Cyclic voltammetry as an indicator of antioxidant activity

Article abstract of DOI:10.1071/CH01193

A series of compounds based around combining the neuroprotective properties of non-competitive N-methyl D-aspartic acid (NMDA) receptor antagonists with antioxidant functionalities have been prepared. The redox chemistry of these compounds has been evalua

Full text of DOI:10.1071/CH01193

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Corrigendum
Aust. J. Chem. 2002, 55, iii
Cyclic Voltammetry as an Indicator of Antioxidant Activity
Alexandra Papanikos,A,B John Eklund,A W. Roy Jackson,C,D Vijaya B. Kenche,A Eva M. Campi,C
Alan D. Robertson,E Bevyn Jarrott,F Philip M. Beart,F Fiona E. MunroF and Jennifer K CallawayF
A School of Chemistry, P.O. Box 23, Monash University, Vic. 3800, Australia.
B Current address: Carlsberg Laboratory, Department of Chemistry, Copenhagen DK-2500, Denmark.
C Centre for Green Chemistry, P.O. Box 23, Monash University, Vic. 3800, Australia.
D Author to whom correspondence should be addressed (e-mail: w.r.jackson@sci.monash.edu.au).
E AMRAD Operations Pty Ltd, Richmond, Vic. 3121, Australia.
F Department of Pharmacology, Monash University, Clayton, Vic. 3800, Australia.
C
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0 193_C
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Aust. J. Chem. 2002, 55, pp. 205–212
Please note the correct affiliation of authors as indicated above.
© CSIRO 2002
0004-9425/02/050iii

Aust. J. Chem. 2002, 55, 205–212
Cyclic Voltammetry as an Indicator of Antioxidant Activity
Alexandra Papanikos,A,B John Eklund,A W. Roy Jackson,C,D Vijaya B. Kenche,A
Eva M. Campi,C Alan D. Robertson,D Bevyn Jarrott,E Philip M. Beart,E
Fiona E. MunroE and Jennifer K CallawayE
A School of Chemistry, P.O. Box 23, Monash University, Vic. 3800, Australia.
B Current address: Carlsberg Laboratory, Department of Chemistry, Copenhagen DK-2500, Denmark.
C Centre for Green Chemistry, P.O. Box 23, Monash University, Vic. 3800, Australia.
D Author to whom correspondence should be addressed (e-mail: w.r.jackson@sci.monash.edu.au).
E AMRAD Operations Pty Ltd, Richmond, Vic. 3121, Australia.
F Department of Pharmacology, Monash University, Clayton, Vic. 3800, Australia.
A series of compounds based around combining the neuroprotective properties of non-competitive N-methyl
D-aspartic acid (NMDA) receptor antagonists with antioxidant functionalities have been prepared. The redox
chemistry of these compounds has been evaluated using cyclic voltammetry, and the results have been compared
with their radical-scavenging properties obtained from two standard biological assays, the inhibition of lipid
peroxidation (thiobarbituric acid reacting substances assay) and the Sapphire colorimetric assay. Results from these
different methods show general concordance. The most effective antioxidants were substituted phenols, e.g.
Trolox®. The antioxidant activity of a series of pyrimidines was shown to be dependent on the presence of three
amino substituents.
Manuscript received: 5 December 2001.
Final version: 4 February 2002.
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Introduction
Novel hybrid molecules based around combining the
neuroprotective properties of derivatives of the
non-competitive NMDA receptor antagonists ifenprodil (1)
and eliprodil[1,2] (2) with different antioxidant
functionalities have been prepared.[3,4] The aim of linking
two differently acting neuroprotective agents together in a
single molecule was to obtain additive or even synergistic
effects on the biological activity. Antioxidant groups used
for structural modifications of ifenprodil fragments were
based on triaminopyrimidines, which have been previously
incorporated into steroids e.g. tirilazad mesylate.[5] This
strategy has led to compounds (3), (4), and (5). In addition,
incorporation of the more commonly used antioxidants,[5],
butylated hydroxytoluene and Trolox®, has led to
compounds (6) and (7). The antioxidant properties of each
of these molecules have been evaluated using cyclic
voltammetry,
inhibition
of
lipid
peroxidation
(thiobarbituric acid reacting substances, TBARS, assay)[6]
and a Sapphire colorimetric assay.[6,7] The compounds have
shown great promise in the management of ischaemic
episodes, such as stroke, and one of them is undergoing
Phase 1 trials in a London hospital. A large number of
compounds with substituent variation throughout
molecules (3)–(7) have been prepared and evaluated. We
felt it necessary to establish an independent, non-biological
method for the evaluation of antioxidant activity for these
molecules. The samples chosen for this study were those
with the highest activity.
© CSIRO 2002
10.1071/CH01193
0004-9425/02/030205

206
A. Papanikos et al.
pyrimidines (9a–c). Reaction with piperazine at 120°C in a
Carius tube using pyridine as a solvent gave (10a–c) in
superior yields to those reported using milder reaction
conditions.[8]. Reaction with α-bromo-p-bromoaceto-
phenone followed by reduction of the intermediate ketones
(11) gave the target amino alcohols (3a–c).
Unsymmetrical (4) and Symmetrical (5)
Ethanolaminopyrimidines
Reaction
of
the
ethanolamine
(12)
with
2,4,6-trichloropyrimidine (8) gave the readily separated
unsymmetrical (13) and symmetrical (14) dichloro-
pyrimidines which, on reaction with pyrrolidine, gave the
target compounds (4) and (5) (Scheme 2).
Scheme
2
Results and Discussion
Chemistry
The t-Butylhydroxytoluene Derivative (6) and the Trolox®
Derivative (7)
Triaminopyrimidines (3a–c)
α-Bromo-p-chloroacetophenone
was
reacted
with
ethylpiperazinecarboxylate and the product decarboxylated
to give (15). This compound was coupled with the
bromomethylphenol (16) and the product reduced to give (6)
(Scheme 3). 1,3-Dicyclohexylcarbodiimide promoted
These compounds were prepared by the method outlined in
Scheme 1. 2,4,6-Trichloropyrimidine (8) was reacted with
the appropriate amine, pyrrolidine, diethylamine, or
morpholine to give mainly the 2,4-diamino-6-chloro-
Scheme
1

Cyclic Voltammetry as an Indicator of Antioxidant Activity
207
even in the absence of compound (3a). It was concluded that
the observed electrochemical process was associated with
oxidation of the acetate buffer.
