Does conjugation of antioxidants improve their antioxidative/anti- inflammatory potential?

Article abstract of DOI:10.1016/j.bmc.2010.10.012

A series of symmetric and asymmetric spermine (SPM) conjugates with all-trans-retinoic acid (ATRA), acitretin (ACI), (E)-3-(trioxsalen-4′-yl) acrylic acid (TRAA) and l-DOPA, amides of ACI, l-DOPA and TRAA with 1-aminobutane, benzylamine, dopamine and 1,12-diaminobutane as well as hybrid conjugates of O,O′-dimethylcaffeic acid (DMCA) with TRAA or N-fumaroyl-indole-3-carboxanilide (FICA) and 2-(2-aminoethoxy)ethanol were synthesized and their antioxidant properties were studied. The reducing activity (RA)% of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay and found to be in the range 0-92(20 min)%/96(60 min)% at 100 μM, the most powerful being the conjugates l-DOPA-SPM-l-DOPA (8, RA = 89%/96%) and l-DOPA-dopamine (13, RA = 92%/92%). Conjugate DMCA-NH(CH₂CH₂O)₂-FICA (14) was the most powerful LOX inhibitor with IC₅₀ 33.5 μM, followed by the conjugates ACI-NHCH₂Ph (10, IC₅₀ 40.5 μM), ACI-SPM-TRAA (7, IC₅₀ 41.5 μM), DMCA-NH(CH₂CH₂O) ₂-TRAA (15, IC₅₀ 65 μM), 13 (IC₅₀ 81.5 μM) and ACI-dopamine (11, IC₅₀ 87 μM). The most potent inhibitors of lipid peroxidation at 100 μM were the conjugates 15 (98%) and ACI-SPM-ACI (4, 97%) whereas all other compounds showed activities comparable or lower than trolox. The most interesting compounds, namely ATRA-SPM-ATRA (3), 4, 10, 11 and 15, as well as unconjugated compounds such as ATRA and dopamine, were studied for their anti-inflammatory activity in vivo on rat paw oedema induced by Carrageenan and found to exhibit, for doses of 0.01 mmol/mL of conjugates per Kg of rat body weight, weaker anti-inflammatory activities (3.6-40%) than indomethacin (47%) with conjugate 3 being the most potent (40%) in this series of compounds. The cytocompatibility of selected compounds was evaluated by the viability of RAMEC cells in the presence of different concentrations (0.5-50 μM) of the compounds. Conjugates 3 (IC₅₀ 2.6 μM) and 4 (IC₅₀ 4.7 μM) were more cytotoxic than the corresponding unconjugated retinoids ATRA (IC₅₀ 18.3 μM) and ACI (IC₅₀ 14.6 μM), whereas conjugate 15 (IC₅₀ 12.9 μM) was less cytotoxic than either DCSP (IC₅₀ 11.3 μM) or the tert-butyl ester of TRAA (IC₅₀ 2.9 μM).

Full text of DOI:10.1016/j.bmc.2010.10.012

Bioorganic & Medicinal Chemistry 18 (2010) 8204–8217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Does conjugation of antioxidants improve their
antioxidative/anti-inflammatory potential?
Dimitra Hadjipavlou-Litina a, , George E. Magoulas b, Stavros E. Bariamis b, Denis Drainas c,
⇑
Konstantinos Avgoustakis d, Dionissios Papaioannou b,
⇑
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
b Laboratory of Synthetic Organic Chemistry, Department of Chemistry, School of Natural Sciences, University of Patras, 26504 Patras, Greece
c Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece
d Laboratory of Pharmaceutical Technology, Department of Pharmacy, University of Patras, 26504 Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of symmetric and asymmetric spermine (SPM) conjugates with all-trans-retinoic acid (ATRA),
acitretin (ACI), (E)-3-(trioxsalen-40-yl)acrylic acid (TRAA) and
L-DOPA, amides of ACI, L-DOPA and TRAA with
Received 16 February 2010
Revised 30 September 2010
Accepted 6 October 2010
Available online 30 October 2010
1-aminobutane, benzylamine, dopamine and 1,12-diaminobutane as well as hybrid conjugates of O,O0-dim-
ethylcaffeic acid (DMCA) with TRAA or N-fumaroyl-indole-3-carboxanilide (FICA) and 2-(2-aminoeth-
oxy)ethanol were synthesized and their antioxidant properties were studied. The reducing activity (RA)%
of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging
Keywords:
Retinoids
Psoralens
Spermine
assay and found to be inthe range 0–92(20 min)%/96(60 min)% at 100
