Synthesis, pulse radiolysis, and in vitro radioprotection studies of melatoninolipoamide, a novel conjugate of melatonin and α-lipoic acid

Article abstract of DOI:10.1016/j.bmc.2006.05.042

A novel conjugate of melatonin 2 and α-lipoic acid 4 has been prepared using DCC mediated coupling. The conjugate named melatoninolipoamide has been assigned its structure 1 on the basis of spectral analysis (UV, IR, NMR, and EI-MS). Pulse radiolysis studies of the conjugate were carried out in aqueous solutions with both oxidizing and reducing radicals. The results indicate that the melatonin moiety of the conjugate reacts preferably with oxidizing radicals and the lipoic acid moiety exhibits preferential reaction with reducing radicals. The in vitro radioprotection ability of 1 was examined by γ-radiation induced lipid peroxidation in liposomes and hemolysis of erythrocytes, and compared the results with those of melatonin and α-lipoic acid. The studies suggest that the conjugate can be explored as a probable radioprotector.

Full text of DOI:10.1016/j.bmc.2006.05.042

Bioorganic & Medicinal Chemistry 14 (2006) 6414–6419
Synthesis, pulse radiolysis, and in vitro radioprotection
studies of melatoninolipoamide, a novel conjugate
of melatonin and a-lipoic acid
a
a,
b
b
S. R. Venkatachalam,a,* A. Salaskar, A. Chattopadhyay, A. Barik, B. Mishra,
*
R. Gangabhagirathic and K. I. Priyadarsinib,*
aBio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
bRadiation and Photochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
cRadiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
Received 27 March 2006; revised 19 May 2006; accepted 20 May 2006
Available online 12 June 2006
Abstract—A novel conjugate of melatonin 2 and a-lipoic acid 4 has been prepared using DCC mediated coupling. The conjugate
named melatoninolipoamide has been assigned its structure 1 on the basis of spectral analysis (UV, IR, NMR, and EI-MS). Pulse
radiolysis studies of the conjugate were carried out in aqueous solutions with both oxidizing and reducing radicals. The results indi-
cate that the melatonin moiety of the conjugate reacts preferably with oxidizing radicals and the lipoic acid moiety exhibits prefer-
ential reaction with reducing radicals. The in vitro radioprotection ability of 1 was examined by c-radiation induced lipid
peroxidation in liposomes and hemolysis of erythrocytes, and compared the results with those of melatonin and a-lipoic acid.
The studies suggest that the conjugate can be explored as a probable radioprotector.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
injury, neurodegeneration, hypertension, and hypergly-
cemia.10,11 a-Lipoic acid has also been identified as a
drug for the treatment of diabetic polyneuropathy,
age-related memory decline, and liver diseases.12–14
The aforementioned bioactivities of both melatonin
and a-lipoic acid prompted us to devise a rational strat-
egy directed toward the synthesis of novel derivatives
possessing enhanced activities compared to the parent
molecules. This report presents our work on the synthe-
sis, characterization, evaluation of antioxidant and
radioprotection activities, and pulse radiolysis of a novel
conjugate of melatonin and a-lipoic acid for which we
have assigned the name melatoninolipoamide (1).
The importance of antioxidants in health and prophy-
laxis is now well established.1 Melatonin is a pineal
gland hormone which regulates circadian rhythms; crit-
ically controlling the sleep–wake cycle.2 It has been iden-
tified as a potent antioxidant as well as radioprotectant.3
Besides, this hormone has importance as a prophylactic
and therapeutic agent. Thus, melatonin has a role in the
prevention of cancer,4 neurodegenerative diseases,4 and
diabetic complications,5 and is a potential therapeutic
agent for breast cancer6 besides it possessing immuno-
modulating activity.7 a-Lipoic acid is an essential coen-
zyme for the activity of several key enzyme complexes.8
Like melatonin, it is also a well-known antioxidant and
radioprotecting agent with implications in prophylaxis
and therapy.9 This coenzyme is reported to play a role
in the prevention of cataract, HIV-activation, radiation
2. Results and discussion
2.1. Synthesis of melatoninolipoamide 1
The conjugate melatoninolipoamide 1 was prepared
starting from melatonin 2 and a-lipoic acid 4 ( )
following a route as shown in Scheme 1. Amide hydro-
lysis of 2 in presence of strong acid, followed by base
treatment, afforded 5-methoxytryptamine 3. The latter
Keywords: Melatoninolipoamide; Melatonin; a-Lipoic acid; Bioconju-
gate; Pulse radiolysis; Lipid peroxidation; Radioprotector.
