Synthesis and study of new phenolic antioxidants with nitroaromatic and heterocyclic substituents

Article abstract of DOI:10.1007/s11172-018-2127-2

New polyfunctional aromatic, nitroaromatic, and heterocyclic compounds linked to the 2,6-di-tert-butylphenol moiety via –NH–, –C(O)NH–, –S–, or–C=N– spacers were synthesized. These structures provide intramolecular charge transfer (ICT) and exhibit antioxidant activity. The structures of the new compounds were established by X-ray diffraction. The novel compounds were evaluated for antioxidant activity using the DPPH assay. The presence of the 2,4,6-trinitrophenyl moiety in combination with the –NH– spacer leads to a considerable increase in the antioxidant activity of 2,6-di-tert-butylphenols. These compounds are also weak lipoxygenase inhibitors. The results of this study provide an opportunity to search for new types of antioxidants with ICT.

Full text of DOI:10.1007/s11172-018-2127-2

Russian Chemical Bulletin, International Edition, Vol. 67, No. 4, pp. 712—720, April, 2018
712
Synthesis and study of new phenolic antioxidants with nitroaromatic
and heterocyclic substituents
O. V. Mikhalev, D. B. Shpakovsky, Yu. A. Gracheva, T. A. Antonenko, D. V. Albov, L. A. Aslanov, and E. R. Milaeva
M. V. Lomonosov Moscow State University,
1, Build. 3, Leninskie Gory, 119991 Moscow, Russian Federation.
Fax: +7 (495) 932 8846. E-mail: mov@med.chem.msu.ru
New polyfunctional aromatic, nitroaromatic, and heterocyclic compounds linked to the
2,6-di-tert-butylphenol moiety via —NH—, —C(O)NH—, —S—, or—C=N— spacers were
synthesized. These structures provide intramolecular charge transfer (ICT) and exhibit anti-
oxidant activity. The structures of the new compounds were established by X-ray diffraction.
The novel compounds were evaluated for antioxidant activity using the DPPH assay. The pres-
ence of the 2,4,6-trinitrophenyl moiety in combination with the —NH— spacer leads to
a considerable increase in the antioxidant activity of 2,6-di-tert-butylphenols. These compounds
are also weak lipoxygenase inhibitors. The results of this study provide an opportunity to search
for new types of antioxidants with ICT.
Key words: 2,6-di-tert-butylphenol, antioxidant activity, DPPH assay, intramolecular charge
transfer, X-ray diffraction analysis.
Oxidative destruction plays a significant role in bio-
chemical processes, oxidative stress being a cause of many
body pathologies. Natural phenolic products are involved
in the antioxidant defense system in the living organism.1
The corresponding phenoxyl radicals are key intermediates
responsible for antioxidant activity of such compounds
that are able to interact with reactive free radicals.
2,6-Dialkylphenols mimic natural systems, in particu-
lar, tocopherols (e.g., vitamin E), tyrosine, or thyroxine.
These compounds have low toxicity and, consequently,
are of interest as biologically active compounds for med-
icine and also as industrially used inhibitors of radical
chain oxidation of organic molecules.2 Sterically hindered
phenols also meet requirements for effective antioxidants
and serve as inhibitors of oxidation of various organic
substrates.3 2,6-Di-tert-butyl-4-methylphenol (ionol,
commercial index E321) is the most widely utilized syn-
thetic antioxidant. The efficacy of 2,6-di-tert-butylphenols
as 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. Groups of different nature and having
different structures, including metal-containing moieties,
can serve as substitutes at the para-position.4—6 A search
for new metal-containing phenolic antioxidants for pre-
vention of oxidative stress-related carcinogenesis in the
cell is currently underway.5,7
cyclooxygenase inhibitors and exhibit pronounced anti-
inflammatory activity.8
The living cells include nucleotides, porphyrins, flavins,
quinones, and peptides as essential low-molecular-weight
components, all of which being characterized by a relatively
low electronic excitation energy, low ionization potentials,
and high electron affinity. Therefore, these reactive com-
pounds can be involved in charge transfer processes.
The energy transfer in the electron transport chain is
associated with the formation of charge transfer complexes
between purines that act as electron donors and riboflavin
(vitamin В2) as an electron acceptor.9
Organic compounds with intramolecular charge transfer
(CICT), comprising donor and acceptor moieties separated
by a group of atoms or a spacer, are of considerable interest
due to specific physicochemical, optical, and biological
properties. The spatial arrangement of the interacting
Various N,S,O-containing amine and amide derivatives
bearing the 2,6-di-tert-butylphenol moiety belong to
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 0712—0720, April, 2018.
1066-5285/18/6704-0712 © 2018 Springer Science+Business Media, Inc.