jugates -DOPA-SPM- -DOPA (8, RA = 89%/96%) and -DOPA-dopamine (13, RA = 92%/92%). Conjugate
DMCA-NH(CH2CH2O)2-FICA (14) was the most powerful LOX inhibitor with IC50 33.5 M, followed by the
conjugates ACI-NHCH2Ph (10, IC50 40.5 M), ACI-SPM-TRAA (7, IC50 41.5 M), DMCA-NH(CH2CH2O)2-TRAA
(15, IC50 65 M), 13 (IC50 81.5 M) and ACI-dopamine (11, IC50 87 M). The most potent inhibitors of lipid
peroxidation at 100 M were the conjugates 15 (98%) and ACI-SPM-ACI (4, 97%) whereas all other com-
lM, the most powerfulbeing the con-
L
L
L
l
l
l
Caffeic acid analogs
l
l
l
L-DOPA
Dopamine
Conjugates
l
pounds showed activities comparable or lower than trolox. The most interesting compounds, namely
ATRA-SPM-ATRA (3), 4, 10, 11 and 15, as well as unconjugated compounds such as ATRA and dopamine,
were studied for their anti-inflammatory activity in vivo on rat paw oedema induced by Carrageenan and
found to exhibit, for doses of 0.01 mmol/mL of conjugates per Kg of rat body weight, weaker anti-inflamma-
tory activities (3.6–40%) than indomethacin (47%) with conjugate 3 being the most potent (40%) in this ser-
ies of compounds. The cytocompatibility of selected compounds was evaluated by the viability of RAMEC
Antioxidant activities
Anti-inflammatory activities
Lipoxygenase inhibitors
Lipid peroxidation
Cytotoxicity
cells in the presence of different concentrations (0.5–50
2.6 M) and 4 (IC50 4.7 M) were more cytotoxic than the corresponding unconjugated retinoids ATRA
(IC50 18.3 M) and ACI (IC50 14.6 M), whereas conjugate 15 (IC50 12.9 M) was less cytotoxic than either
DCSP (IC50 11.3 M) or the tert-butyl ester of TRAA (IC50 2.9 M).
lM) of the compounds. Conjugates 3 (IC50
l
l
l
l
l
l
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
rancidity in foods and oxidant damages in vivo, relating to diseases
such as cancer, diabetes, and cardiovascular, Alzheimer’s, and
Parkinson’s diseases. Persistently high levels of ROS can modify
essentially biological molecules, such lipids, proteins and DNA. It
is consistent that rates of ROS production are increased in most
diseases. Oxidative stress has been associated with several human
diseases such as cancer, neurodegenerative syndromes and
inflammation.
Spermine (SPM) is a natural antioxidant, which is found in all
living organisms. Research shows that SPM has an important func-
tion in those areas of the body that are exposed to a high level of
oxygen usage. This pertains to the skin, the brain, sperm cells
and the lungs in particular. Spermine’s role is that of protecting
cells against free radicals and their destructive effects.3
Hydroxycinnamic acid compounds are widely distributed in the
plant kingdom as esters of organic acid or glycosides and they are
bound to protein and other cell wall polymers. They present poten-
tial antioxidative properties involving free radical-scavenging
activity, metal ion chelation, and inhibitory actions on specific
enzymes that induce free radical and lipid hydroperoxide forma-
tion.1,2 Therefore, their antioxidant actions could prevent oxidant
⇑
Corresponding authors. Tel.: +30 2310997627; fax: +30 2310997679 (D.H.-L.);
tel.: +30 2610962954; fax: +30 2610962956 (D.P.).
E-mail addresses: hadjipav@pharm.auth.gr (D. Hadjipavlou-Litina), dapapaio@-
chemistry.upatras.gr (D. Papaioannou).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.012

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8205
Today, there is an increase interest in the combination of two
pharmacophores on the same scaffold. This procedure leads to hy-
brid molecules or conjugates. Hybrid drugs, just as their name im-
plies, combine two drugs in a single molecule with the goal of
creating a chemical entity more medically effective than its indi-
vidual components.4 These combo drugs can indeed be more pow-
erful than either of their precursors.