*
Corresponding authors. Tel.: +91 22 2559 5422; fax: +91 22 2550
5151 (A.C.); e-mail: achat@apsara.barc.ernet.in
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.042

S. R. Venkatachalam et al. / Bioorg. Med. Chem. 14 (2006) 6414–6419
6415
MeO
(CH2)2NHCOMe
i, ii
MeO
(CH2)2NH2
NH
NH
2
3
S
S
iii
(CH2)4COOH
MeO
(CH2)2NHCO(CH2)4
3
+
1
4
S
S
NH
Scheme 1. Reagents and conditions: (i) 10% H2SO4, heat; (ii) 20% NaOH; (iii) DCC, CHCl3/THF (8:1), rt.
on condensation with 4 in presence of DCC15 afforded 1
in good yield. The product was purified by thin-layer
chromatography (silica gel, 35% EtOAc in CHCl3) and
characterized by spectral analysis. Mass spectrum
showed the molecular ion peak at m/e 378. The signal
at 1648 cmꢀ1 in its IR spectrum indicates the presence
of amide functionality which was further confirmed
from the signal at 178 in its 13C NMR spectrum. There
is a good resemblance of its UV spectrum with that of
melatonin. Its 1H- and 13C-spectra gave evidence for
aromatic and aliphatic signals attributed to its melato-
nin and lipoic acid segments, respectively (see Section 4).
radical is known to react with organic compounds by
different reaction channels (abstraction, addition, and
electron transfer). In order to differentiate various
pathways of ÅOH radical reaction, pulse radiolysis
Åꢀ
studies were carried out with Br2 radical, which is a
specific one-electron oxidant with EoðBr2Åꢀ=2Brꢀ ¼
1:7 VÞ.21 The transient spectrum on oxidation by
Åꢀ
Br2 radical is given in Figure 1b. Comparing the two
spectra it can be seen that the transient formed by
one-electron oxidation shows two distinct peaks one at
330 nm region and the other at 520–530 nm region., which
mainly correspond to the one-electron oxidized species
from the melatonin moiety, which is an indolyl-type rad-
ical.18 The broad, featureless absorption in the 350–
450 nm region in Figure 1a therefore may correspond to
either hydroxyl radical adducts or the sulfur centered rad-
icals formed by the oxidation of the a-lipoic acid moiety.
The reaction with other specific one-electron oxidant,
2.2. Pulse radiolysis study of the conjugate 1
Reactions of radiolytically produced oxidizing and
reducing radicals with 1 were studied and wherever re-
quired, the results were compared with those of melato-
nin and lipoic acid, and related compounds which have
been published in the literature extensively.16–20
CCl3 O2 , also produced similar type22 of transient as seen
Å
by the transient spectrum in Figure 1c. The bimolecular
rate constant for the reaction of the conjugate with
Åꢀ
Å
2.2.1. Oxidizing radicals. For these studies, hydroxyl
Br2
and CCl3O2 radical was determined to be
radicals (ÅOÅH), Br2 radicals, and CCl3O2 radicals were
employed. OH radical reaction with 1 at neutral pH
produced a transient with absorption spectrum as given
in Figure 1a. It shows broad absorption-maximum
1.3 · 1010 Mꢀ1 sꢀ1 and 4.4 · 108 Mꢀ1 sꢀ1, respectively.
Åꢀ
Å
In order to further confirm the nature of oxidation,
the transient spectra were followed as a function of
pH in the pH range from 2 to 7. Figure 2 gives the
Å
around 480–520 nm and a sharp band at 330 nm. OH
20.0
9.0
8.0
15.0
7.0
15.0
6.0
5.0
4.0
a
a
10.0
5.0
2
3
4
5
6
7
10.0
pH
c
b
5.0
0.0
b
c
0.0
300
350
400
450
500
550
600
300
350
400
450
500
550
600
Wavelength, nm
Wavelength, nm
Figure 2. Transient absorption spectra of 1 (2.12 · 10ꢀ4 M) on pulse
radiolysis of N2O-saturated aqueous solution and in presence of 0.1 M
Brꢀ at pH 3 (a), pH 4.7 (b), and pH 6.8 (c). Inset shows the variation of
transient absorption of 1 (2.12 · 10ꢀ4 M) as a function of pH at
550 nm.
