.V.V., analyzing spectroscopy data, writing manuscript. L.V.I., electrochemical experiments,
analyzing data, writing manuscript. V.N.K., concept, supervision, writing manuscript. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).