Kukoamines A (KukA) (Fig. 1) and B (KukB) are SPM alkaloids,
actually SPM conjugates with dihydrocaffeic acid (DHCA), which
were isolated from the medicinal plant Lycium chinense.5 More re-
cently, alkaloids of the kukoamine type, for example, KukA, were
identified in a range of solanaceous species, including potato.6
KukA shows hypotensive activity,5a and is a potent and selective
inhibitor of trypanothione reductase (TryR),7 a crucial enzyme for
the survival of pathogenic trypanosomatid parasites from oxida-
tive stress.
of compounds were examined for their antioxidative properties,
namely analogs of the antipsoriatic drugs trioxsalen (TRX) and aci-
tretin (ACI) (Fig. 1). In the former family of compounds, the most po-
tent LOX inhibitor was the analog tert-butyl 3-(trioxsalen-40-yl)
acrylate (TRAB) which also showed high lipid peroxidation inhibi-
tion (80% at 100 lM using the AAPH assay) and almost identical
anti-inflammatory activity to indomethacin.9 In the latter family
of compounds, although ACI showed a very low LOX activity, it
inhibited at 85% (at 100 lM) lipid peroxidation induced by AAPH
and was the most powerful anti-inflammatory agent in the series
with activity higher (ca. 1.3 times) than indomethacin. Similar inhi-
bition of lipid peroxidation to acitretin was presented by the analog
N-fumaroylindole-3-carboxanilide (FICA) (Fig. 1), which however
had a much more powerful effect in inhibiting LOX.10
There is an increasing interest in antioxidants, particularly in
those intended to prevent the presumed deleterious effects of free
radicals in the human body, and to prevent the deterioration of fats
and other constituents of foodstuffs. Taking into consideration that
conjugation of caffeic acid analogs8 or acidic retinoids like all-
trans-retinoic acid (ATRA) and ACI11 with SPM results in molecular
entities with altered or superior biological properties compared to
the combination of the unconjugated molecules, we decided to
examine the effect of conjugation of various bioactive molecules
on their antioxidative activity. It should be noted that attachment
of lipophilic moieties on the amino function(s) of PAs usually leads
We have recently examined the antioxidative properties of a ser-
ies of KukA analogs and found that KukA was the most potent inhib-
itor of soybean lipoxygenase (LOX) whereas its analog di(O,O0-
dimethylcaffeoyl)spermine (DCSP, Fig. 1), a conjugate of SPM with
O,O0-dimethylcaffeic acid (1), inhibited 100% the lipid peroxidation
at 100 lM using the water-soluble azo compound2,2-azobis(2-ami-
dinopropane) dihydrochloride (AAPH) assay. Both compounds had
almost identical anti-inflammatory activity, which was comparable
to that of the usual standard indomethacin.8 In addition, other types
O
RO
NH2
HO
HO
OH
R
H
RO
CA : R = H
1 : R = Me (DMCA)
:
L-DOPA R = CO2H
2 : R = H
H
N
H2N
N
H
NH2
SPM
O
O
OH
OH
MeO
ATRA
ACI
R
O
H
N
N
OH
O
O
O
O
O
TRX : R = H
t
t
FICA
TRAA : R = CH=CHCO2H
TRAB : R = CH=CHCO2tBu
OH
OH
O
H
N
H
HO
N
N
N
H
H
O
HO
KukA
OMe
OMe
O
H
N
H
MeO
N
N
N
H
H
O
MeO
DCSP
Figure 1. Structures of compounds encountered in the present work.

8206
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
to increased toxicity (SPM to a greater extent than SPD).12 How-
ever, we have chosen to use SPM over spermidine (SPD) as the
PA because the SPD conjugates we have tested so far were invari-
ably less active than the corresponding SPM conjugates8,11 and on
the other hand PAs present antioxidative activity which is corre-
lated to the number of the amino groups in the molecule (SPM
more efficient than SPD).13 For this purpose, we have synthesized
symmetric conjugates of SPM with (a) the acidic retinoids ATRA
and ACI (conjugates 3 and 4, respectively),11 (b) the trioxsalen ana-
log 3-(trioxsalen-40-yl)acrylic acid (TRAA) (conjugate 5)14 and (c)
possible lipoxygenase (LOX) activity of these compounds, pre-
sented in Figures 2 and 3, their potential to inhibit lipid peroxida-
tion of biological membranes and to act as anti-inflammatory
agents as well as evaluate their cytocompatibility. Through these
compounds, we could hopefully determine (a) the effect of conju-
gation of polyamines or simpler amines, such as the benzyl and bu-
tyl amine, dopamine, 1,12-diaminododecane, 2-(2-aminoethoxy)
ethanol, with biologically active molecules, such as retinoids and
analogs, psoralen analogs, caffeic acid analogs and L-DOPA, on the
biological activity and (b) the particular structural characteristics
which might possibly lead to increased antioxidative and/or anti-
inflammatory activities and reduced cytotoxicity.