Further cyclic-voltammetric studies were thus carried out
in either 0.1 M aqueous KCl solution or in CH3CN with
Bu4NPF6 as the electrolyte. The latter medium was used for
bulk electrolysis experiments. The peak oxidative potentials
recorded at a scan rate of 100 mV s−1 were similar in both
solvent systems.
Scheme
3
coupling of (15) with the Trolox® acid (17), and reduction of
the resulting amide with LiAlH4/AlCl3 gave (7) (Scheme 3).
The Hydroxyethylpyrimidine (20)
6-Methyl-2,4-bis(pyrrolidin-l-yl)pyrimidine
(18)
was
lithitated by heating under reflux with BunLi in the presence
of N,N,N´,N´-tetramethylethylenediamine to form the
lithium salt (19) which was reacted with p-bromo-
benzaldehyde to give (20) (Scheme 4).
Fig. 1. Cyclic voltammogram of compound (4).
The three closely related compounds (3a–c) exhibited
similar voltammetric behaviour and the results are
Cyclic Voltammetry Studies
summarized in Table 1. An irreversible anodic oxidation
ox
pl
(peak oxidative potential, E ) was evident at scan rates
Initial attempts to study the cyclic voltammograms of the
above compounds involved the use of conditions previously
described for recording the cyclic voltammograms of the
between 10 and 2000 mV s–1, indicating that the
cation-radical formed initially decomposed within the
time-scale of these voltammetric experiments. The oxidation
potential of the three compounds (3a–c) showed some
21-aminosteroid containing
a
dipyrrolidinopiperazo-
pyrimidine, Tirilazad mesylate (U74-006F), an antioxidant
described by the Upjohn Company.[9] The voltammogram
was recorded for solutions in 30% (v/v) aqueous ethanol
variation, with the pyrrolidinyl compound (3a) being the
ox
pl
strongest antioxidant (lowest E at 0.85 V). The redox
process was found to be diffusion controlled as the peak
oxidative current (Iopx) scaled linearly with the square root of
the scan rate.[10,11] Evidence for a small amount of
absorption onto the electrode surface was found in a few
cases at high scan rates due to observed decreases in the peak
oxidative current. A voltammogram for the closely related
compound (4), which does not contain the piperazine ring of
(3a–c), is shown in Figure 1 and closely resembles those for
(3a–c). In addition to the irreversible oxidation at 0.94 V,
with 0.2 M sodium acetate as buffer. A peak oxidative
ox
potential (E ) of 0.23 V was recorded (versus Ag/AgCl as
pl
1
the reference electrode, scan rate 100 mV s– ) using a glassy
carbon working electrode and a Pt auxiliary electrode. The
voltammogram of compound (3a), which bears the same
antioxidant moiety as U74-006F, was recorded using the
conditions described above. The initial voltammogram
appeared to show that compound (3a) exhibited identical
voltammetric behaviour to that of U74-006F, but it was later
revealed that the same voltammogram could be produced
evidence for
a
reversible reduction at −0.12
V
Scheme
4

208
A. Papanikos et al.
Table 1. Comparison of electrochemical and biological evaluations of antioxidant activity
ox
pl
Cpd
No.
E
TBARS assay
Sapphire assay
Fe3+ (100 mM)A
IC50 (mM)B maxC
(V)
Fe3+ (100 mM)A
IC50 (mM)B
Fe3+ (1000 mM)A
IC50 (mM)B
maxC
maxC
(3a)
(3b)
(3c)
(4)
(5)
(6)
0.85
0.90
0.98
0.94
0.99
0.78
24 (7–44)
90
63
63
49
71
95
97
62
70E
55
81
59
40
29
72
91
70
123 (87–180)
559 (444–864)
393 (298–528)
147 (62–528)
902 (788–1175)
62 (46–82)
87
76
68
39
51
81
95
41
146 (55–443)
386 (319–469)
427 (288–718)
800 (626–1497)
50 (39–64)
3 (1.5–5)
191 (35–3533)
542 (463–634)
700 (510–1305)
839 (559–990)
37 (22–53)
(7)
(20)
0.50
19 (9–40)
> 1000
4 (2.5–5.5)
> 1000
D
> 1000
A
B
C
Concentration of Fe3+ used to stimulate lipid peroxidation.
95% confidence limits in parentheses.
Maximum
percentage inhibition. D No oxidative behaviour. E Confidence limits not calculated.
(Erped), associated with reduction of the pyrimidinium salts,
was observed. The mechanism of the electrochemical
reduction of pyrimidinium salts has been studied
previously.[12]
µM). The results for these assays alongside the voltammetric
data described above are summarized in Table 1.
In general there is good concordance between the three
sets of results. The biological evaluation of compound (20)
with only two nitrogen substituents on the pyrimidine ring
gave IC50 values > 1000 µM, consistent with the lack of
electrochemical behaviour. All the triamino-substituted
pyrimidines (3a–c), (4), and (5) showed mild antioxidant
activity relative to the phenolic compounds (6) and (7).
Surprisingly, the pyrrolidinyl compound (3a) showed higher
activity than (3b), (3c), (4), and (5) in both biological and
electrochemical evaluations.
In order to identify a minimum structural element
required for antioxidant activity, the electrochemical
behavior of the isolated fragments, 2,4-diamino-
6-chloropyrimidine, piperazine, α-bromo-p-bromaceto-
phenone, and compound (20) (which lacks the nitrogen
substitution at C(6) of the pyrimidine ring compared with
(3a)) was investigated. All these compounds showed no
oxidation potential. This result strongly suggests that the
minimum structural element required for antioxidant activity
is a 2-amino-substituted pyrimidine (as in (5)), or a
6-amino-substituted pyrimidine (as in (3) and (4)), but more
likely triamino-substitution of the pyrimidine is essential.
This concordance provides an independent, non-
biological method for the evaluation of antioxidant activity.