Figure 1. Transient absorption spectra of 1 (2.12 · 10ꢀ4 M) on pulse
radiolysis of N2O-saturated aqueous solution at pH 6.8 (a), in presence
of 0.1 M Brꢀ (b). Spectrum (c) is transient absorption spectra of 1
(2.12 · 10ꢀ4 M) on pulse radiolysis of aerated aqueous solution of
CCl4 and isopropanol mixture.

6416
S. R. Venkatachalam et al. / Bioorg. Med. Chem. 14 (2006) 6414–6419
CH3O
transient spectra at three different pH 3.0,Åꢀ4.7, and
6.8, where the oxidation is induced by Br2 radical
reaction. As the spectra significantly differ with the
pH, to evaluate the radical pKa, the absorption chang-
es (inset of Fig. 2) at 430 and 550 nm were followed
as a function of pH from 2 to 7, which showed an
inflection point due to the pKa of the transient at
5.02. This pKa matches well with that of the indolyl
radical formed by the one-electron oxidation of mela-
tonin and other indolic derivatives.16–18
.
+
oxidising
radicals
NH
CO
N
H
CH3O
indolyl
radical
NH
CO
N
H
S
S
reducing
radicals
S
S
CH3O
NH
CO
N
H
The above results suggest that the reaction of oxidizing
radicals preferentially takes place on the melatonin moi-
ety of the conjugate. This was also confirmed by inde-
Åꢀ
pendently studying the reactions of Br2 radicals with
_
S
melatonin and a-lipoic acid. At pH 7, the one-electron
oxidation potential of a-lipoic acid is 1.1 V versus
NHE,19 while that of melatonin is 0.95 V versus
NHE,16 therefore the melatonin moiety in the conjugate
should undergo oxidation preferentially over the lipoic
acid moiety. The indolyl radicals of melatonin show sim-
ilar type of transient spectra as that of the complex, but
.
disulphide
anion radical
S
Scheme 2.
the disulfide bond of a-lipoic acid forming anion radical,
while melatonin is less reactive toward hydrated elec-
tron.16,19,20 Based on these oxidation and reduction
studies, it can be inferred that 1 has two parts, of which
one is reactive toward the oxidizing radicals, that is, the
melatonin moiety, while the other part undergoes reac-
tion with reducing radical, that is, lipoic acid moiety.
The results are therefore encouraging to predict that 1
has the ability to react with both oxidizing and reducing
free radicals. The radicals generated during oxidative
stress are mostly oxidizing radicals.23 However, some
reducing type of radicals may attack the lipoic acid moi-
ety forming dihydrolipoic acid, which is an efficient
scavenger of superoxide radicals.24 However, the relative
reactivity by different moieties in the conjugate will be
decided by the one-electron reduction potentials for
the formation of radical anions of melatonin, a-lipoic
acid, and the conjugate. The literature reported value
for the reduction of a-lipoic acid of ꢀ0.29 V however
corresponds to two-electron process,25 while many free
radicals react by one-electron process. Therefore, accu-
rate estimation of one-electron reduction potentials un-
der physiological conditions is necessary, our future
experiments will be directed toward this. However based
on these preliminary pulse radiolysis studies, it can be
concluded that the conjugate 1 can be effective toward
most of the free radicals produced. The overall reaction
of oxidizing and reducing radicals with 1 is summarized
in Scheme 2.
a-lipoic acid shows altogether different transient spectra
Åꢀ
on reaction with Br2
maximum at ꢁ425 nm.19
radical, having absorption
2.2.2. Reactions of reducing radicals. Pulse radiolysis
studies of the conjugate 1 were carried out with hydrated
electrons (eaqꢀ) at neutral pH in the presence of tert-bu-
Å
tanol, which scavenges OH radical produced by radiol-
ysis. The transient spectrum having absorption
maximum at 400 nm, obtained from the reaction of 1
with eaqꢀ, is shown in Figure 3, which is very similar
to the a-lipoic acid anion radical. A small shoulder at
330 nm may correspond to the anion radicalꢀof melato-
nin. The rate constant for the reaction of eaq with a-li-
poic acid is reported to be 1.5 · 1010 Mꢀ1 sꢀ1, while that
of melatonin is 4.2 · 108 Mꢀ1 sꢀ1
.