New phenolic antioxidants
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
713
donor and acceptor groups and the character of charge
transfer are determined by the spacer (bridge) type and
length. The following three cases can be distinguished: (1) the
acceptor and the donor are not involved in conjugation, (2)
the spacer is involved in conjugation with the acceptor or the
donor, and (3) the donor is conjugated with the acceptor.10
Organic compounds with intramolecular charge trans-
fer can be classified according to the number of atoms
comprising the spacer (one-atom, two-atom, etc.). De-
rivatives of the picryl series containing donor groups
of different nature and donor strength were synthe-
sized.11—13 An example is a compound containing the
aminomethoxyquinoline moiety, which is present in the
known antimalarial drugs, such as pentaquine and prima-
quine.14 In picryl derivatives, the overlap of -orbitals of
benzene rings along the conjugation chain is insignificant
and depends on the angle of rotation of the donor ring
plane with respect to the acceptor moiety. In any electronic
donor-acceptor system, the electron transfer can be
considered as the electron density redistribution from
the highest occupied molecular orbital (HOMO) of the
donor moiety to the lowest unoccupied molecular orbital
(LUMO) of the acceptor. Intramolecular charge-transfer
complexes include molecules, in which HOMO and
LUMO are strictly localizes on the corresponding donor
and acceptor moieties of the molecule, with no direct
conjugation between their -electron systems, for example,
if the donor and the acceptor are separated by at least one
sp3-hybridized carbon atom.12,15 The steric factor plays
an important role in the formation of CICTs. Some of such
compounds are characterized by the alternating arrange-
ment of the donor and acceptor rings in parallel planes.16
The introduction of substituents, which hinder coplanar-
ity of the donor and acceptor rings (alkyl substituents in
the ortho position), decreases the constant for the forma-
tion of 1 : 1 charge transfer complexes. Molecular CICTs
can be used as ligands for the preparation of coordination
compounds that hold promise in studying metal-contain-
ing donor-acceptor systems.
The construction of polyfunctional antioxidants is
a new promising approach to search for physiologically
active compounds, because a combination of the antioxi-
dant 2,6-dialkylphenol group and different functional
moieties allows the targeted design of antioxidants with
controlled activity.
The goal of this study is to find new antioxidants. For
this purpose, we synthesized compounds 1—6 based on
2,6-di-tert-butylphenol (Scheme 1). Antioxidant activity
of compounds 1—6 was evaluated by spectrophotometry
using the phenolic hydrogen transfer to the stable radical
2,2-diphenyl-1-picrylhydrazyl (DPPH) and the enzymatic
oxidation of linoleic acid by the enzyme lipoxygenase.
Results and Discussion
The known nucleophilic substitution of halogen in
activated 2,4,6-trinitrochlorobenzene in the reactions with
N- or S-nucleophiles affords arylation products, viz.,
derivatives 1 and 2 containing the picryl moiety. We syn-
thesized compound 117 by a modified procedure, which
made it possible to substantially increase the yield of the
target product and decrease the reaction time.
Scheme 1
R =

714
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Mikhalev et al.
A
nitrobenzene that is often masked by absorption of the
solvent, the electronic absorption spectra of compounds
1 and 2 show bands assigned to charge transfer from the
donor aryl moiety to the acceptor picryl group via a spacer
(—NH—) and also a band arising from through-space
intramolecular charge transfer (ICT) in a stacking interac-
tion mode between one of the ortho-nitro groups of the
acceptor picryl moiety and the -electron system of the
donor.12,14,18
The electronic absorption spectra of all the synthesized
compounds are characterized by a long-wavelength max-
imum undergoing a bathochromic shift with increasing do-
nor strength of the aryl moiety. The long-wavelength maxi-
mum at 360—390 nm should be assigned to the charge
transfer from the donor moiety of the molecule to the planar
ortho-nitro group. Then, with increasing excitation energy, the
para-transition should be observed, appearing as an inflection
point or a maximum at 330—340 nm. Since compounds 1
and 2 are quite intensely colored, which is an evidence of
a long-wavelength charge transfer band, we compared the
electron transfer energies (EICT) for related compounds
of a picryl series. These energies can be evaluated by the
equation EICT = 1239.8/max (see Ref. 19). For compound 1
(max = 397 nm) containing the—NH— spacer, EICT is
3.12 eV; for compound 2 ( max = 402 nm), 3.08 eV (Fig. 1).
The molecular structures of compounds 1, 2, 3, and 5
were determined by X-ray diffraction. The crystallographic
characteristics and selected bond lengths and bond angles
for these compounds are summarized in Tables 1 and 2.
The molecular structures of compounds 1 and 2 are shown
in Fig. 2. In structure 1, the N(1) atom forms the intra-
2.0
1.6
1.2
0.8
0.4
1
2
300
400
500
600 /nm
Fig. 1. Absorption spectra of compounds 1 (20 mol L–1) and 2
(100 mol L–1) in MeCN.
With the aim of varying the spacer type, we synthesized
Schiff bases 3 and 4 with the RCH=N group (R = 3,5-di-
tert-butyl-4-hydroxyphenyl). The reaction of 5-amino-
quinoline with 3,5-di-tert-butyl-4-hydroxybenzoyl chlor-
ide in CH2Cl2 in the presence of NEt3 gave the acylation
product, amide 5 containing the phenol group and the
—CONH— spacer. 3,5-Di-tert-butyl-4-hydroxybenzo-
hydrazide was used to synthesize derivative 6. Air- and
solution-stable compounds 1—6 were characterized by IR,
UV, and NMR spectroscopy and elemental analysis.
Compounds 1—4 are brightly colored substances,
as opposed to colorless derivatives 5 and 6 with the
—CONH— spacer. Apart from the absorption band of
O(11)
a
b
O(12)
N(1)
C(7)
S(1)
C(8)
C(18)
C(5)
C(1)
C(4)
C(9)
C(19)
C(6)
O(31)
O(3)
O(22)
C(10)
N(2)
C(12)
C(17)
N(19)
O(6)
C(3)
C(2)
N(3)
O(32)
C(11)
C(20)
C(36)
C(16)
C(14)
C(13)
C(12)
O(1)
O(8)
N(1)
O(9)
O(21)
C(24)
C(11)
C(20)
C(4)
C(34)
C(33)
C(15)
C(10)
C(16)
C(43)
C(42)
N(21)
C(22)
C(14)
O(41)
C(15)
C(5)
C(17)
N(23)
C(43)
C(18)
O(13)
C(25)
O(7)
O(1)
C(32)
C(31)
C(35)
Fig. 2. Molecular structures of compounds 1 (а) and 2 (b) with thermal ellipsoids drawn at 50% and 35% probability level for 1 and 2,
respectively. Hydrogen atoms are not shown.