Experimental
The symmetrically substituted compound (5) had a
Syntheses
ox
slightly higher oxidation potential (E 0.99 V) than the
pl
Melting points (m.p.) are uncorrected. Infrared spectra were recorded
on a Perkin–Elmer 1600 FT-IR spectrophotometer. 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded using Bruker
AC-200, AM-300 and DRX-400 spectrometers for solutions in
base-washed CDCl3 unless otherwise stated. Electrospray ionization
mass spectra (ESI+) were measured on a Bruker BioApex 47 e– FTMS
(Fourier transform mass spectrometer) with a 4.7 Tesla magnet and an
Analytica electrospray source. Electron impact mass spectra (EI) were
recorded on a VG TRIO-1 Quadrupole Mass Spectrometer at 70 eV
with a source temperature of 200°C. Microanalyses were performed by
Chemical and Microanalytical Services Pty Ltd, Melbourne. Merck
Silica Gel 60 (230–400 mesh, no. 9385) was used for flash
chromatography. Light petroleum (b.p. range 60–80°C) was used for
chromatography. High-performance liquid chromatography (HPLC)
was performed on a Waters Model 6000A (Column Deltapak C18-100
Å, 19 mm by 30 cm, 15 µ or Column HAIsil C18-300 Å, 25 mm by 30
cm, 5 µ), Waters gradient program Model 660 and Waters Model 481
detector. Solvent systems of acetonitrile/water were used.
ox
pl
unsymmetrical analogue (4) (E 0.94 V). Bulk electrolysis
of (4), followed by liquid chromatography mass
spectrometry of the resulting solution, showed an oxidized
species with m/z [M–2]+ as the major product, suggesting
loss of hydrogen from a radical cation initially formed on the
N atom at C(6) or on one of the N-atoms in the pyrimidine
ring.
The compounds (6) and (7), which contain phenolic
antioxidants, both showed irreversible oxidative peak
ox
pl
potentials (E 0.78 and 0.5 V, respectively) indicative of
strong and very strong antioxidant activity, respectively. The
voltammogram of the Trolox® containing compound (7)
showed a second irreversible oxidation which was attributed
to oxidation of one of the readily formed decomposition
products of the original molecule.
2,4-Bis-1-amino-6-chloropyrimidines (9a–c)
The three diamines were prepared by the literature method.[13]
Biological Evaluation of Antioxidant Activity
Compounds (3)–(7) and (20) were examined using the
TBARS assay[6] and the Sapphire colorimetric assay.[6,7] The
results from the TBARS and Sapphire assays represent 50%
inhibition (IC50 values) of malondialdehyde (MDA)
formation in rat-brain homogenates obtained from
concentration response curves. Lipid peroxidation was
stimulated using two concentrations of Fe3+ (100 and 1000
2,4-Bis(pyrrolidin-1-yl)-6-chloropyrimidine (9a). (1.23 g, 89%
had m.p. 76.8–77.2°C (lit.[13] 77–79°C) and identical spectroscopic
data to those recorded previously.
2,4-Bis(N,N-diethylamino)-6-chloropyrimidine (9b). 2,4,6-Tri-
chloropyrimidine (8) (1.0 g, 5.5 mmol) was reacted with diethylamine
(10 mL) and pyridine (10 mL) for 5 days at ambient temperature
(23°C). Chromatography (ethyl acetate/light petroleum, 1:19) gave the

Cyclic Voltammetry as an Indicator of Antioxidant Activity
209
title compound (9b) as a colourless liquid (1.14 g, 81%), b.p. 85°C
(oven)/0.2 mm (Found: C, 56.2; H, 8.4; N, 21.8%. C12H21ClN4 requires
C, 56.1; H, 8.2; N, 21.8%). νmax (neat) 2974s, 2932s, 1578s (br), 1509s,
1491s, 1458s, 1431s, 1376s, 1362s, 1329s, 1261s, 1186s, 1130s, 1081s,
H3´, H5´; 2.66–2.73, m, 2H, NCH2CH(OH); 3.32–3.61, m, 12H,
NCH2CH2, H2´, and H6´; 4.65, dd, J 10.0, 3.8 Hz, 1H, NCH2CH(OH);
4.79, s, 1H, H5; 7.19, d, J 8.4Hz, 2H and 7.39, d, J 8.4 Hz, 2H, ArH. 13
C
NMR δ (75 MHz) 25.3; 25.5; 44.5; 46.0; 46.2; 52.85; 66.1; 68.2; 72.5;
121.2; 127.5; 131.4; 141.2; 160.2; 162.5; 164.0. Mass spectrum (ESI,
CH3OH) m/z 501.2 [M(79Br)+H]+, 503.2 [M(81Br)+H]+.
1
966s, 806s, 782s cm–1. H NMR δ (200 MHz) 1.16, t, J 7.1 Hz, 12H,
NCH2CH3; 3.42, q, J 7.0 Hz, 4H and 3.55, q, J 7.0 Hz, 4H, NCH2CH3;
5.70, s, 1H, H5. 13C NMR δ (50 MHz) 13.0; 13.3; 41.6; 42.2; 89.3;
159.6; 160.3; 162.0. Mass spectrum (ESI, CH3CN) m/z 259
[M(37Cl)+H]+, 257 [M(35Cl)+H]+.
6-{4-[2-(4-Bromophenyl)-2-hydroxy]ethylpiperazin-1-yl}-2,4-bis-
(N,N-diethylamino)pyrimidine
(3b). Chromatography
(ethyl
acetate/light petroleum, 2:5) followed by semi-preparative HPLC
(Waters 6000A HPLC, column HAIsil C18-300 Å, 25 mm by 30 cm,
5 µ) gave (3b) as colourless crystals (44%) (Found: [M+H]+ 505.2274.
C24H3779BrN6O requires [M+H]+ 505.2290). νmax (CH2Cl2) 3054s,
2976s (br), 2931s, 2834s, 1566s (br), 1488s, 1451s, 1425s, 1375s,
1325 s, 1270s, 1239s, 1216s, 1165s, 1141s, 1114s, 1080s, 1010s,
1001s, 896s, 790s, 758s (br) cm–1. 1H NMR δ (300 MHz) 1.15, t, J 7.0
Hz, 12H, NCH2CH3; 2.41–2.56, m, 4H, H3´, H5´; 2.74–2.81, m, 2H,
NCH2CH(OH); 3.43, q, J 7.1 Hz, 4H, NCH2CH3 3.48–3.63, m, 8H,
H2´, H6´ and NCH2CH3; 4.73, dd, J 10.1, 3.7 Hz, 1H, NCH2CH(OH);
4.97, s, 1H, H5; 7.26, d, J 8.5 Hz, 2H and 7.47, d, J 8.5 Hz, 2H, ArH.