This difference in
17,19
rate constants indicates that the hydrated electron pref-
erentially attacks the lipoic acid part of 1. It is reported
in the literature that the hydrated electron reacts with
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
2.3. In vitro radioprotection studies with conjugate 1
2.3.1. Radiation induced hemolysis of erythrocytes. Mela-
tonin has been reported to protect RBC against super-
oxide radical26 and peroxynitrite mediated hemolysis
in bovine erythrocytes.27 This has been explained on
the basis of its radical scavenging ability. Since ÅOH rad-
icals and other free radicals are implicated in radiation
induced erythrocyte damage, we expect that the conju-
gate may also exhibit similar protection to RBC against
radiation induced hemolysis. For this, the reduction in
300
350
400
450
500
550
Wavelength, nm
Figure 3. Transient absorption spectra of 1 (2.12 · 10ꢀ4 M) and on
pulse radiolysis of N2-saturated aqueous solution and in presence of
0.3 M tert-butanol at pH 6.8.

S. R. Venkatachalam et al. / Bioorg. Med. Chem. 14 (2006) 6414–6419
6417
1.6
Melatonin
Conjugate
Lipoic acid
control
80
60
40
20
0
Melatonin
Lipoic acid
conjugate
control
1.2
0.8
0.4
25
[Substrate],μM
0
50
100
0.0
50
100
μM
0
Figure 4. Percentage hemolysis in human erythrocytes after irradiating
with c-rays (75 Gy) in the absence and presence of different concen-
trations of melatonin, a-lipoic acid, and the conjugate. All the
solutions contain 0.5% ethanol and control indicates percentage
hemolysis after irradiation of erythrocytes containing 0.5% ethanol
(p < 0.001 compared to control).
[Substrate],
Figure 5. Inhibition of c-radiation induced lipid peroxidation in
liposomes (1 mg/ml) in presence and absence of melatonin, a-lipoic
acid, and their conjugate 1. (absorbed dose 280 Gy)
in formation of malonaldehyde, which reacts with thio-
barbituric acid to produce TBARS.23,28 At a constant c-
radiation dose of 280 Gy, inhibition of peroxidation was
followed at 50 lM and 100 lM. The TBARS formation
was found to decrease with increasing concentration of 1
as shown in Figure 5. It protected the liposomes from
peroxidation by 54.1% and 70.0% at 50 lM and
100 lM concentration, respectively. At the same concen-
trations, a-lipoic acid was found to inhibit the peroxida-
tion by 61.8% and 73.4%, while melatonin protected the
liposomes by 40.9% and 51.8% at 50 lM and 100 lM,
respectively. The studies showed that 1 protects lipo-
somes from peroxidation more than that by melatonin
but its activity is less compared to a-lipoic acid.
absorbance at 540 nm due to the released hemoglobin
was monitored in presence of melatonin, a-lipoic acid,
and 1 by independent experiments. Figure 4 gives per-
centage hemolysis in human erythrocytes in the absence
and presence of the added substrates, 1 or melatonin or
a-lipoic acid. In the same figure, percentage hemolysis
on irradiation of erythrocytes in presence of 0.5% etha-
nol is given as control. Both melatonin and a-lipoic acid
inhibited hemolysis, respectively, by 32.9% and 44.5% at
50 lM and 45.9%, and 54.1% at 100 lM concentration.