New phenolic antioxidants
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
715
Table 1. Crystallographic characteristics of compounds 1—3 and solvates 5•MeCN and 5•Me2CO
Compound
1
2
3
5•MeCN
5•Me2CO
Molecular formula
C20H24N4O7
C23H23N3O7S
C24H28N2O
C24H28N2O2•
•MeCN
C24H28N2O2•
•Me2CO
434.56
Molecular weight
Crystal system
Space group
a/Å
432.43
Monoclinic
P21/n
5.7884(5)
11.6766(6)
32.854(2)
90.00
91.292(6)
90.00
293(2)
485.53
Monoclinic
C2/c
34.836(4)
7.5321(6)
18.923(2)
90.00
102.747(10)
90.00
293(2)
360.48
417.54
Triclinic
Tricli nic
Triclinic
–
–
–
P1
P1
P1
6.5791(10)
11.4699(18)
14.432(3)
85.177(14)
80.374(13)
83.385(13)
293(2)
9.046(3)
10.164(3)
13.588(4)
93.01(2)
99.31(2)
102.99(2)
293(2)
9.4903(4)
9.8061(5)
13.6912(6)
102.941(4)
92.855(4)
101.787(4)
293(2)
b/Å
c/Å
/deg
/deg
/deg
T/K
V/Å3
2220.0(3)
4
4842.8(9)
1
1064.2(3)
2
1196.2(6)
2
1209.48(10)
2
Z
dcalc/g cm–3
Radiation
(K)/mm–1
-Angle range/deg
h, k, l ranges
1.258
1.352
1.125
1.159
1.193
CuK
MoK
MoK
MoK
CuK
0.575
0.183
0.069
0.074
0.613
4—68.8
2—25.5
3—30
3.5—28
5—70
–6 ≤ h ≤ 3,
–13 ≤ k ≤ 13,
–36 ≤ l ≤ 39
0.10.10.1
3088
–42 ≤ h ≤42,
–6 ≤ k ≤ 9,
–22 ≤ l ≤ 20
0.20.20.2
4421
–9 ≤ h ≤ 3,
–16 ≤ k ≤ 14,
–20 ≤ l ≤ 20
0.30.20.2
6090
–11 ≤ h ≤ 11,
–13 ≤ k ≤ 13,
–8 ≤ l ≤ 17
0.20.20.2
5468
–11 ≤ h ≤ 11,
–11 ≤ k ≤ 11,
0 ≤ l ≤ 16
0.30.20.2
4158
Crystal size/mm
Total number of reflections
Number of unique reflections
GООF
1803
2.304
1051
0.846
683
0.414
1306
0.552
2463
0.850
R1/wR2 (I ≥ 2(I))
Residual electron density
(max/min)/e Å–3
0.1712/0.3844
0.591/–0.654
0.1491/0.3270
0.694/–0.548
0.0363/0.0810
0.095/–0.126
0.0402/0.0676
0.112/–0.134
0.0834/0.0834
0.178/–0.171
molecular N(1)—H(1)...O(1) and N(1)—H(1)...O(9)
hydrogen bonds with oxygen atoms of two nitro groups
(H(1)...O(1) and H(1)...O(9) are 2.657 and 3.543 Å, re-
spectively). In structure 2, the S(1) atom is linked to ad-
jacent nitro oxygen atoms through the intermolecular
S(1)...O(11) contact (2.744 Å) and two short intermo-
lecular S(1)...O(11) and S(1)...O(31) contacts (3.117 and
3.110 Å, respectively). The O(11) and O(31) atoms form
short intermolecular contacts with one another (3.039 Å).
The phenol oxygen atom is involved in the intermolecular
O(1)—H(1)...O(41) hydrogen bond (2.331 Å) with the
oxygen atom of the acetone molecule.
Evaluation of antioxidant activity. Due to the presence
of the 2,6-di-tert-butylphenol moiety, the synthesized
compounds would be expected to exhibit antioxidant activ-
Scheme 2
Compound 3 (Fig. 3) was found to be a covalent mono-
mer stabilized by the intermolecular O(1)—H(1)...N(1)
hydrogen bond (H(1)...N(1), 2.284 Å).
Compound 5 showed a tendency to form solvates. The
structures of the solvates with the solvents acetonitrile
(5•MeCN) and acetone (5•Me2CO) were determined by
X-ray diffraction. In the structure of 5•MeCN (see Fig. 3),
there are the intermolecular N(11)—H(11)...N(31) and
O(1)—H(1)...N(1) hydrogen bonds with the H(11)...N(31)
and H(1)...N(1) distances of 2.107 and 2.275 Å, respec-
tively. The structure of 5•Me2CO is stabilized by the
intermolecular N(11)—H(11)...O(3) and O(1)—H(1)...N(1)
hydrogen bonds with the H(11)...O(3) and H(1)...N(1),
distances of 2.139 and 2.193 Å, respectively.

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Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Mikhalev et al.
