Novel (3,5-di-tert-butyl-2-hydroxy-phenylcarbamoyl)-alkanoic acids as potent antioxidants

Article abstract of DOI:10.1016/j.bmcl.2004.06.003

A series of novel phenolic antioxidants of amphiphilic structure has been synthesized. Investigations into the influence of aliphatic spacer length and nature of a hydrophilic anchor on the antioxidant activity allowed elucidating certain structure requirements for the membrane-addressed antioxidant designing.

Full text of DOI:10.1016/j.bmcl.2004.06.003

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4253–4256
Novel (3,5-di-tert-butyl-2-hydroxy-phenylcarbamoyl)-alkanoic
acids as potent antioxidants
Vladimir I. Lodyato,a,b Irina L. Yurkova,a Viktor L. Sorokin,a Oleg I. Shadyro,a
Vladimir I. Dolgopaletsb and Mikhail A. Kiselb,*
aDepartment of Chemistry of the Belarusian State University, Skaryna av., 4, 220050 Minsk, Belarus
bInstitute of Bioorganic Chemistry of the Belarus National Academy of Sciences, Kuprevich st., 5/2, 220141 Minsk, Belarus
Received 12 March 2004; revised 21 May 2004; accepted 3 June 2004
Available online 26 June 2004
Abstract—A series of novel phenolic antioxidants of amphiphilic structure has been synthesized. Investigations into the influence of
aliphatic spacer length and nature of a hydrophilic anchor on the antioxidant activity allowed elucidating certain structure
requirements for the membrane-addressed antioxidant designing.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Uncontrolled oxidative damage of biomolecules is one
of the important factors causing occurrence and devel-
opment of numerous degenerative diseases (rheumatoid
arthritis, atherosclerosis, cardiac infarction, stroke,
Alzheimer’s and Parkinson’s diseases, etc.).1 Therefore,
mitigation and removal of the oxidative stress by means
of using natural2 and synthetic antioxidants3 is regarded
as a practicable strategy in prevention and treatment of
the named diseases.4 Thus, there is evidence of a pro-
tective effect of a-tocopherol in cases of reperfusional
syndrome5a and atherosclerosis.5b A synthetic antioxi-
dant of phenolic nature, BO-653 (1) (Fig. 1), which has
been developed by means of a rational designing strat-
A very promising approach is also combining antioxi-
dant properties with other types of biological activity
within a single chemical structure. For example,
piperazine, AM-36 (2) (Fig. 1), being simultaneously a
sodium channel blocker and an antioxidant, diminishes
the extent of cardiac infarction and decreases the level
of reactive oxygen species within the ischemic tissue.7a
As shown by the studies performed in animal models, a
neuronal synthase inhibitor, BN 80933 (3) (Fig. 1),
containing an a-tocopherol residue in its molecular
structure, decreases brain damage in cases of cranial
injuries and ischemic conditions.7b In that way, an-
tioxidants are promising therapeutic agents, and eluci-
dation of factors that optimize their anti-radical
activity appears to be an important target for investi-
gation.
egy,6a manifests
a marked anti-atherogenic effect
in vivo6b and is proposed as an agent for prevention of
atherosclerosis.6c
Figure 1. Drug candidates bearing antioxidant properties.
Keywords: Membrane-addressed antioxidants; Lipid peroxidation.
* Corresponding author. Tel.: +375-17-263-7131; fax: +375-17-264-8761; e-mail: kisel@iboch.bas-net.by
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.003