Under similar conditions, 1 showed significant reduction
in hemolysis of irradiated samples. It imparted up to
52% and 68% protections at 50 lM and 100 lM concen-
trations. If we compare the percentage protection at
similar concentrations of 1, or melatonin or a-lipoic
acid, the protection imparted by the conjugate was
much higher than either a-lipoic acid or melatonin
alone. However, since the conjugate is a combination
of two molecules, we compared the percentage protec-
tion of hemolysis at 25 lM of 1, which is half the origi-
nal concentration (i.e., 50 lM). It can be seen that at
half the concentration, the protection rendered by the
conjugate is in between that of melatonin and a-lipoic
acid. This indicates that the conjugate can inhibit the
hemolysis induced by radiation at similar molar concen-
tration, which may be due to the fact that it may tempo-
rarily increase the resistance of erythrocyte membrane to
free radical aggression, induced by c-radiation. The con-
jugate therefore seems to be a promising candidate for
use as a radioprotector and also as a protector against
free radical mediated erythrocytic damage.
3. Conclusions
A new conjugate of melatonin and a-lipoic acid was syn-
thesized, purified, and characterized as 1. Both melato-
nin and a-lipoic acid are known to exhibit good
radioprotecting ability and are excellent free radical
scavengers. It is therefore expected that the conjugate
1 formed by the amide linkage of the two parent residual
moieties can show good radioprotecting ability. Pulse
radiolysis induced one-electron oxidation and reduction
of 1 showed that the melatonin moiety in the conjugate
is more susceptible for the oxidation, while the lipoic
acid moiety for the reduction. Thus, the conjugate can
react with all types of free radicals formed during the
radiation injury. It was found to be superior in protect-
ing erythrocytes from radiation induced hemolysis and
is a moderate inhibitor of radiation induced lipid perox-
idation. These preliminary studies indicate that 1 can be
regarded as a promising radioprotector. In view of re-
cent finding that conjugates of L-Dopa and Dopamine
and (R)-a-lipoic acid are multifunctional co-drugs with
antioxidant properties,30 the present work provides
another example of lipoic acid conjugates possessing
similar kind of biological potential.
2.3.2. c-Radiation induced lipid peroxidation in liposomes.
The extent of c-radiation induced lipid peroxidation in
liposomes was monitored as TBARS28,29 in the absence
and presence of 1 and compared with that in presence of
melatonin and a-lipoic acid individually. In such condi-
tions, the radicals formed by radiolysis of water, mainly
ÅOH radicals, induce peroxidation in the lipids, resulting

6418
S. R. Venkatachalam et al. / Bioorg. Med. Chem. 14 (2006) 6414–6419
4. Experimental
radical reactions, 1 was dissolved in the matrix, which
is given below. The pH of the solution was adjusted
by phosphate buffers. Nanopure water from Millipore
system was used for preparing the solutions and freshly
prepared solutions were used for each experiment. Elec-
tron pulses from 7 MeV linear electron accelerator were
used for irradiation.32 The absorbed dose was measured
Chemicals used as starting materials are of Aldrich
make and were used without further purification. All
solvents used for extraction and chromatography were
distilled twice at atmospheric pressure prior to use. Tet-
rahydrofuran was dried by heating over LiAlH4. The IR
spectra were recorded with a Perkin-Elmer spectropho-
Åꢀ
using thiocyanate dosimeter by monitoring the ðSCNÞ
2
1
tometer model 837. The H and 13C NMR spectra were
species at 500 nm.33 Typical dose/pulse used for these
studies varied from 15 to 16 Gy.
scanned with a Brucker Ac-200 (200 MHz) instrument
in CDCl3. Electron impact (EI) mass spectrum was
recorded on a ShimadzuQP5050A mass spectrometer.
The organic extracts were desiccated over dry Na2SO4.
Å
Radiolysis of water34,35 produces eaqꢀ, HÅ and OH rad-
icals in addition to the molecular products H2, H2O2,
and H3O+.
4.1. Synthesis of melatoninolipoamide 1
H2O ! eꢀaq; HÅ; ÅOH;H2; H2O2; H3Oþ
ð1Þ
Melatonin 2 (464 mg, 2 mmol, Acros Organics, USA,
99%) was refluxed with 10% aq H2SO4 (25 ml) over a
period of 8 h. The mixture was cooled, treated with
20% aq NaOH solution till basic, and then extracted
with EtOAc. The combined organic extract was washed
with water, brine and dried. Solvent removal under re-
duced pressure afforded a yellow solid residue. This
was purified by column chromatography (neutral alumi-
na, 0–5% MeOH in CHCl3) to afford 5-methoxytrypta-
mine 3 (265 mg, 69.7%).31a
The reaction of ÅOH radical was studied in N2O-saturat-
ꢀ
ed condition where all the eaq is quantitatively convert-
ed into OH radical.