Table 2. Selected bond lengths and bond angles in compounds 1—3 and solvates 5•MeCN and 5•Me2CO
Bond
d/Å
Angle
/deg
Compound 1
N(1)—C(20)
N(1)—C(10)
O(1)—C(18)
O(3)—N(19)
O(6)—N(19)
O(7)—N(23)
O(9)—N(23)
O(8)—N(21)
O(13)—N(21)
C(11)—N(21)
C(12)—N(19)
C(14)—N(23)
1.352(11)
1.417(11)
1.389(10)
1.241(10)
1.198(10)
1.287(14)
1.210(12)
1.194(11)
1.230(13)
1.448(12)
1.468(13)
1.408(12)
C(20)—N(1)—C(10)
C(4)—C(10)—N(1)
N(1)—C(10)—C(15)
N(1)—C(20)—C(12)
N(1)—C(20)—C(14)
O(1)—C(18)—C(22)
O(6)—N(19)—O(3)
O(8)—N(21)—O(13)
O(9)—N(23)—O(7)
C(5)—C(18)—O(1)
O(13)—N(21)—C(11)
O(9)—N(23)—C(14)
128.1(9)
121.9(10)
118.0(8)
123.4(10)
122.0(9)
118.0(8)
124.3(9)
124.4(10)
121.5(10)
120.2(9)
115.7(9)
120.5(11)
Compound 2
S(1)—C(7)
1.738(7)
1.750(5)
1.384(7)
1.436(8)
1.405(9)
1.452(8)
1.210(8)
1.202(8)
1.232(7)
1.268(7)
1.172(7)
1.234(9)
C(7)—S(1)—C(4)
C(3)—C(4)—S(1)
C(5)—C(4)—S(1)
C(12)—C(7)—S(1)
C(8)—C(7)—S(1)
C(6)—C(1)—O(1)
O(1)—C(1)—C(2)
O(11)—N(1)—O(12)
O(21)—N(2)—O(22)
O(32)—N(3)—O(31)
N(1)—C(8)—C(7)
N(2)—C(10)—C(11)
104.1(3)
121.9(5)
117.4(5)
128.2(4)
120.1(5)
117.9(6)
118.0(6)
22.5(6)
19.8(7)
24.8(6)
119.1(6)
20.0(6)
S(1)—C(4)
O(1)—C(1)
C(8)—N(1)
C(10)—N(2)
C(12)—N(3)
N(1)—O(11)
N(1)—O(12)
N(2)—O(21)
N(2)—O(22)
N(3)—O(32)
N(3)—O(31)
Compound 3
N(1)—C(2)
N(1)—C(9)
C(5)—N(11)
N(11)—C(12)
C(16)—O(1)
O(1)—H(1)
C(12)—C(13)
C(5)—C(10)
C(5)—C(6)
1.302(6)
1.388(6)
1.424(6)
1.271(5)
1.369(6)
0.75(5)
1.458(6)
1.431(6)
1.352(6)
C(2)—N(1)—C(9)
N(1)—C(2)—C(3)
N(11)—C(5)—C(10)
N(1)—C(9)—C(8)
N(1)—C(9)—C(10)
N(11)—C(12)—C(13)
C(16)—O(1)—H(1)
116.7(5)
125.3(6)
117.4(5)
117.8(5)
121.7(5)
124.2(5)
117.0(4)
Compound 5•MeCN
N(1)—C(2)
N(1)—C(9)
C(5)—N(11)
N(11)—C(12)
N(11)—H(11)
C(12)—O(2)
C(16)—O(1)
O(1)—H(1)
N(31)—C(32)
1.319(4)
1.374(3)
1.435(4)
1.367(3)
0.920(2)
1.229(3)
1.370(3)
0.780(3)
1.103(4)
C(2)—N(1)—C(9)
N(1)—C(2)—C(3)
C(6)—C(5)—N(11)
C(10)—C(5)—N(11)
N(1)—C(9)—C(8)
N(1)—C(9)—C(10)
O(2)—C(12)—N(11)
C(16)—O(1)—H(1)
116.8(3)
124.7(3)
119.2(3)
119.8(3)
118.0(3)
122.8(3)
120.2(3)
119.0(2)
Compound 5•Me2CO
N(1)—C(2)
N(1)—C(9)
C(5)—N(11)
N(11)—C(12)
N(11)—H(11)
C(12)—O(2)
C(16)—O(1)
O(1)—H(1)
C(32)—O(3)
1.311(3)
1.374(3)
1.423(2)
1.353(3)
0.821(17)
1.209(2)
1.353(2)
0.740(4)
1.199(3)
C(2)—N(1)—C(9)
N(1)—C(2)—C(3)
C(6)—C(5)—N(11)
C(10)—C(5)—N(11)
N(1)—C(9)—C(8)
N(1)—C(9)—C(10)
O(2)—C(12)—N(11)
C(16)—O(1)—H(1)
118.53(19)
124.0(2)
120.0(2)
119.12(18)
119.87(18)
121.05(19)
122.51(18)
125.0(3)

New phenolic antioxidants
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
717
a
b
C(25)
C(25)
C(23)
O(1)
O(1)
C(22)
C(26)
C(24)
C(23)
C(21)
C(22)
C(21)
C(19)
C(16)
C(19)
C(15)
C(26)
C(16)
C(17)
C(18)
C(17)
C(18)
C(15)
C(20)
C(24)
C(20)
C(32)
C(14)
C(14)
C(13)
C(12)
C(13)
C(12)
C(33)
N(31)
N(11)
C(5)
N(11)
O(2)
C(5)
C(4)
C(4)
C(10)
C(6)
C(10)
C(6)
C(3)
C(2)
C(3)
C(9)
C(7)
C(9)
C(7)
C(8)
C(8)
C(2)
N(1)
N(1)
Fig. 3. X-ray molecular structures of compounds 3 (а) and 5•Me2CO (b) with thermal ellipsoids drawn at 50% probability level.