the known powerful antioxidant L-DOPA (conjugate 8), as well as
hybrid conjugates of TRAA with the afore mentioned acidic reti-
noids (conjugates 6 and 7, respectively) (Fig. 2). An antioxidant ef-
fect of dopamine has been reported15,16 in the literature on lipid
peroxidation (LPO) in rat brain homogenates. L-DOPA and many
other catechol compounds, including norepinephrine, alpha-meth-
yldopamine, and catechol itself, but not monophenols, exhibit a
2. Results and discussion
2.1. Chemistry
The conjugates 3 and 411 and 5 and 714 were synthesized accord-
ing to published procedures. Conjugate 6 was prepared in analogous
manner to conjugate 7, that is by first coupling compound 16
(TRAA)14 with N1,N4,N9-tris(tert-butoxycarbonyl)spermine (Boc3-
SPM),18 in the presence of O-benzotriazole-N,N,N0,N0-tetramethyl-
uronium hexafluoro-phosphate (HBTU), and then removal of the
Boc protecting groups by treatment with trifluoroacetic acid (TFA)
to obtain the monoacylated SPM derivative 17, as the corresponding
tris-trifluoroacetate salt. This was then selectively acylated by the
succinimidyl ‘active’ ester 1811 (Scheme 1) to give conjugate 6 in
45% overall yield.
similar property. L-DOPA was found to be potent in free radical-
scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radicals. Hydroxyl radicals (OH) and superoxide anion radicals
were effectively scavenged by L
-DOPA.17
In addition, in an effort to examine the significance of the sec-
ondary free amino groups in the conjugates, we thought of includ-
ing in our study the conjugate 9 (Fig. 3), considered as an analog of
SPM conjugate 3. Furthermore, we included in the present study
simpler conjugates/amides of ACI, such as compounds 10 and 11,
and of TRAA (compound 12), the conjugate 13 of L-DOPA and dopa-
mine (2) and finally the hybrid conjugates 14 and 15 (Fig. 3) of 1 (a
constituent of SPM conjugate DCSP) and the ACI analog FICA or the
TRX derived acid TRAA. We considered of interest to examine the
The synthesis of conjugate 8 started with the preparation of
N-Trt-L-DOPA (19), following a literature procedure for the synthe-
O
H
N
H
N
N
H
N
H
O
3
4
OMe
O
O
H
N
H
N
N
H
N
H
O
MeO
O
O
O
H
N
H
N
N
H
N
O
H
O
O
5
O
12
2
3
O
O
1
4
29
35
5
28
O
30
31
O
11
6
15
H
N
H
25
1'
3'
4'
19
21
6'
9'
23
7
20
33
24
34
18
N
10
17
27
9 O 8
32
N
H
N
H
16
13
2'
7'
8'
10'
26
5'
22
O
36 37
14
6
OMe
O
O
H
N
H
N
N
H
N
H
O
O
7
8
OH
OH
NH2
H
O
9
8
H
N
H
N
3
1'
4'
3'
4
2
HO
HO
1
N
H
N
H
2'
5'
7
5
H
O
H2N
6
Figure 2. Structures of spermine conjugates synthesized in the context of the present work.

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8207
18
3
17
7
20
19
10
O
H
N
9
5
5'
3'
1'
14
13
15
12
1
N
H
8
6
2'
4
6'
2
4'
O
11
16
9
20
3
19
18
15
O
9
5
23
21
17
22
10
7
24
25
1
14
N
H
8
6
4
2
13
11
16
MeO
12
10
25
28
26
20
OH
18
15
19
7
24
23
22
O
9
5
21
17
10
11
3
27
1
14
13
N
H
OH
8
6
4
2
16
MeO
12
11
12
5
2
3
O
16
13
1
3a
H
H
3b
4
15
14
OH
OH
17
12
O
O
9
6
O
10
11
15
8
7
6
2
HO
HO
18
20
7
1
4
5
N
H
10
11
17
9O
13
16
N
H
19
21
H
H2N
8
14
13
12
28
27
26
25
29
30
O
O
14
3
H
N
9
6
10
13
16
22
8
7
4
MeO
MeO
17
21
O
N
20
19
1
23
24
N
H
O
11
2
12
18
15
5
O
O
14
28
23
24
O
22
25
26
27
O
1
O
O
3
16
30
13
9
10
21
17
8
4
MeO
MeO
O
N
H
O
14
20
2
11
12
15
O19
29
5
7
18
6
15
Figure 3. Structures of other types of conjugates synthesized in the context of the present work.
sis of
L
-amino acids N-protected with the triphenylmethyl (trityl,
-DOPA with Me3SiCl,
2.2. Antioxidant and anti-inflammatory activity
2.2.1. In vitro antioxidant activity studies
Trt) group.19 It involved treatment of
L
followed by N-tritylation and carboxyl group deprotection with
methanolysis. The product 19 was obtained pure as the corre-
sponding diethylammonium salt in 81% yield. Compound 19 was
then converted to the corresponding isolable benzotriazolyl (Bt)
ester (20), according to a literature procedure for the activation
of N-tritylamino acids,20 in 85% yield. This ester was then coupled
to N4,N9-ditritylspermine (N4,N9-Trt2-SPM),21 N-deprotected with
TFA and finally treated with 1.4 M HCl/EtOH to give conjugate 8,
as the corresponding tetrakis-hydrochloride salt in 39% overall
yield.