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V. I. Lodyato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4253–4256
Syntheses of homologous acids 4a–f have been per-
formed according to an approach that we have described
earlier8 (Scheme 1). 2-Amino-4,6-di-tert-butylphenol (6),
readily obtained from catechol, was regioselectively
acylated with acyl chlorides of the respective dicarbox-
ylic acid monomethyl esters 9a–f. Treatment of the ob-
tained amides with LiOH in aqueous methanol, followed
by acidification of the reaction mixture with 2 N HCl, led
to the target acids 4a–f9 in high yields. Monomethyl-
succinate (9a) was synthesized in high yields by treating
succinic anhydride with methanol at room temperature.
Monomethyl esters 9b–f were prepared by monohydro-
lysis of readily available dimethyl esters 8b–f in the sys-
tem KOH–MeOH–MeCN–Et2O (Scheme 1). The
modified procedure that we have developed, which
consisted in increasing v/v percentage of a more polar
solvent (MeCN) in cases of more lipophilic diesters, 9e–f,
or increasing that of less polar solvent (Et2O) in cases of
less lipophilic diesters, 9b,c, resulted in higher yields of
products 9b–f, as compared to the original procedure.10
Figure 2. Chemical structures of new membrane-addressed antioxi-
dants.
In our previous paper,8 we have reported about syn-
thesizing a new amphiphilic antioxidant, 11-(3,5-di-tert-
butyl-2-hydroxyphenylcarbamoyl)undecanoic acid (4e)
(Fig. 2). Designing of this lipid peroxidation (LPO)
inhibitor has been performed on the basis of the mem-
brane-addressing concept. The essence of this concept is
introduction of a hydrophilic group into the terminal
part of molecular side chain of an antioxidant. The
hydrophilic group plays the role of an ‘anchor’ on the
lipid membrane surface, while the phenolic moiety
penetrates deeply into the lipid bilayer, where just the
LPO chain reaction takes place. Owing to such an
‘addressed’ delivery of the phenolic pharmacophore to a
definite area of the membrane, a significant increase of
inhibitory efficacy of membrane-addressed antioxidants
with respect to LPO is observed, as compared to their
nonamphiphilic analogues.8
Compound 59 was prepared by acylation of aminophe-
nol 6 with 11-bromoundecanoic acid acyl chloride fol-
lowed by quaternization with trimethylamine in the
system DMFA–CHCl3–EtOH for 5 days (Scheme 1).
Antioxidant activity of compounds 4a–f and 5 was
evaluated by their influence on the radiation-induced
peroxidation of phosphatidyl choline in multilamellar
liposomes.11 The extent of LPO was determined by the
accumulation of conjugated diene products (CDP)12 and
2-thiobarbituric acid reactive substances (TBARS)13
(Table 1).
It should be expected that the nature of terminal sub-
stituent and length of the aliphatic spacer between the
hydrophilic anchor and the phenolic pharmacophore
would exert a substantial influence on activity of mem-
brane-addressed antioxidants. Besides, there are nega-
tively charged phosphate groups on the surface of a
membrane, therefore, it should be of interest to follow
changes in antioxidant activity on changing from an
anionic anchor to a cationic one. In this paper, we de-
scribe syntheses of homologues 4a–f and an analogue 5
(Fig. 2) with a cationic hydrophilic group, and discuss
the structure–antioxidant activity relationship in the
series of compounds prepared.
Lipophilicity (log P) is also an important characteristic
of membrane-addressed antioxidants, because log P
characterizes affinity of a compound toward the lipid
phase. The log P values for compounds 4a–f and 5 were
calculated by means of CLOGP14 (Table 1). The log P
values for the acids, 4a–f, correspond to anionic forms
of the compounds, because just anionic forms will pre-
vail at physiologic pH values.
Scheme 1. Reagents and conditions: (a) MeOH (50 equiv), rt, 12 h. (b) KOH (1.15 equiv), MeOH–MeCN–Et2O, 0 ꢁC, 54 h. (c) (i) (COCl)2 (1.1 equiv),
toluene, rt, 4 h; (ii) 6 (1 equiv), Et3N (1.1 equiv), THF, 0 ꢁC fi rt, 30 min; (iii) LiOH (5 equiv), MeOH–H2O, 5 ꢁC, 20 h; (iv) HCl 2 N. (d) (i) (COCl)2
(1.1 equiv), toluene, rt, 4 h; (ii) 6 (1 equiv), Et3N (1.1 equiv), THF, 0 ꢁC fi rt, 30 min; (iii) Me3N (5 equiv, 20 wt% in EtOH), DMFA–CHCl3–EtOH, rt,
120 h.