13C NMR δ (50 MHz) 13.3; 13.6; 41.5; 42.0; 44.5; 52.9; 66.1; 68.1;
71.2; 121.2; 127.5; 131.4; 141.1; 160.5; 162.8; 164.5. Mass spectrum
(EI) m/z 505 [(M(81Br)]+, (18%), 503 (11), 502 (18), 425 (13), 320 (12),
319 (41), 251 (16), 250 (100), 237 (47), 56 (10).
2,4-Bis(morpholino)-6-chloropyrimidine (9c). The compound
was prepared as for (9a) as a white solid (1.33 g, 86%), m.p. 145–146°C
(Found: C, 50.7; H, 6.1; N, 19.6%. C12H17ClN4O2 requires C, 50.6; H,
6.0; N, 19.7%). νmax (Nujol) 2930s (br), 2856s, 1579s (br), 1546s (br),
1465s (br), 1378s, 1366s, 1305s, 1242s, 1164s, 1114s, 1006s, 966s,
953s cm–1. 1H NMR δ (200 MHz) 3.53–3.58, m, 4H, NCH2CH2O;
3.64–3.78, m, 12H, NCH2CH2O and NCH2CH2O; 5.87, s, 1H, H5. 13
C
NMR δ (50 MHz) 44.2; 44.3; 66.5; 66.8; 91.1; 160.6; 160.8; 163.4.
Mass spectrum (ESI, CH3CN) m/z 287 [M(37Cl)+H]+, 285
[M(35Cl)+H]+.
2,4-Bis(diamino)-6-piperazin-1-ylpyrimidines (10a–c)
2,4-Bis(pyrrolidin-l-yl)-6-piperazin-1-ylpyrimidine (10a).
This
was prepared as described by Jacobsen[13] as a white solid (1.02 g,
85%), m.p. 180°C (dec.) (lit.[13] 177–178°C) with identical
spectroscopic values to the literature.
6-{4-[2-(4-Bromophenyl)-2-hydroxy]ethylpiperazin-1-yl}-2,4-bis-
(morpholino)pyrimidine (3c). Chromatography (ethyl acetate/light
petroleum, 4:1) followed by recrystallization (ethyl acetate/light
petroleum, 1:3) gave (3c) as a white solid (64%), m.p. 188.7–189.7°C
(Found: C, 54.0; H, 6.2; N, 15.8%. C24H33BrN6O3 requires C, 54.0; H,
6.2; N, 15.8%). νmax (CH2Cl2) 3054s, 2987s, 1564s (br), 1422s, 1266s
(br), 1230s (br), 1212s, 1119s, 1010s, 1002s, 896s, 754s, 668s cm–1. 1H
NMR δ (200 MHz) 2.44–2.57, m, 4H, H2´, H6´; 2.74–2.81, m, 2H,
NCH2CH(OH); 3.48–3.51, m, 4H, H3´, H5´; 3.56–3.80, m, 16H,
NCH2CH2O and NCH2CH2O; 3.98, br s, 1H, OH; 4.72, dd, J 9.4, 3.9
Hz, 1H, NCH2CH(OH); 5.10, s, 1H, H5; 7.24, d, J 8.4 Hz, 2H and 7.46,
d, J 8.4 Hz, 2H, ArH. 13C NMR δ (50 MHz) 44.4; 44.8; 52.8; 66.0; 66.7;
67.0; 68.2; 73.3; 121.3; 127.5; 131.5; 141.0; 161.2; 164.5; 164.9. Mass
spectrum (ESI, CH3OH) m/z 535 [M(81Br)+H]+, 533 [M(79Br)+H]+.
2,4-Bis(N,N-diethylamino)-6-piperazin-1-ylpyrimidine (10b). This
was prepared as a white solid (2.29 g, 96%), m.p. 184.3–185.5°C (dec.)
(Found: [M+H]+ 307.2598. Calc. for C16H31N6: [M+H]+ 307.2619).
νmax (neat) 3300s (br), 2964s (br), 2844s (br), 1574s (br), 1538s (br),
1455s (br), 1372s (br), 1322s, 1272s, 1267s, 1238s, 1225s, 1211s,
1190s, 1164s, 1080s (br), 1032s, 998s, 888s (br), 813s, 790s, 772s, 673s
cm–1. 1H NMR δ (400 MHz) 1.12, t, J 7.0 Hz, 12H, NCH2CH3; 2.90, t,
J 5.1 Hz, 4H, H3´, H5´; 3.41, q, J 7.0 Hz, 4H, NCH2CH3; 3.45, t, J 5.1
Hz, 4H, H2´, H6´; 3.53, q, J 7.0 Hz, 4H, NCH2CH3; 4.94, s, 1H, H5.
13C NMR δ (100 MHz) 13.3; 13.6; 41.5; 42.0; 45.6; 45.9; 71.2; 160.5;
162.8; 164.8. Mass spectrum (ESI, CH3OH) m/z 307 [M+H]+. The 1H
NMR data were consistent with literature data.[8]
2,4-Bis(morpholino)-6-piperazin-1-ylpyrimidine
(10c).[8] This
2-Amino-1-(4-bromophenyl)ethanol (12)
was prepared as a pale yellow solid (0.96 g, 82%), m.p. 219–220°C
(dec.) (Found: [M+H]+ 335.2184. Calc. for C16H26N6O2: [M+H]+
335.2195). νmax (CH2Cl2) 3054s, 2987s, 1564s (br), 1426s, 1365s (br),
1306s, 1268s (br), 1229s, 1212s, 1195s, 1119s, 1008s, 896s, 790s, 746s
(br) cm–1. 1H NMR δ (200 MHz) 2.92–2.97, m, 4H, H3´, H5´;
3.40–3.56, m, 8H, H2´, H6´ and NCH2CH2O; 3.61–3.79, m, 13H,
NCH2CH2O, NCH2CH2O and NH; 5.10, s, 1H, H5. 13C NMR δ (50
MHz) 44.4; 44.5; 44.9; 51.3; 66.8; 67.1; 73.4; 161.3; 164.7; 165.0.
Mass spectrum (ESI, CH3CN) m/z 335 [M+H]+.