In the present investigation, the conjugates 3–15 as well as the
parent molecules comprising them were studied with regard to
their antioxidant ability as well as to their ability to inhibit soy-
bean LOX. Taking into account the multifactorial character of oxi-
dative stress and inflammation, we decided to evaluate the
in vitro antioxidant activity of the synthesized molecules using
two different antioxidant assays: (a) interaction with the stable
free radical 1,l-diphenyl-2-picrylhydrazyl radical (DPPH), (b) inter-
action with the water-soluble azo compound AAPH.
Conjugate 9 was readily obtained in 90% yield through coupling
the active ester 18 with 1,12-diaminododecane, followed by rou-
tine flash column chromatography (FCC) purification. Conjugates
10 and 11 were obtained in 68% and 85% yield, respectively, by
coupling active ester 2111 with either benzyl amine or dopamine,
whereas conjugate 12 was obtained in 78% yield through coupling
acid 16 with 1-butylamine. Conjugate 13 was obtained in 85% yield
by coupling active ester 20 with dopamine followed by FCC purifi-
cation. Finally, the conjugates 14 and 15 were obtained by first
coupling the active ester 2222 with 2-(2-aminoethoxy)ethanol to
give amide 23 in 88% yield. This compound was then condensed
with either FICA10 or TRAA14 under Mitsunobu reaction conditions
to afford conjugates 14 and 15 in 64% and 68% yields, respectively,
following FCC purification.
Doth require a spectrophotometric measurement and a certain
reaction time in order to obtain reproducible results.23 DPPH has
been used in a radical-scavenging measuring method. DPPH is a
stable free radical in an ethanolic solution. In its oxidized form,
the DPPH radical has an absorbance maximum centred at about
517 nm.24 The DPPH method is described as a simple, rapid and
convenient method independent of sample polarity for screening
many samples for radical-scavenging activity.25 These advantages
made the DPPH method interesting for testing our compounds.
The use of the free radical reactions initiator AAPH is recom-
mended as more appropriate for measuring radical-scavenging
activity in vitro, because the activity of the peroxyl radicals pro-
duced by the action of AAPH shows a greater similarity to cellular
activities such as lipid peroxidation.26

8208
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
12
xi
O
O
O
O
O
O
i, ii
H
N
NH2
OH
O
N
H
N
H
O
O
16
18
17
O
17/iii
N
6
O
O
viii
13
9
xiii
3a
3Hb
4
5
N
O
N
N
O
13
H
vi, vii
9
6
HO
HO
8
7
2
HO
HO
v
8
12
1
OH
O
H
0
1
11
HN
X
H
19
HN
Trt
[
]
20
:
X = H
L-DOPA
iv
19:
X = Trt
O
O
x
ix
N
11
10
O
O
MeO
21
15
xv
O
O
O
1
9
6
3
10
13
8
4
MeO
MeO
O
xii
MeO
MeO
N
N
OH
2
12
11
O
H
5
O
7
23
xiv
22
14
Scheme 1. Synthesis of conjugates 6, 8 and 9–15. Reagents and conditions: (i) Boc3-SPM, TEA, HBTU, DMF, 25 °C, 45 min, 61%; (ii) TFA/CH2Cl2 (1:1), 25 °C, 2 h; (iii) DIEA,
CHCl3/DMF (1:1), 25 °C, 5 h, 61%; (iv) (a) Me3SiCl, TEA, CHCl3, 61 °C, 1 h; (b) i-PrOH/CH2Cl2 (1:3), TEA, 0 °C, 15 min then Trt-Cl, 25 °C, 1 h; (c) MeOH, 25 °C, 30 min, 81%; (v)
HOBt, DCC, THF, 0–25 °C, 2 h, 85%; (vi) N4,N9-Trt2-SPM, DIEA, CH2Cl2, 25 °C, 2 h, 47%; (vii) TFA/CH2Cl2 (2:8), 0 °C, 2 h then 1.4 M HCl/EtOH, 83%; (viii) 1,12-diaminododecane,
TEA, CHCl3, 40 °C, 2 h, 90%; (ix) benzylamine, DIEA, CH2Cl2, 25 °C, 15 min, 68%; (x) dopamineꢀHCl, DIEA, CHCl3/DMF (5:1), 0–25 °C, 2 h, 85%; (xi) 1-butylamine, DIEA, HBTU,
DMF, 25 °C, 15 min, 78%; (xii) 2-(2-aminoethoxy)ethanol, TEA, DMF, 0–25 °C, 45 min, 88%; (xiii) (a) dopamineꢀHCl, DIEA, CHCl3/DMF (6:1), 0–25 °C, 30 min, 53%; (b) TFA/
CH2Cl2 (3:7), 25 °C, 30 min, 85%; (xiv) FICA, TPP, DIAD, THF, 0–25 °C, 40 min, 64%; (xv) TRAA, TPP, DIAD, THF, 0 to 25 °C, 45 min, 68%.