V. I. Lodyato et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4253–4256
Table 1. Antioxidant activity and log P of compounds 4a–f and 5
4255
Compda
Inhibition of CDP formationb (%)
Inhibition of TBARS formationb (%)
Calcd log Pc
1.4 kGy
2.4 kGy
1.4 kGy
2.4 kGy
4a, n ¼ 2
4b, n ¼ 4
4c, n ¼ 6
4d, n ¼ 8
4e, n ¼ 10
4f, n ¼ 12
5, n ¼ 10
a-T
50.2 2.2
56.9 3.0
62.1 1.5
75.7 2.4
53.1 3.1
43.5 2.0
38.2 2.8
20.6 3.0
44.6 2.3
48.0 2.8
55.2 4.1
72.7 3.6
47.5 3.4
43.0 2.0
35.5 1.7
21.8 3.1
51.8 2.2
59.0 2.8
63.4 3.0
79.7 3.3
67.5 3.7
43.4 3.5
38.1 2.4
24.6 3.4
34.5 3.3
42.2 2.4
45.0 2.0
66.9 3.6
57.3 2.1
53.7 1.9
36.7 3.1
20.4 2.7
1.10
0.47
1.50
2.56
3.62
4.67
2.56
12.0
a The investigated compounds and a-tocopherol (a reference compound) were taken in the 0.1 mM concentration; the phosphatidyl choline con-
centration was 20 mM.
b Percent inhibition with respect to a blank experiment, without antioxidant. The confidence interval was calculated for n ¼ 3, p ¼ 0:95 using
Student’s t-distribution.
c The log P values of compounds 4a–f were calculated for anionic forms.
Experimental results obtained in the liposomal model of
LPO (Table 1) show that antioxidant activity of the
compounds prepared depends to a significant extent on
the aliphatic spacer length and nature of the membrane
anchor. On examining changes in antioxidant properties
of homologues 4a–f, one can mark out a range of in-
creased activity (compounds 4a–d), a range of decreased
activity (compounds 4d–f), and a peak of activity cor-
responding to compound 4d.
An illustrative example is comparison of activities of
compounds 4a and 4b (Table 1). The anion of succinic
acid derivative 4a is more lipophilic than the anion of
compound 4b owing to the possibility of intramolecular
hydrogen bond formation. At the same time, amide 4a is
inferior to compound 4b as to antioxidant activity. This
indicates that the aliphatic spacer length is a more
important parameter determining inhibitory properties
of an antioxidant than lipophilicity.
Such character of the structure–activity relationship can
be explained from the standpoint of the membrane-
addressing concept. In cases of short-chain homologues,
the immersion depth of phenolic moiety into the mem-
brane is small, and the antioxidant pharmacophore is
situated above the membrane area where LPO takes
place (methylene groups of the 1,4-pentadiene system
belonging to polyunsaturated acid residues). Therefore,
in the case of a short spacer, protective role of membrane-
addressed antioxidants consists mainly in neutralization
of radical species that penetrate into the membrane from
the aqueous phase and initiate LPO; hence, antioxidant
activity in this case is not particularly high. When the side
chain length is gradually built up, the radical-scavenging
part moves closer to the region where free-radical LPO
processes take place, and antioxidant activity increases.
With a spacer length of eight methylene units (compound
4d), maximum activity is observed. To all appearances,
such a spacer provides localization of the phenolic
pharmacophore just within the area of development of
free-radical LPO processes causing damage to the lipid
membrane (C7–C11 atoms of polyunsaturated fatty acid
residues). In this case, along with neutralization of radi-
cals initiating the LPO process, compound 4d breaks up
the chain of its development most efficiently, and this
produces a high antiperoxidant effect. Further increase of
the spacer length leads to a gradual decrease in activity,
because the radical-scavenging region falls through into
the depth of the lipid bilayer. Antioxidants now practi-
cally do not protect the membrane any longer from
radical species penetrating from the aqueous phase.
However, since the aliphatic spacer is a flexible structure,
and the phenolic hydroxyl group can reside for sometime
within the area of LPO occurrence, compounds 4e–f still
retain a sufficient inhibitory activity.
Quaternary ammonium salt 5, with its side chain length
close to an optimum (n ¼ 10) and having a considerable
lipophilicity, is, nevertheless, the least active of the group
of antioxidants studied. This may be explained by the
circumstance that compound 5 causes disruption of the
lipid bilayer structure, leading to intensification of LPO.
In conclusion, synthesis of new membrane-addressed
antioxidants 4a–f and 5 has been described in this paper,
and structure–antioxidant activity relationship in the
series of compounds prepared has been discussed. The
side chain consisting of eight methylene groups has been
shown to provide maximum antioxidant activity. It has
also been demonstrated that replacement of a carboxyl
group in an amphiphilic antioxidant structure by a
quaternary nitrogen leads to a considerable deteriora-
tion of inhibitory properties. As a result of this work, an
effective antioxidant (I4d=Ia-T ¼ 3:5) has been found that
can be regarded as a primary structure in designing drug
products for prevention and treatment of diseases
caused by the oxidative stress.
Acknowledgements
The work was supported, in part, by ISTC Grant B-434.
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