Å
eaꢀq þ N2O H!2O N2 þ OHꢀ þ ÅOH
ð2Þ
More selective oxidizing radicals, Br2 radicals, were
Åꢀ
Å
generated by the reaction of OH radicals (in N2O-satu-
rated solutions) with KBr (0.1 M) according to the reac-
tions and are briefly described below:
Brꢀ + ÅOH ! BrÅ + OHꢀ
BrÅ þ Brꢀ ! BrÅ2ꢀ
ð3Þ
A mixture of a-lipoic acid 4 (250 mg, 1.21 mmol, Al-
drich, 98%, ) and DCC (248 mg, 1.2 mmol, Aldrich,
99%) in a solvent mixture of CH2Cl2/THF (8:1, 50 ml)
was stirred at ambient temperature for 4 h. To this,
amine 3 (200 mg, 1.05 mmol) was added and stirring
was continued overnight. Solvent was evaporated under
reduced pressure to give a reddish mass which was puri-
fied by preparative thin-layer chromatography over sili-
ca gel plate (35:65 EtOAc in CHCl3) to yield 1 as a pale
yellowish semi-solid (178 mg, yield 45%).31b IR (CHCl3)
3420, 3310, 1648 cmꢀ1; UV (kmax): 300, 275 nm. 1H
NMR (CDCl3): 1.3–1.7 (m, 6H), 1.84 (m, 1H), 2.07 (t,
J = 7.2 Hz, 2H), 2.32 (m, 1H), 2.90 (t, 6.4 Hz, 2H),
3.0–3.1 (m, 2H), 3.40–3.56 (m, 3H), 3.80 (s, 3H), 5.91
(br s, 1H), 6.81 (d, J = 6 Hz, 1H), 6.93 (m, 2H), 7.19
(d, J = 6.8 Hz, 1H), 8.79 (s, 1H). 13C NMR (CDCl3):
25.09, 28.60, 34.29, 36.22, 38.20, 39.49, 39.92, 55.75,
56.22, 100.20, 100.80, 111.97, 122.95, 127.45, 131.45,
153.58, 172.87, 211.04. EI-MS: 378 (M+), 173 (100%),
100 (30%), 145 (6%). Anal. Calcd for C19H26O2N2 S2:
C, 60.28; H, 6.92; N, 7.40%. Found: C, 60.55; H, 6.68;
N, 7.59%.
ð4Þ
ꢀ
Chloroperoxyl radicals were generated by allowing eaq
to react with CCl4 (4% by volume) in aerated or oxygen-
saturated solution. 2-Propanol (48% by volume) reacts
with OH and HÅ to give the reducing acetone ketyl
Å
Å
radical, which in turn reduces CCl4 compounds. CCl3
reacts with oxygen to form CCl3 O2 .22
Å
CCl4 þ eꢀaq ! ÅCCl3 þ Clꢀ
ð5Þ
Å
(CH3)2CHOH + HÅ/ÅOH ! (CH3)2C OH + H2/ H2O
ð6Þ
Å
Å
(CH3)2C OH + CCl4 ! CCl3 + (CH3)2CO + HCl ð7Þ
ÅCCl3 + O2 ! CCl3OOÅ
ð8Þ
4.2. Pulse radiolysis studies
Å
ꢀ
eaq reaction can be studied by scavenging OH radical
by adding 0.3 mol dmꢀ3 tert-butanol under N2-saturat-
ed condition.
As the conjugate 1 is sparingly soluble in water, it was
first dissolved in some organic solvent like acetonitrile.