Antioxidants are defined as substances that even at low concen-
tration significantly delay or prevent oxidation of easy oxidizable
substrates. Antioxidants with high antioxidant potential activity
will therefore be beneficial for clinical applications. It is well-
known that free radicals play an important role in the inflamma-
tory process.27 Many non-steroidal anti-inflammatory drugs have
been reported to act either as inhibitors of free radical production
or as radical scavengers.28 Consequently, compounds with antiox-
idant properties could be expected to offer protection in rheuma-
toid arthritis and inflammation and to lead to potentially
effective drugs. Thus, we tested the present conjugates with regard
to their antioxidant ability and in comparison to well-known anti-
oxidant agents, for example, CA, ascorbic acid, NDGA and trolox.
The interaction/reducing activity (RA) of the examined com-
pounds with the stable, long-lived, free radical DPPH is shown in
Table 1. This interaction indicates their radical-scavenging ability
in an iron-free system. In the DPPH assay, the dominant chemical
reaction involved is the reduction of the DPPH radical by a single
electron transfer (ET) from the antioxidant.29 Particularly effective
such antioxidants are the phenoxide anions from phenolic com-
pounds like catechol and derivatives. The highest interactions in
the present set of compounds were observed for compounds 8
and 13 (values higher than those for NDGA and comparable to
ascorbic acid) and compound 11 (value comparable to NDGA but
lower than ascorbic acid). These compounds are characterized by
the presence of either
L-DOPA (conjugate 8) or dopamine

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8209
Table 1
Interaction-reducing activity % with DPPH (RA %); in vitro inhibition of soybean lipoxygenase (LOX) (IC50); % inhibition of lipid peroxidation (AAPH %); inhibition % of
Carrageenan-induced rat paw oedema (ICPE %)
Compda
C log P35 RA % 0.1 mM 20/60 min
IC50
l
M or % LOX Inh. @ 0.1 mM/0.05 mV ( SD)b AAPH % @ 0.1 mM ( SD)b ICPE % 0.01 mmol/Kg
( SD)b
( SD)d
CA
0.82
0.45
1.90
ꢁ2.33
ꢁ1.79
1.42
3.42
5.03
5.5/6 0.3
100/100 3.8
9/10 0.2
600
l
V
15
17.5 0.8
47 1.3
56 1.6
na
72 2.3
100 6.9
73 5.3
80 6.2
85 5.8
65 1.2
DHCA8,c
DMCA
SPM8
16/na 0.2
23/na 0.4
na/na
4/4 0.5
KukA8
DCSP8
TRAA9
TRAB9
FICA10
96/100 2.9
52/58 3.2
11/11 0.9
11/11 0.7
1/na 0.05
76#/76 4.3
86#/86 5.7
11/23 0.8
na/na
na/5 0.3
26/38 1.4
41/54 2.8
11/38 0.4
na/na
89/96 6.7
na/13.4 0.8
na/2 0.008
79/79 1.4
na/8.4 0.7
92/92 4.6
na/12 1.0
na/15 0.8
81/83 2.4
9.5
62
l
V
1.1
3.2
43* 0.6
42** 2.6
l
M
9/na 0.8
9.4 3.1
52.4 2.6
23/na 1.3
100 4.5
na/na
l
M
lM
46** 1.8
11* 0.7
2.66
ꢁ2.82
L
-DOPA
2 (Dopamine) 0.17
lV
43 2.7
75 3.5
85 1.6
58 4.5
97 3.8
33 2.2
45 2.8
31 1.9
71 4.1
41 2.3
45 1.1
10 0.7
66 1.6
na
13* 0.8
11* 0.8
63** 3.4
40** 2.8
19* 1.2
ATRA
6.74
6.07
¥
¥
¥
¥
¥
¥
ACI10
1/na 10ꢁ3
76/na 3.8
75/na 6.7
57/na 2.2
43/na 3.1
41.5
32/na 1.2
23/na 0.8
3
4
5
6
7
8
9
10
11
12
13
14
15
NDGA
lV
2.9
¥
¥
¥
40.5
87
24/na 1.4
lV
3.5
6.2
3.6* 0.8
l
M
13.7* 1.0
4.72
ꢁ0.31
¥
81.5
33.5
l
M
5.2
2.4
2.7
1.3
lV
na
98 4.6
¥
65
28
l
l
V
V
9* 0.3
Trolox
63 1.2
Ascorbic acid
Indomethacin
99/100 1.8
47** 3.1
*
**
Each value represents the mean obtained from 6 animals in two independent experiments (n = 6 ꢂ 2). In all cases, significant difference from control: p <0.1, p <0.01
(Student’s t-test); ¥, calculated values are too high to be realistic; na, no activity under the reported experimental conditions; #, immediately, both compounds presented the
same high antioxidant activity at 0.05 mM.