The reaction of 2-(4-bromophenyl)-2-hydroxyacetonitrile (1.84 g,
8.7 mmol) in dry tetrahydrofuran (30 mL) with lithium aluminium
hydride in tetrahydrofuran (1 M) (17.4 mL, 17.4 mmol) gave a yellow
solid. Trituration with ether gave (12) as a white solid (0.90 g, 77%),
m.p. 113.4–114.1°C (lit.[14] 112–113.5°C) (Found: C, 44.4; H, 4.7; N,
6.5%. Calc. for C8H10BrNO: C, 44.5; H, 4.7; N, 6.5%). νmax (Nujol)
1
2926s (br), 2855s, 1459s (br) cm–1. H NMR δ (400 MHz) 1.82, br s,
3H, OH, NH2; 2.75, dd, J 12.7, 7.8 Hz, 1H and 3.00, dd, J 12.7, 3.9 Hz,
1H, H2; 4.58, dd, J 7.8, 3.9 Hz, 1H, H1; 7.24, d, J 8.3 Hz, 2H and 7.47,
d, J 8.3 Hz, 2H, ArH. 13C NMR δ (50 MHz) 49.1; 73.4; 121.2; 127.6;
131.5; 141.5. Mass spectrum (ESI, CH3CN) m/z 216 [M(79Br)+H]+,
218 [M(81Br)+H]+.
6-{4-[2-(4-Bromophenyl)-2-hydroxy]ethylpiperazin-1-yl}-2,4-
bis(diamino)pyrimidines (3a–c)
A solution of 2,4´-dibromoacetophenone (1.5 mmol), (10a–c) (1.5
mmol), and triethylamine (10 mL) in tetrahydrofuran (20 mL) was
heated under reflux for 4 h. The reaction mixture was cooled, filtered
and the filtrate concentrated under vacuum. The crude material was
redissolved in dichloromethane (30 mL), washed with water (20 mL)
followed by saturated NaCl (20 mL) and dried (MgSO4). After
evaporation of the solvent, the crude compound was reduced with
lithium aluminium hydride (1.5 mmol) in tetrahydrofuran (20 mL) at
0°C under N2 for 4 h. The mixture was quenched with Na2SO4·10H2O
until the evolution of hydrogen ceased. The mixture was then filtered
and the solvent removed under vacuum. Flash chromatography on silica
gel, with ethyl acetate/light petroleum as eluent, gave the pyrimidines
(3a–c) as white solids.
6-[(4-Bromophenyl)-2-hydroxyethylamino]-2,4-dichloropyrimidine
(13) and 2-[(4-Bromophenyl)-2-hydroxyethylamino]-4,6-
dichloropyrimidine (14)
A solution of 2,4,6-trichloropyrimidine (0.85 g, 4.63 mmol) in dioxan
(10 mL) was added dropwise at ambient temperature to a stirred
solution of (12) (1.0 g, 4.63 mmol) in dioxan (20 mL). Once the
addition was complete, sodium bicarbonate (2.33 g, 27.8 mmol) was
added and the resulting mixture was heated to reflux for 7 h. The
mixture was cooled, quenched with water (40 mL) and the pH was
adjusted to 12 by addition of a 2 M solution of NaOH. The solution was
extracted with dichloromethane (3 × 40 mL) and the combined extracts
were dried (MgSO4) and evaporated to give an orange gum (1.87 g).
The 1H NMR spectrum of the crude product indicated that (13) and (14)
were present in a ca. 1:1 ratio. Chromatography (diethyl ether/light
petroleum, 1:1) gave (14) and (13) as white solids (0.87 g, 51%) and
(0.81 g, 47%) respectively.
6-{4-[2-(4-Bromophenyl)-2-hydroxy]ethylpiperazin-1-yl}-2,4-bis-
(pyrrolidin-l-yl)-pyrimidine (3a). (70%) m.p. 168–169°C (Found:
[M+H]+ 501.1976. C24H3479BrN6O [M+H]+ requires 501.1978). 1H
NMR δ (300 MHz) 1.79–1.87, m, 8H, NCH2CH2; 2.37–2.48, m, 4H,

210
A. Papanikos et al.
Compound (13). m.p. 130.0–131.4°C (Found: C, 39.6; H 2.8; N,
1-[2-(4-Chlorophenyl)-2-hydroxy]ethyl-4-[3,5-bis(1,1-dimethylethyl)-
4-hydroxyphenyl]methylpiperazine (6) and 1-[2-(4-Chlorophenyl)-2-
hydroxy]ethyl-4-[6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-1-
benzopyran-2-yl]methylpiperazine (7)
11.6%. C12H10BrCl2N3O requires C, 39.7; H, 2.8; N, 11.6%). νmax
(Nujol) 3301s, 3244s (br), 3156s, 3104s, 2925s (br), 2855s, 1604s (br),
1579s, 1508s, 1500s, 1459s (br), 1376s, 1361s, 1288s, 1274s, 1204s,
1
1125s, 1076s, 1045s, 1010s, 981s, 823s cm–1. H NMR δ (200 MHz,
A solution of 2-bromo-4´-chloroacetophenone (0.7 g, 3 mmol), ethyl
1-piperazinecarboxylate (0.47 g, 3 mmol), and triethylamine (0.42 mL,
3 mmol) in diethyl ether (10 mL) was heated under reflux for 3 h
(reaction was monitored by thin-layer chromatography, TLC). The
reaction mixture was cooled and further diluted with ethyl acetate and
washed with water, brine and dried over Na2SO4. After evaporation of
the solvent under vacuum, the crude viscous liquid was treated with
concentrated. HCl (15 mL) at refluxing temperature for 48 h. The
cooled reaction mixture was made alkaline by addition of 50% NaOH
and extracted with ethyl acetate. The combined extracts were washed
with water, brine and dried over Na2SO4. Evaporation of the solvent
CD3COCD3) 2.89, br s, 1H, OH or NH; 3.40–3.59, m, 1H (3.45, dd, J
13.6, 7.7 Hz, 1H on D2O exchange), H1´a; 3.71–3.80, m, 1H (3.64, dd,
J 13.6, 4.4 Hz, 1H on D2O exchange), H1´b; 4.83–4.95, m, 2H (4.83,
dd, J 7.5, 4.4 Hz, 1H on D2O exchange), H2´ and NH or OH; 6.63, s,
1H, H5; 7.42, d, J 8.5 Hz, 2H and 7.51, d, J 8.5 Hz, 2H, ArH. [1H NMR
(200 MHz, CDCl3) 6.30, br s, H5.] 13C NMR δ (100 MHz, CD3COCD3)
49.2; 72.3; 103.7; 121.5; 129.0; 132.0; 143.2; 158.9; 160.5; 165.6. Mass
spectrum (ESI, CH3OH) m/z 362, 364, 366 and 368 [M+H]+.