Hydrogen atoms are not shown.
ity. We evaluated the antioxidant activity of the synthesized
compounds using the model reaction (DPPH assay) and
the inhibition of linoleic acid oxidation by lipoxygenase.
Evaluation of antioxidant activity of compounds using the
DPPH assay. The activity of 2,6-di-tert-butylphenols as
free-radical scavengers was evaluated using a known
method20 based on the ability of the compounds to reduce
the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)
(Scheme 2).
2,2-Diphenyl-1-picrylhydrazyl is one of a few stable
N-centered organic radicals. Its solution is deep violet in
color. The absorbance (A) of 0.1 mM DPPH solutions in
ethanol containing the test compounds at different con-
centrations was measured for 30 min at a wavelength cor-
responding to the absorption maximum of DPPH. By
comparing the effective concentrations of compounds 1—6
that lead to 50% reduction of the initial DPPH concentra-
tion (EC50, see Fig. 4), the following conclusion was made:
the nature of the spacer and the number of phenol groups
considerably affect the antioxidant activity of compounds.
The presence of the amide moiety CONH leads to a de-
crease in the antioxidant activity due apparently to the
absence of conjugation between the donor phenol group
and the acceptor moiety.
Inhibition of the enzyme lipoxygenase. Lipoxygenase
(LOX), which is of interest as a pharmaceutical target,
catalyzes the oxidation of polyunsaturated fatty acids. This
oxidation process affords reactive oxygen metabolites as
by-products that cause oxidative stress.21 The oxida-
tion of linoleic acid catalyzed by soybean lipoxygenase
(LOX-1-B) was performed at room temperature. The
course of the reaction was monitored by measuring the
increase in absorbance of the reaction mixture at a wave-
length of 234 nm corresponding to the absorption maxi-
mum of the reaction products, isomeric 9-hydroperoxy-
trans-10,cis-12- and 13-hydroperoxy-cis-9,trans-11-octa-
decadienoic acids (LOOH). An increase in the concentra-
tion of isomeric linoleic acid hydroperoxides in the
presence of different compounds is shown in Fig. 5.
EC50/mol L–1
460 37
500
400
300
200
121
9
70
4
50
Compounds 4 and 6 containing two phenol groups are
much more active than the standard antioxidant ionol.
Compound 1 containing the one-atom NH spacer is the
most effective antioxidant. The presence of the acceptor
picryl moiety conjugated with the phenol group leads to
an increase in stability of the resulting phenoxyl radical.
61
3
4
100
32
3
20
2
1
2
3
4
5
6
Ionol
Fig. 4. Effective concentrations EC50 (mol L–1) for compounds
1—6 and ionol evaluated using the DPPH assay.

718
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Mikhalev et al.
C(LOOH)/mol L–1
45
hydrazide26 were synthesized by known procedures. The solvents
(EtOH (95%), CHCl3, CH2Cl2, MeOH, MeCN, toluene,
acetone, hexane (all of reagent grade) and petroleum ether
(b.p. 40—70 C)) were used as-received.
The IR spectra were recorded on an IR200 Fourier-transform
IR spectrophotometer (Thermo Nicolet) as KBr pellets. The
NMR spectra were measured on a Bruker AMX-400 spectrometer
in CDCl3 (1H, 400 MHz; 13C, 100 MHz). The electronic absorp-
tion spectra were recorded on Evolution 300 (Termo Scientific)
and Zenyth200rt (Anthos) spectrophotometers.
30
15
N-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2,4,6-trinitrophenyl-
amine (1). A solution of picryl chloride (247 mg, 1 mmol) in
ethanol (5 mL) was added dropwise to a solution of 3,5-di-tert-
butyl-4-hydroxyphenylamine hydrochloride (258 mg, 1 mmol)
in ethanol (5 mL). The mixture was refluxed for 30 min, cooled
to room temperature, concentrated in vacuo to 3 mL, and kept
until crystals formed. The dark-red crystals were filtered off,
washed with ethanol, and dried in vacuo at 80 C. The crystals
were studied by X-ray diffraction. The yield was 374 mg (86%).
M.p. 190—191 C (decomp.) (cf. Ref. 17: m.p. 187—189 C
(propan-1-ol)). IR, /cm–1: 3619 (OH); 3335 (NH); 3102; 3088
(CH arom); 2963; 2877 (CH); 1624; 1591; 1534 (NO2); 1437;
1356; 1338; 1287. 1H NMR, : 1.43 (s, 18 H, But); 5.36 (s, 1 H,
OH); 6.88 (s, 2 H, CH arom); 9.06 (s, 2 H, CH arom (picryl));
10.40 (s, 1 H, NH). 13C NMR, : 29.9 (C(CH3)3); 34.5 (C(CH3)3);
118.3; 120.6; 127.3; 129.0; 134.7; 137.5; 139.5; 153.2 (Carom).
Found (%): C, 55.40; H, 5.50; N, 12.76. C20H24N4O7. Calculat-
ed (%): C, 55.55; H, 5.59; N, 12.95. UV (MeCN), λmax/nm (ε):
239 (15100); 397 (11600).
1
2
3
4
5
6
Control
Fig. 5. Accumulation of linoleic acid hydroperoxides after 5 min
oxidation of linoleic acid by lipoxygenase LOX 1-В in the pres-
ence of compounds 1—6 at a concentration of 100 mol L–1
;
control, in the absence of the compounds.