a
Full structures of all compounds in this column are presented in Supplementary data section (Table 1).
Values are means SD of three or four different determinations, significant differences from control values *p <0.05.
DHCA stands for dihydrocaffeic acid.
The effect on oedema is expressed as percent of weight increase of hind paw (and as percent of inhibition of oedema) in comparison to controls.
b
c
d
(conjugate 11) or both substructures (conjugate 13), for which high
interaction values were already observed when used alone (76%/
76% for L-DOPA and 86%/86% for dopamine). The interaction, for
ities like the ones (two catechol subunits) present in NDGA which is
used as a reference compound. Regarding the effect of conjugation
on the interaction values of the conjugates compared to the uncon-
jugated molecules, it is apparent that conjugation of ATRA with SPM
or 1,12-diaminododecane (conjugates 3 and 9) leads to lower inter-
action values than the unconjugated retinoid, an effect which is
however reversed with the more electron-rich and thus more easily
oxidizable aromaticmolecules ACI (conjugate 4), TRAA (conjugate 5)
these compounds remains, with the exception of 8, highly constant
after 60 min. It is worth noting that, obviously, the activity of con-
jugate 11 comes from its dopamine substructure alone as ACI itself
presented zero interaction. Packer30 also earlier did not find any
interaction with DPPH for ACI. The high interaction of compounds
8, 11 and 13 with the DPPH radical should be attributed to the
presence of the easily oxidizable catechol subunits present in the
molecule, whereas the higher values for compounds 8 and 13 com-
pared to 11 might be further attributed to the presence of the free
amino function(s). DHCA as well as Kuka, incorporating the cate-
chol unit, also presented very high interaction values, whereas
for DMCA as well as DCSP, in which the hydroxyl groups of the cat-
echol unit have been converted into methyl ether groups, the
reducing ability has been greatly decreased. Interestingly, DMCA
interacts more effectively with DPPH than CA, in which the cate-
chol subunit is retained. It therefore seems that the presence of a
double bond in position p to one of the hydroxyl groups has an
unfavourable effect on the interaction with DPPH and that in such
cases better interaction is secured when the hydroxyl groups are
masked as methyl ethers.
and L-DOPA (conjugate 8). Interestingly, conjugate 9, lacking the sec-
ondary amino functions in its chain, interacts with DPPH slightly
better than conjugate 3. Furthermore concerning the hybrid conju-
gates 6 and 7, although conjugation of ATRA and TRAA through
SPM (conjugate 6) caused essentially no improvement in the antiox-
idative properties of the unconjugated molecules, the conjugation of
ACI to TRAA (conjugate 7) resulted in complete loss of activity.