A known amount of this solution is taken in a flask
and the solvent was evaporated on a rotary evaporator
Å
ÅOH + (CH3)3COH ! CH2 (CH3)2COH + H2O ð9Þ
Å
to form a thin film. For OH radical reaction, the film
was incubated and sonicated with aqueous buffer solu-
4.3. Studies on hemolysis of erythrocytes
tion and the saturated solution was used for the tran-
Åꢀ
Human RBCs were procured from venous blood and
washed five times with phosphate-buffered saline (PBS)
solution (pH 7.4) at 4 ꢁC. They were then irradiated in
hypotonic saline to an absorbed dose of 75 Gy in the
sient studies. For reactions with Br2 radicals, a
known amount of acetonitrile solution of 1 was taken
and then diluted with buffer solution such that the
Å
acetonitrile content does not exceed 10%. For CCl3 O2

S. R. Venkatachalam et al. / Bioorg. Med. Chem. 14 (2006) 6414–6419
6419
12. Ou, P.; Tritschler, H. J.; Wolff, S. P. Biochem. Parmacols.
1995, 50, 123–126.
13. Stoll, S.; Rostock, A.; Bartsch, R.; Korn, E.; Meichelbock,
A.; Mueller, W. E. Ann. N. Y. Acad. Sci. 1994, 717,
122–128.
14. Arderwald, C.; Koca, G.; Fuernsinn, C.; Roden, M. Liver
2002, 22, 356–362.
15. Adlam, G.; Blagbrough, I. S.; Taylor, S.; Latham, H. C.;
Roger, A. Bioorg. Med. Chem. Lett. 1994, 4, 2435–2440.
16. Mahal, H. S.; Sharma, H. S.; Mukherjee, T. Free Radical
Biol. Med. 1999, 26, 557–565.
absence and in the presence of different concentrations
of melatonin, a-lipoic acid, and 1 (25, 50, and
100 lM). The compounds were dissolved in ethanol
such that the final concentration of ethanol was less than
0.5% during irradiation with appropriate controls. The
suspensions were centrifuged and the absorbance in
the supernatant was measured at 540 nm to analyze
the extent of hemolysis in each sample.26 The statistical
significance of the difference in the parameters between
the drug containing samples and the appropriate con-
trols was assessed by Student’s ‘t’ test and the errors cor-
respond to the standard deviation.
17. Roberts, J. E.; Hu, D. N.; Wishrat, J. F. J. Photochem.
Photobiol. 1998, B. 42, 125–132.
18. Naik, G. H.; Priyadarsini, K. I.; Maity, D. K.; Mohan, H.
J. Phys. Chem. A 2005, 109, 2062–2068.
4.4. Lipid peroxidation
ˇ ´
19. Bonifacic, M.; Asmus, K. D. J. Chem. Soc., Perkin Trans.
2 1986, 1805–1809.
Lipid peroxidation studies were carried out in phospha-
tidylcholine liposomes prepared according to the proce-
dure given in reference.36 Lamellar liposomes of 150 nm
size suspended in pH 7.4, phosphate buffer were subject-
ed to c-radiation. c-Radiolysis was carried out using
60Co c-source with a dose rate of 48 Gy minꢀ1 as mea-
sured by standard Fricke dosimetry.37 It was followed
at different absorbed doses in N2O/O2-purged liposomal
solution in absence and presence of the compounds
(melatonin, a-lipoic acid, and 1) at pH 7.4 (phosphate
buffer). The detailed methodology used in the lipid per-
oxidation is given in earlier references.28,29 The extent of
lipid peroxidation was estimated in terms of thiobarbitu-
ric acid reactive substances (TBARS) using 15% w/v tri-
chloroacetic acid, 0.375% w/v TBA, 0.25 N hydrochloric
acid, and 0.05% w/v BHT as TBA reagent measuring the
absorbance at 532 nm (e532 = 1.56 · 105 Mꢀ1 cmꢀ1).
ˇ ´
20. Go¨bl, M.; Bonifacic, M.; Asmus, K. D. J. Am. Chem. Soc.
1986, 106, 5984–5988.
21. Wardman, P. J. Phys. Chem. Ref. Data 1989, 18,
1637–1723.
22. Packer, J. E.; Wilson, R. L.; Bahnemann, D.; Asmus, K.
D. J. Chem. Soc., Perkin Trans. 2 1980, 296–299.
23. Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology
and Medicine, 2nd ed.; Clarendon Press: Oxford, 1989.