A comparative analysis of the inhibition of linoleic acid
oxidation by compounds 1—6 demonstrates that they are
weak inhibitors of linoleic acid oxidation.
The 2,6-di-tert-butylphenol moiety imparts inhibitory
activity to the compounds. Schiff base 4 exhibits the high-
est activity. The nature of the spacer also affects the degree
of inhibition of lipoxygenase by the compounds under
examination.
The development of potential pharmacological agents
is aimed at reducing the side effect on healthy cells. For
this purpose, we propose to introduce groups that have
proven antioxidant activity. Sterically hindered phenols
are mimetics of vitamin E and inhibitors of free-radical
oxidation processes. We synthesized aromatic and hetero-
cyclic compounds containing 2,6-di-tert-butylphenol
moieties. The molecular structures in the crystals were
studied by X-ray diffraction.
3,5-Di-tert-butyl-4-hydroxyphenyl 2,4,6-trinitrophenyl sulfide
(2). A solution of picryl chloride (248 mg, 1 mmol) in ethanol
(5 mL) was added to a solution of 2,6-di-tert-butyl-4-mercapto-
phenol (238 mg, 1 mmol) in ethanol (5 mL). The mixture was
refluxed for 10 min, cooled to room temperature, and kept for
16 h. The orange crystals that formed were filtered off, washed
with cold ethanol, dried in vacuo, and recrystallized from acetone.
The crystals were studied by X-ray diffraction. The yield was
399 mg (89%). M.p. 179 C (decomp.). IR, /cm–1: 3629 (OH);
3105; 3078 (CH arom); 2960; 2876 (CH); 1599; 1537 (NO2);
1
1427; 1337. H NMR, : 1.41 (s, 18 H, But); 5.57 (s, 1 H, OH);
The synthesized compounds were evaluated for anti-
oxidant activity using the hydrogen transfer reaction
(DPPH assay) and enzymatic oxidation of linoleic acid.
It was demonstrated that the introduction of the 2,4,6-tri-
nitrophenyl moiety into the molecule in combination with
the —NH— spacer leads to a significant increase in anti-
oxidant activity. The synthesized compounds proved to be
weak lipoxygenase inhibitors. The results of this study
provide an opportunity to search for new types of anti-
oxidants.
7.14 (s, 2 H, CH (arom)); 8.71 (s, 2 H, CH arom (picryl)).
13C NMR, : 29.9 (C(CH3)3); 34.5 (C(CH3)3); 118.3; 122.6 (2 C);
131.5 (2 C); 137.8; 140.8; 144.0; 151.0; 156.1 (Carom). Found (%):
C, 53.46; H, 5.20; N, 9.12; S, 7.05. C20H23N3O7S. Calculated (%):
C, 53.44; H, 5.16; N, 9.35; S, 7.13. UV (MeCN), max/nm ():
317 (5300); 402 (4800).
2,6-Di-tert-butyl-4-[(quinolin-5-ylimino)methyl]phenol (3).
3,5-Di-tert-butyl-4-hydroxybenzaldehyde hemihydrate (1.12 g,
5 mmol) and several crystals of p-toluenesulfonic acid were
added to a solution of 5-aminoquinoline (0.72 g, 5 mmol) in
ethanol (30 mL). The mixture was refluxed for 1 h, the solvent
was removed in vacuo to one-third of the initial volume, toluene
(15 mL) was added to the residue, and the mixture was refluxed
for 6 h. Then petroleum ether (35 mL, 15 mL in total) was
added, the mixture was refluxed for 5 min, and the insoluble
residue was filtered off. The solution was cooled to 5 C, and the
orange crystals that formed were washed with petroleum ether
and dried in vacuo (8 Torr). The yield was 1.41 g (79%). M.p.
197 C. IR, /cm–1: 3621 (OH free); 3029; 2870 (CH); 1620
(C=N); 1569; 1467; 1427; 1211. 1H NMR, : 1.55 (s, 18 H, But);
5.74 (s, 1 H, OH); 7.12 (d, 1 H, quinol, 3Jнн = 8.1 Hz); 7.41—7.45
Experimental
The following commercially available reactants were used:
3,5-di-tert-butyl-4-hydroxybenzaldehyde hemihydrate (99%,
Sigma-Aldrich), 5-aminoquinoline (97%, Sigma-Aldrich), and
Et3N (99%, Sigma-Aldrich). 2,4,6-Trinitrochlorobenzene (picryl
chloride),22 3,5-di-tert-butyl-4-hydroxybenzoyl chloride,23
2,6-di-tert-butyl-4-aminophenol hydrochloride,24 2,6-di-tert-
butyl-4-mercaptophenol,25 and 3,5-di-tert-butyl-4-hydroxybenzo-

New phenolic antioxidants
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
719
(m, 1 H, quinol); 7.71 (t, 1 H, quinol, 3Jнн = 8.0 Hz); 7.89 (s, 2 H,
1.5 mmol) in toluene (30 mL) was refluxed for 3 h with a Dean—
Stark trap. Then the mixture was cooled to room temperature,
and the solution was concentrated in vacuo to a small volume.
The pale-beige precipitate that formed was filtered off, washed
with hexane, dried in air, and recrystallized from a CHCl3—hex-
ane mixture. The yield was 493 mg (68%), colorless powder. M.p.