Compound (14). m.p. 143.1–143.7°C (Found: C, 39.7; H 2.7; N,
11.6%. C12H10BrCl2N3O requires C, 39.7; H, 2.8; N, 11.6%). νmax
(Nujol) 3474s (br), 3279s, 3122s, 2924s (br), 2854s, 1594s, 1581s,
gave (15) as
a semisolid, which was reacted without further
1
1568s, 1521s, 1456s (br), 1420s, 1378s, 1068s, 560s cm–1. H NMR
purification. 1H NMR δ (300 MHz) 2.54, m, 4H, NCH2CH2NH; 2.93,
m, 4H, NCH2CH2NH; 3.69, s, 2H, CH2CO; 7.35, d, J 8.6 Hz, 2H and
7.88, d, J 8.6 Hz, 2H, ArH.
δ (200 MHz) 2.97, d, J 3.5 Hz, 1H, OH or NH; 3.51, ddd, J 14.2, 7.7,
5.3 Hz, 1H (3.49, dd, J 14.2, 7.7 Hz, 1H on D2O exchange), H1´a; 3.83,
ddd, J 14.2, 7.0, 3.6 Hz, 1H (3.81, dd, J 14.2, 3.6 Hz, 1H on D2O
exchange), H1´b; 4.89–4.96, m, 1H, H2´; 5.86, br s, 1H, NH or OH;
6.64, s, 1H, H5; 7.29, d, J 8.4 Hz, 2H and 7.50, d, J 8.4 Hz, 2H, ArH.
13C NMR δ (100 MHz) 49.0; 72.8; 109.7; 122.0; 127.6; 131.8; 140.5;
161.8; 162.0. Mass spectrum (ESI, CH3OH) m/z 362, 364, 366 and 368
[M+H]+. Note that the 13C NMR spectrum of the symmetrical isomer
(14) shows two quaternary carbons for the pyrimidine ring where the
unsymmetrical isomer (13) shows three such carbons thus confirming
the structural assignment.
A
solution of (15) (0.48 g, 2 mmol) and 4-(bromo-
methyl)-2,6-bis(1,1-dimethylethyl)phenol (16) (0.55 g, 2 mmol) in
tetrahydrofuran (10 mL) was stirred at ambient temperature for 4 h
(reaction monitored by TLC). The reaction mixture was concentrated,
treated with aqueous sodium bicarbonate and extracted with ethyl
acetate. The combined extracts were washed with water, then with
brine, and dried with Na2SO4. After evaporation of the solvent, the
crude compound was reduced with LiAlH4 (1.25 mmol) using diethyl
ether (10 mL) as solvent at 0°C under N2 atmosphere (2 h). The reaction
was quenched by the dropwise addition of ice-cold water (2 mL) and
Na2SO4. The reaction mixture was further diluted with ethyl acetate
(50 mL) and filtered. The filtrate was washed with water, then with
brine, and dried over Na2SO4. After evaporation of the solvent under
vacuum, the crude product was subjected to flash chromatography on
silica gel. Elution with 70% ethyl acetate/light petroleum gave (6) as a
white solid (0.83 g 90%), m.p. 149–150°C (Found: C, 70.4; H, 8.6; N,
6-[2-(4-Bromophenyl)-2-hydroxyethylamino]-2,4-bis(pyrrolidin-1-yl)-
pyrimidine (4)
The pyrimidine (13) (0.47 g, 1.3 mmol) was heated to reflux with
pyrrolidine (10 mL) for 4 h. The mixture was cooled to ambient
temperature, diluted with dichloromethane (20 mL) and washed with
saturated NaHCO3 (3x 20 mL). The organic phase was dried (MgSO4),
filtered and the solvent was removed under vacuum. Recrystallization
(ethyl acetate/light petroleum, 3:2) gave (4) as a white solid (0.41 g,
73%), m.p. 180°C (dec.) (Found: C, 55.9; H, 5.8; N, 15.9%.
C20H26BrN5O requires C, 55.6; H, 6.1; N, 16.2%). νmax (CH2Cl2)
3055s, 2987s, 1593s, 1570s, 1476s, 1458s, 1422s, 1270s (br), 896s,
1
6.3%. C27H39ClN2O2 requires C, 70.4; H, 8.6; N, 6.3%). H NMR δ
(300 MHz) 1.4, s, 18H, But; 2.43–2.83, m, 10H, NCH2CH2 and
NCH2CH(OH); 3.51, s, 2H, NCH2Ar; 4.71, dd, J 10.3, 3.4 Hz, 1H,
NCH2CH(OH); 5.17, s, 1H, OH; 7.10, s, 2H and 7.30, s, 4H, ArH. Mass
spectrum (EI) m/z 458, 317, 219 (100%).
The ditartrate salt was prepared by adding a solution of tartaric acid
(2 equiv.) in ethanol to a solution of (6) (1 equiv.) in ethyl acetate. The
homogenous solution was stirred at 60°C for 15 min. The ditartrate salt
precipitated out of the solvent and was filtered off and dried under
vacuum, m.p. 170–172°C (1H NMR 4.16, s, integrated to 4H compared
with But, 1.4, s, 18H).
4-Chloro-2-(piperazin-1-yl)acetophenone (15) was also coupled
with 6-hydroxy-2,5,6,8-tetramethylchroman-2-carboxylic acid (17)
using 1,3-dicyclohexylcarbodiimide under standard conditions. The
resulting oxo amide was reduced as described above using LiAlH4 in
tetrahydrofuran in the presence of AlCl3 (ca. 0.3 equiv.) to give (7)
(Found: [M+H]+ 459.2410. C26H3635ClN2O3 requires [M+H]+
459.2414). 1H NMR δ (300 MHz) 1.23, s, 3H, CH3CO; 1.65–1.8, m, 1H
and 1.9–2.05, m, 1H, CH2CO; 2.08, s, 3H, 2.11, s, 3H and 2.16, s, 3H,
CH3Ar; 2.4–2.9, m, 14H, NCH2CH2, NCH2CH(OH), NCH2CO and
CH2Ar; 4.71–4.77, m, 1H, NCH2CH(OH); 7.35, s, 4H, ArH. The
ditartrate salt was prepared as described above, m.p. 88–90°C (1H NMR
4.20, s, integrated to 4H compared with 2.1, 3 × CH3Ar, 9H).