24. Packer, L.; Witt, E. H.; Tritschler, H. J. Free Radical Biol.
Med. 1995, 19, 227–250.
25. Datta, G.; Bera, T. Biochim. Biophys. Acta 2001, 1512,
149–157.
26. Kwak, C. S.; Mun, K. C.; Suh, H. I. Transplant Proc.
2002, 34, 2654–2655.
27. Wrobel, A.; Lukaszynska, B.; Kedzierska, J. Cell Mol.
Biol. Lett. 2003, 8, 455–460.
28. Sreejayan, N.; Rao, M. N. A.; Priyadarsini, K. I.;
Devasagayam, T. P. A. Int. J. Pharmac. 1997, 151,
127–130.
29. Khopde, S. M.; Priyadarsini, K. I.; Guha, S. N.; Satav, J.
C.; Venkatesan, P.; Rao, M. N. Biosci. Biotech. Biochem.
2000, 64, 503–509.
References and notes
30. Stefano, A.; Sozio, P.; Cocco, A.; Iannitelli, A.; Santucci,
E.; Coosta, M.; Pecci, L.; Nasuti, C.; Cantalmessa, F.;
Pinnen, F. J. Med. Chem. 2006, 49, 1486–1493.
31. (a) He, L.; Li, J. L.; Zhang, J. J.; Su, P. Synth. Commun.
2003, 33, 741–747; (b) The same DCC mediated coupling
reaction between 4 and the crude hydrolysis product
obtained from 2 was also performed to produce 1 in 30%
yield.
1. Packer, L.; Fuchs, J. In Handbook of Antioxidants;
Cadenas, E., Packer, L., Eds.; Marcel Decker Inc: New
York, 1996; pp iii–v.
2. Zisapel, N. Drug Dev. Res. 2000, 50, 226–234.
3. Vijayalaxmi, M.; Reiter, R. J.; Herman, T. S.; Meltz, M.
L. Mutat. Res. 1996, 371, 221–228.
4. Cavalieri, E. L.; Rogan, E. PCT. Int. Appl.
WO2003077900, A1, 25 September, 2003.
5. Baydas, G.; Canatan, H.; Turkoglu, A. J. Pineal Res.
2002, 32, 225–230.
32. Guha, S. N.; Moorthy, P. N.; Kishore, K.; Naik, D. B.;
Rao, K. N. Proc. Indian Acad. Sci. (Chem. Sci.) 1987, 99,
261–271.
33. Fielden, E. M. In The Study of Fast Processes and
Transient Species by Electron Pulse Radiolysis. Baxendale,
J. H., Busi, R. D., Eds.; Reidel publishing Co: London,
1984; p 59.
34. Buxton, G. V.; Greenstock, C. L.; Helman, W. P.; Ross,
A. B. J. Phys. Chem. Ref. Data 1988, 17, 513–886.
35. Neta, P.; Huie, R. E.; Ross, A. B. J. Phys. Chem. Ref.
Data 1988, 17, 1027–1284.
36. Pandey, B. N.; Mishra, K. P. Radiat. Phys. Chem. 1999,
54, 481–489.
37. Spinks, J. W. T.; Woods, R. J. An introduction to radiation
chemistry, third ed.; Weily Interscience Publication, 1990,
pp 95–101.
6. Cos, S.; Sanchez, B. E. Histol. Histopathol. 2000, 15,
637–647.
7. Cerea, G.; Vaghi, M.; Ardizzoia, A.; Villa, S.; Bucovec,
R.; Mengo, S. Anticancer Res. 2003, 23, 1951–1954.
8. Wada, M.; Yasuno, R.; Jordan, S. W.; Croman, J. E.
Plant Cell Physiol. 2001, 42, 650–656.
9. Packer, L.; Witt, E. H.; Tritschler, H. J. In Handbook of
Antioxidants; Cadenas, E., Packer, L., Eds.; Marcel
Dekker Inc: New York, 1996; pp 545–591.
10. Packer, L.; Tritschler, H. J.; Wessel, K. Free Radical Biol.
Med. 1997, 22, 359–378.
11. El Midaouri, A.; de-Champlen, J.; Elimadi, A.; Wu, L.;
Haddad-Pierre, S. Am. J. Hypertension 2003, 16, 173–179.