290 C. IR, /cm–1: 3635 (OH free); 3205 (NH); 3041; 2856
3
CH arom.); 7.97 (d, 1 H, quinol, Jнн = 8.0 Hz); 8.50 (s, 1 H,
3
CH=N); 8.74 (d, 1 H, quinol, Jнн = 8.6 Hz); 8.96 (dd, 1 H,
quinol, Jнн = 4.0 Hz, Jнн = 2.5 Hz). 13C NMR, : 30.2 (C(CH3)3);
34.5 (C(CH3)3); 113.1 (CH quinol); 120.6 (CH quinol); 124.3;
126.4 (CH quinol); 126.5 (2 C); 127.8; 129.6 (CH quinol); 132.8
(CH quinol); 136.6 (2 C); 148.6; 149.9; 150.7 (CH quinol); 157.5;
161.7 (C=N). Found (%): C, 79.76; H, 7.67; N, 7.64. C24H28N2O.
Calculated (%): C, 79.96; H, 7.83; N, 7.77. UV (CHCl3),
max/nm (): 246 (20029); 296,5 (9301); 342 (11394).
1
(CH); 1648 (C=O); 1537; 1462; 1375; 1306; 1238. H NMR, :
1.45 (s, 18 H, But); 1.48 (s, 18 H, But); 5.51 (s, 1 H, OH); 5.62
(s, 1 H, OH); 7.57 (s, 2 H); 7.72 (s, 2 H); 8.47 (br.s, 1 H, CH=N);
9.46 (br.s, 1 H, NH). 13C NMR, : 30.2 (C(CH3)3); 30.2
(C(CH3)3); 34.4 (C(CH3)3); 34.4 (C(CH3)3); 124.5; 125.1 (2 C);
127.7; 136.3 (2 C); 146.0; 150.0; 156.2; 157.1; 162.5; 164.9 (C=O).
Found (%): C, 75.07; H, 9.25; N, 5.81. C30H44N2O3. Calculat-
ed (%): C, 75.00; H, 9.16; N, 5.83. UV (MeCN), max/nm ():
316 (26919).
4-{(3,5-Di-tert-butyl-4-hydroxyphenyl)imino]methyl}-2,6-
di-tert-butylphenol (4). 2,6-Di-tert-butyl-4-aminophenol hydro-
chloride (309 mg, 1.2 mmol) and Et3N (0.167 mL, 1.2 mmol)
were added to a solution of 3,5-di-tert-butyl-4-hydroxybenz-
aldehyde hemihydrate (244 mg, 1 mmol) in ethanol (15 mL)
under argon atmosphere on heating to 40 C. The mixture was
stirred for 30 min and then cooled to –18 C. The yellow-orange
precipitate that formed was filtered off, washed with hexane, dried
in vacuo (8 Torr), and recrystallized from ethanol. The yield was
324 mg (71%). M.p. 227 C (with decomp.). IR, /cm–1: 3604
br. (OH free); 3000; 2858 (CH); 1620 (C=N); 1462; 1377; 1236.
1H NMR, : 1.50 (s, 18 H, But); 5.14 (s, 1 H, OH); 5.57 (br.s,
1 H, OH); 7.10 (s, 2 H); 7.74 (s, 2 H); 8.40 (br.s, 1 H, CH=N).
13C NMR, : 30.2 (C(CH3)3); 30.3 (C(CH3)3); 34.4 (C(CH3)3);
34.5 (C(CH3)3); 117.7; 125.9; 127.7; 128.3; 136.3; 136.6;
152.0; 158.8 (C=N). Found (%): C, 76.68; H, 10.05; N, 3.01.
C29H43NO2•H2O. Calculated (%): C, 76.44; H, 9.95; N, 3.07.
UV (CHCl3), max/nm (): 287 (12730); 339.5 (12172).
X-ray diffraction study. X-ray diffraction intensities for the
compounds were measured on a STOE StadiVari Pilatus100K
diffractometer, (CuK) = 1.5418 Å, (MoK) = 0.71073 Å,
-scanning technique.27 The X-ray diffraction data sets were
processed with the WinGX suite.28 All subsequent calculations
were performed using the SHELX-97 program package.29 The
crystal structures were solved by direct methods and then refined
with anisotropic displacement parameters for all nonhydrogen
atoms. The hydrogen atoms were positioned geometrically and
refined isotropically using a riding model. The structure drawings
were prepared with the MERCURY CSD 3.1 program.30 The
atomic coordinates and other crystal structure parameters of
compounds 1—3 and solvates 5•MeCN and 5•Me2CO were
deposited with the Cambridge Crystallographic Data Centre
(CCDC 1575906 (1), 1575907 (2), 1582462 (3), 1582463 (5•MeCN),
and 1582464 (5•Me2CO)) and are available at www.ccdc.cam.
ac.uk/data_request/cif. The X-ray diffraction data and the prin-
cipal crystallographic characteristics for compounds 1—3 and
solvates 5•MeCN and 5•Me2CO are given in Tables 1 and 2.
Evaluation of antioxidant activity of compounds using the
DPPH assay. The antioxidant activity was evaluated using the
stable radical 2,2-diphenyl-1-picrylhydrazyl (Sigma-Aldrich)20
by spectrophotometry at max = 517 nm. The known procedure31
was modified for a microplate spectrophotometer. The reaction
was performed in plate wells (96 wells). The reaction mixture
contained DPPH (0.1 mL, 0.2 mmol L–1) and a solution of the
test compound at different concentrations (0.01, 0.02, 0.05, and
0.1 mmol L–1) in EtOH (0.1 mL). The experiments were per-
formed in three parallel runs. The reaction was accomplished at
25 C for 30 min. The data were processed and the EC50 values
were calculated using the Microsoft Excel 2010 and GraphPad
Prism 5 programs. The antioxidant activity I (%) was calculated
according to the formula
N-(Quinolin-5-yl)-3,5-di-tert-butyl-4-hydroxybenzamide (5).