1
758s (br) cm–1. H NMR δ (300 MHz) 1.89–1.95, m, 8H, NCH2CH2;
3.38–3.47, m, 5H (3.42, dd, J 14.7, 6.2 Hz, 1H on D2O exchange)
NCH2CH2 and H1´a; 3.72, ddd, J 14.6, 6.1, 2.2 Hz, 1H (3.70, dd, J 14.6,
2.2 Hz, 1H on D2O exchange) H1´b; 4.41, t, J 5.7 Hz, 1H, NH; 4.71, s,
1H, H5; 4.82, dd, J 6.1, 1.9 Hz, 1H (4.81, dd, J 6.2, 2.1 Hz, 1H on D2O
exchange) H2´; 7.24, d, J 8.4 Hz, 2H and 7.43, d, J 8.4 Hz, 2H, ArH;
7.95, br s, 1H, OH. 13C NMR δ (100 MHz) 25.3; 25.5; 46.0; 46.4; 50.6;
72.9; 75.0; 120.6; 127.9; 131.2; 142.7; 159.3; 161.4; 163.4. Mass
spectrum (ESI, CH3OH) m/z 432 [M(79Br)+H]+, 434 [M(81Br)+H]+.
2-[2-(4-Bromophenyl)-2-hydroxyethylamino]-4,6-bis(pyrrolidin-1-yl)-
pyrimidine (5)
The pyrimidine (14) (0.30 g, 0.8 mmol) was heated to reflux with
pyrrolidine (10 mL) for 4 h as described above. Recrystallization (ethyl
acetate/light petroleum, 3:1) gave (5) as a white solid (0.28 g, 79%)
m.p. 178.5–179.2° (Found: C, 55.6; H, 6.1; N, 16.2%. C20H26BrN5O
requires C, 55.6; H, 6.1; N, 16.2%). νmax (CH2Cl2) 3054s, 2986s, 2522s,
1589s (br), 1557s, 1522s, 1484s, 1471s (br), 1460s, 1422s, 1352s,
1263s (br), 790s, 718s (br) cm–1. 1H NMR δ (200 MHz) 1.91–1.98, m,
8H, NCH2CH2; 3.35–3.42, m, 8H, NCH2CH2; 3.48, ddd, J 14.8, 6.3, 2.2
Hz, 1H (3.47, dd, J 14.8, 6.6 Hz, 1H on D2O exchange), H1´a; 3.61, ddd,
J 14.8, 6.3, 2.2 Hz, 1H (3.60, dd, J 14.8, 2.2 Hz, 1H on D2O exchange),
H1´b; 4.67, s, 1H, H5; 4.85–4.89, m, 2H, H2´ and NH or OH; 7.28, d, J
8.3 Hz, 2H and 7.44, d, J 8.3 Hz, 2H, ArH; 7.92, br s, 1H, OH or NH.
13C NMR δ (100 MHz) 25.3; 46.3; 50.9; 73.7; 76.0; 120.6; 128.0;
131.1; 142.8; 161.1; 162.6. Mass spectrum (ESI, CH3OH) m/z 432
[M(79Br)+H]+, 434 [M(81Br)+H]+.
6-Methyl-2,4-bis(pyrrolidin-1-yl)pyrimidine (18)
Pyrrolidine (10 mL) was added dropwise to 6-methyl-2,4-
dichloropyrimidine (1.00 g, 6.13 mmol) at 0°C. The reaction mixture
was allowed to warm to ambient temperature and was then heated at
reflux for 4 h. Once complete, the reaction was diluted with CH2Cl2 (30
mL) and washed with saturated aqueous NaHCO3 (3 × 20 mL). The
organic phase was collected, dried (MgSO4), filtered and the solvent
removed under vacuum. The crude material was recrystallized from
ethanol/water (3:1) to yield (18) as a white solid (1.17 g, 82%) m.p.
77–78°C (Found: C, 67.2; H, 8.7; N, 24.1%. C13H20N4 requires C, 67.2;

Cyclic Voltammetry as an Indicator of Antioxidant Activity
211
H, 8.8; N, 24.2%). νmax (Nujol) 2928s (br), 1580s (br), 1456s, 1420s,
1376s, 1345s, 789s cm–1. 1H NMR δ (200 MHz) 1.88–1.97, m, 8H,
NCH2CH2; 2.24, s, 3H, CH3; 3.23–3.59, m, 8H, NCH2CH2; 5.51, s, 1H,
H5. 13C NMR δ (50 MHz) 24.5; 25.3; 25.6; 45.9; 46.3; 91.4; 160.7;
161.2; 164.9. Mass spectrum (ESI, CH3CN) m/z 233 [M+H]+.
the electrodes were rinsed with nano-pure water and then dried with
clean tissue paper. All electrochemical experiments were carried out for
5 × 10–4 M concentrations at 20 2°C.
Bulk electrolysis experiments were carried out using a Bioanalytical
Systems 100 (BAS 100) electrochemical Analyser with a standard
three-electrode arrangement. A glassy carbon working electrode with a
large surface area was employed. A larger surface area platinum gauze
auxiliary electrode was separated from the solution containing the drug
by a salt bridge with a fritted glass disk. The reference electrode was the
non-aqueous Ag/Ag+ (saturated AgNO3). Electrolyses were conducted
using approximately 0.01 mmol of the compound in acetonitrile over a
period of 3 to 4 h until the current was constant. Nitrogen was purged
in the electrochemical cell throughout the entire process. The potential
for bulk electrolysis was selected on the basis of the peak oxidative
potential of each drug at which potential the working-electrode
potential was held 100 mV more positive than the peak potential.
Samples of the solution were evaluated using liquid-chromatography
mass spectroscopy.