3,5-Di-tert-butyl-4-hydroxybenzoyl chloride (1.35 g, 5 mmol)
was added with stirring to a solution of 5-aminoquinoline (0.72 g,
5 mmol) in CH2Cl2 (5 mL) cooled to 5—10 C. The mixture was
stirred for 10 min at room temperature and then refluxed for
30 min. The red-brown precipitate that formed on cooling was
filtered off. The crystals were washed with acetone (33 mL) and
dried in air. The precipitate was dissolved in water and treated
with NaHCO3 (0.42 g, 5 mmol). The colorless precipitate that
formed was filtered off, washed with water, and recrystallized
from an H2O—EtOH mixture. The yield was 1.75 g (83%). M.p.
235 C. IR, /cm–1: 3551 (OH free); 2958; 2874 (CH); 1703
(C=O); 1697; 1600; 1304; 1238; 1103. 1H NMR, : 1.26 (t, 3 H,
3
CH3CH2, Jнн = 4.0 Hz); 1.50 (s, 18 H, But); 3.74 (q, 2 H,
CH3CH2, 3Jнн = 4.0 Hz); 5.74 (s, 1 H, OH); 7.43—7.45 (m, 1 H,
3
quinol); 7.74 (t, 1 H, quinol, Jнн = 8.0 Hz); 7.83 (s, 2 H, CH
arom.); 7.86 (d, 1 H, quinol, 3Jнн = 8.0 Hz); 8.04 (d, 1 H, quinol,
3Jнн = 8.0 Hz); 8.13 (br.s, 1 H, NH); 8.26 (d, 1 H, quinol,
3Jнн = 8.0 Hz); 8.94—8.97 (m, 1 H, quinol). 13C NMR, : 30.1
(C(CH3)3); 34.6 (C(CH3)3); 121.0; 123.4; 123.6; 124.6 (2 C);
125.3 127.7; 129.2; 130.5; 133.1; 136.3; 148.8; 150.4; 157.5; 167.3
(C=O). Found (%): C, 73.56; H, 8.03; N, 6.32. C24H28N2O2•
•C2H5OH. Calculated (%): C, 73.90; H, 8.11; N, 6.63. Single
crystals suitable for X-ray diffraction were obtained by slow
evaporation of the solvent from a solution in MeCN (5•MeCN)
or acetone (5•Me2CO). Found (%): C, 74.29; H, 7.39; N, 9.66.
C24H28N2O2•CH3CN. Calculated (%): C, 74.79; H, 7.48; N, 10.06.
Found (%): C, 74.85; H, 7.73; N, 6.58. C24H28N2O2•(CH3)2CO.
Calculated (%): C, 74.62; H, 7.89; N, 6.45.
I = (A0 − A1)/A0•100,
where A0 is the absorbance of the control DPPH solution, A1 is
the absorbance of the reaction mixture in the presence of the test
compound after 30 min.
Effect of compounds on enzymatic oxidation of linoleic acid by
lipoxygenase. The lipoxygenase activity was evaluated by spec-
trophotometry32 with a Zenyth200rt 96-well plate spectropho-
tometer (Anthos) using lipoxygenase (LOX I-B, Sigma-Aldrich,
15 MU) and linoleic acid (99%, Sigma-Aldrich). The concentra-
tions of linoleic acid oxidation products, isomeric hydroperoxides,
N´-[(3,5-Di-tert-butyl-4-hydroxyphenyl)methylidene]-4-hy-
droxy-3,5-di-tert-butylbenzohydrazide (6). A mixture of 3,5-di-
tert-butyl-4-hydroxybenzaldehyde hemihydrate (368 mg, 1.5 mmol)
and 3,5-di-tert-butyl-4-hydroxybenzohydrazide (400 mg,

720
Russ. Chem. Bull., Int. Ed., Vol. 67, No. 4, April, 2018
Mikhalev et al.
were measured at max = 234 nm ( = 25000 L mol–1 cm–1).33
The analyte mixture contained a linoleic acid solution (2 mL,
0.3 mmol L–1), borate buffer (0.89 mL), pH 9.0, and a solution
of the test compound in DMSO (0.01 mL). The reaction was
initiated by the addition of 0.1 mL of the enzyme solution (500 U).
The measurements were performed for 5 min at 20 C with dif-
ferent concentrations of the test compounds. All experiments
were performed in three parallel runs.
15. I. G. Il´ina, O. V. Mikhalev, Russ. J. Org. Chem., 1997, 33,
1424—1426.
16. G. V. Gridunova, V. N. Petrov, Yu. T. Struchkov, I. G. Il´ina,
O. V. Mikhalev, Kristallografiya [Sov. Phys. Crystallogr.], 1990,
35, 59—62 (in Russian).
17. E. Manda, T. Shimura, Bull. Chem. Soc. Jpn., 1976, 49,
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18. V. V. Redchenko, S. G. Semenov, O. V. Mikhalev, I. G. Il´ina,
Zh. Obshch. Khim. [J. Gen. Chem. USSR], 1987, 57,
2599—2606 (in Russian).
19. L. A. Kazitsina, N. B. Kupletskaya, Primenenie UF-, IK-,
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Chemistry], Izd-vo MGU, Moscow, 1979, 240 pp. (in Russian).
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This study was financially supported by the Russian
Foundation for Basic Research (Project No. 17-03-01070)
using facilities of the Center for Collective Use of the
M. V. Lomonosov Moscow State University.
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Received December 25, 2017;
in revised form February 9, 2018;
accepted February 17, 2018