6-[2-(4-Bromophenyl)-2-hydroxy]ethyl-2,4-bis(pyrrolidin-1-yl)-
pyrimidine (20)
A solution of n-butyllithium in hexane (2.05 mL, 1.3 M, 2.6 mmol) was
reacted with N,N,N´,N´-tetramethylethylenediamine (0.39 mL, 2.6
mmol) and (18) (0.50 g, 2.2 mmol) in diethyl ether (20 mL) under an
inert atmosphere. The resulting mixture was heated under reflux for 1 h,
cooled to ambient temperature and a solution of 4-bromobenzaldehyde
(0.80 g, 4.3 mmol) in diethyl ether (10 mL) was added. The reaction
mixture was stirred at ambient temperature for 1 h before it was poured
onto ice (20 g) and aqueous sulfuric acid (1 M, 20 mL), and further
diluted with diethyl ether (20 mL). The organic layer was separated; the
aqueous layer was basified to pH 14 by addition of aqueous sodium
hydroxide (4 M) and extracted with dichloromethane (3 × 50 mL). The
combined organic extracts were dried (MgSO4) and the solvent
removed under vacuum to leave a pale-brown coloured gum. Flash
chromatography (ethyl acetate/light petroleum, 1:1) gave (20) as a pale
yellow solid (0.67 g, 74%). A small amount was recrystallized (diethyl
ether/light petroleum, 1:1 and charcoal) and gave (20) as a white solid
m.p. 145.0–146.9°C (Found: C, 57.5; H, 6.0; N, 13.4%. C20H25BrN4O
requires C, 57.6; H, 6.0; N, 13.4%). νmax (Nujol) 2924s (br), 2855s (br),
1589s (br), 1559s, 1495s, 1474s, 1456s, 1416s, 1346s cm–1. 1H NMR δ
(200 MHz) 1.91–1.97, m, 8H, NCH2CH2; 2.76, br s, 2H, H1´;
3.43–3.71, m, 8H, NCH2CH2; 4.99, dd, J 7.1, 4.7 Hz, 1H, H2´; 5.42, s,
1H, H5; 7.33, d, J 8.4 Hz, 2H and 7.45, d, J 8.4 Hz, 2H, ArH; 7.71, br s,
1H, OH. 13C NMR δ (100 MHz) 25.3; 25.5; 44.4; 46.0; 46.4; 72.7; 91.6;
120.7; 127.7; 131.3; 143.7; 159.1; 161.1; 165.4. Mass spectrum (ESI,
CH3CN) m/z 419 [M(81Br)+H]+, 417 [M(79Br)+H]+.
Thiobarbituric Acid Reacting Substances (TBARS) Assay[6]
Rat-brain 10% homogenates and various concentrations of the test
compound were incubated for 10 min at 37°C. An aqueous solution of
100 or 1000 µM Fe3+ was added to stimulate lipid peroxidation, and the
homogenates were further incubated for 30 min at 37°C. The reaction
was stopped by the addition of sodium dodecylsulfate and acetic acid.
The precipitated proteins were then removed by centrifugation.
Aliquots of the clear supernatants were heated with an equal volume of
thiobarbituric acid (TBA) solution. Samples were cooled and
absorbances were read at 532 nm. Blank values containing no
homogenate or drug were subtracted from these values. Each value was
expressed as a percentage of that for samples containing homogenate
and Fe3+ only (i.e. no drug).
Sapphire Assay[6,7]
Cyclic Voltammetry
The procedures outlined in the Sapphire LPO-586 kit were carried
out with the following modifications. Briefly, rat-brain 10%
Reagents and Solvents
homogenates
were
prepared
in
aqueous
20
mM
The potassium chloride (Aldrich) and tetrabutylammonium
hexafluorophosphate (Bu4NPF6) (BDH) used to prepare the electrolyte
solutions were of analytical grade and were used as supplied. The
solvents used for cyclic voltammetry were triply distilled water, and
acetonitrile (CH3CN) (Chromatographic 99.99% HPLC grade
(Mallinckrodt)), which was passed through activated neutral alumina
under nitrogen and stored over 4 Å molecular sieves.
Solutions were prepared just prior to use by dissolution of the
compound of interest as either the hydrochloride or tartrate salt in
aqueous 0.1 M in KCl using nano-pure water. The solutions were
deoxygenated by bubbling nitrogen for ten min prior to each
voltammetric experiment. For the bulk electrolysis experiments, the
compound of interest was dissolved in acetonitrile, which contained
0.01 M tetrabutylammonium hexafluorophosphate (Bu4NPF6).
tris(hydroxymethyl)aminomethane–HCI buffer, pH 7.4 media using
Polytron homogenizer. Assays contained 100 µL of rat-brain
homogenate and various concentrations of test compound (10 µL) and
were incubated for 10 min at 37°C. Lipid peroxidation was then
stimulated by the addition of 10 µL of 100 µM Fe3+ solution and the
homogenates were incubated for
a further 30 min at 37°C.
Chromogenic reagent (1-methyl-2-phenylindole, 325 µL) at the
concentration of 10.3 mM in acetonitrile was added, and the reaction
was initiated by the addition of 75 µL of 37% aqueous HCl to each tube.
The reaction mixture was incubated for 60 min at 45°C, followed by
being cooled on ice, centrifuged at 5000 g for 7 min. 200 µL of each
sample was pipetted into a 96-well microplate and absorbances read at
586 nm using
a Ceres UV900C microplate reader. Following
subtraction of blank values, analysis was performed as for the TBARS
assay.
Methodology
All cyclic and steady-state voltammograms were acquired using a
Cypress Systems Model CS-1090 Computer Controlled
Electroanalytical system in the cyclic staircase mode (with a 2 mV
potential step). The standard three-electrode arrangement was
employed. In all cases a Pt wire auxiliary electrode was used. The
working electrodes of choice for cyclic voltammetry were either a 3.0
mm diameter glassy carbon disc or a 1.0 mm diameter polished
platinum disc. For all aqueous measurements, an aqueous Ag/AgCl
(sat. KCl) reference electrode was used, separated from the solution by
a salt bridge containing an aqueous 0.1 M NaCl solution. For
non-aqueous measurements, the reference electrode was separated
from the main solution by a salt bridge containing 0.01M Bu4NPF6 in
acetonitrile.
Acknowledgments
This project has been supported by AMRAD Operations Pty
Ltd, including the provision of a post-graduate scholarship
(to A.P.). We also thank Dianne Watkins for her assistance in
the biological testing program.
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