In our studies, AAPH was used as a free radical initiator to follow
oxidative changes of linoleic acid to conjugated diene hydroperox-
ide. Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for free radical production studies
in vitro.31 The water-soluble azo compound AAPH has been exten-
sively used as a clean and controllable source of thermally produced
alkyl peroxyl free radicals. In the AAPH assay, the highly reactive
alkylperoxyl radicals are intercepted mainly by a hydrogen atom
transfer (HAT) from the antioxidant.29 Therefore, particularly effec-
tive HAT agents are compounds with high hydrogen atom donating
ability, that is compounds with low heteroatom-H bond dissociation
energies and/or compounds from which hydrogen abstraction leads
Compounds 3–7, 9, 10, 12, 14 and 15 presented low, if any, inter-
action to DPPH at 100 lM after 20 min whereas a small increase in
the interaction is generally observed in most cases after 60 min. This
can be attributed to the absence of any easily oxidizable functional-

8210
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
to sterically hindered radicals as well as compounds from which
abstraction of hydrogen leads to C-centred radicals stabilized by res-
onance. With the exception of compounds 13 and 14 which showed
no inhibition, all other compounds caused inhibition of lipid perox-
idation (LPO). In particular, compounds 3, 5–7 and 9–11 presented
inhibition values (10–58%) lower than the common standard trolox
(63%). On the other hand, compounds 4, 8, 12 and 15 presented val-
ues (66–98%) higher than trolox, with most potent inhibitors being
the compounds 4 (97%) and 15 (98%). Although the conjugates 8
(71%) and KukA (72%) might exert their antioxidative effect through
their easily oxidizable catechol units, the antioxidative effect of the
other three compounds should be attributed to other factors. For
example, conjugate 4 like ACI may exert its inhibitory effect either
by hydrogen atom abstraction from the Me groups of the polyene
chain and stabilization of the resulting new C-centred radical
through resonance or through interception of the alkylperoxyl rad-
icals by their conjugated tetraene chain. Similar considerations
might be applicable to compound 12 and in particular compound
15. It is interesting to note that the compounds incorporating free
phenolic functions are not the most powerful antioxidants in the
AAPH assay with their activity ranging from 0 (conjugate 13) to
72% (KukA). On the contrary, extremely high inhibitory values were
obtained for compounds bearing masked phenolic functions as
methyl ethers, such as conjugates DCSP (100%), 4 (97%) and 15 (98%).
Regarding the effect of conjugation on the ability of the conju-
gates to inhibit LPO, the two most powerful and almost equipotent
inhibitors of LPO (compounds 4 and 15) include the structural
characteristics of the compounds ACI the former and of DCSP and
TRAB the latter, which all have high inhibition values (80–100%).
Although, DMCA is not particularly effective (56%), its conjugate
DCSP with SPM exhibit the highest anti-LPO activity in the present
set of compounds. Also, the conjugate of DHCA with SPM, namely
KukA, shows much lower inhibitory activity than DCSP although
the unconjugated molecule DHCA shows inhibitory activity (47%)
similar to that for DMCA. Since CA itself shows a particularly low
inhibitory activity (17.5), it seems that unsaturation of the side-
chain and replacement of the hydroxyl groups by methyl ether
groups, in the above mentioned cases, improves significantly the
inhibitory activity towards LPO. This is indeed apparent when
the inhibitory activities of CA and DMCA on one hand and of KukA
and DMCA on the other hand are compared. Striking is however
the difference when structural characteristics of the molecules
DSCP and FICA are combined (conjugate 14), which both also have
very high inhibitory values (100% and 85%, respectively). In that
case, complete loss of the inhibitory activity is observed. The same
ate stimulation of neutrophils, arachidonic acid (AA) is cleaved
from membrane phospholipids and can be converted into leukotri-
enes (LTs) through 5-lipoxygenase (5-LOX). Leukotriene B4 (LTB4)
is a potent mediator of inflammation, amplifying recruitment and
activation of inflammatory cells. LTB4 generation is considered to
be important in the pathogenesis of neutrophil-mediated inflam-
matory diseases32 with a marked relation to the severity of cardio-
vascular diseases and cancer. Inhibitors of LOX have attracted
attention initially as potential agents for the treatment of inflam-
matory and allergic diseases, certain types of cancer and cardiovas-
cular diseases.33,34 Goldreich et al.35 examined the effect of retinol,
all-trans-retinoic acid and 13-cis-retinoic acid on the activity of
lipoxygenase-1 and lipoxygenase-2 towards linoleic acid. All-
trans-retinoic acid and 13-cis-retinoic acid inhibited lipoxyge-
nase-1 activity competitively, whereas retinol inhibited lipoxyge-
nase-1 activity in a mixed manner. These findings suggest that
retinoids may bind to the active site of the enzyme or simulta-
neously act as antioxidants.
In this context, we decided to further evaluate the synthesized
conjugates for their ability to inhibit soybean LOX by the UV absor-
bance based enzyme assay.8 Most of the LOX inhibitors are antiox-
idants or free radical scavengers.36 LOXs contain a ‘non-haem’ iron
per molecule in the enzyme active site as high-spin Fe2+ in the na-
tive state and the high spin Fe3+ in the activated state. Some studies
suggest a relationship between LOX inhibition and the ability of
the inhibitors to reduce Fe3+ at the active site to the catalytically
inactive Fe2+. This inhibition is related to their ability to reduce
the iron species in the active site to the catalytically inactive fer-
rous form,34 whereas several LOX inhibitors are excellent ligands
for Fe3+. For the sake of comparison, the inhibitory values of the
conjugates Kuka and DCSP as well as of ‘parent’ unconjugated mol-
ecules are included in the study (Table 1). NDGA a known inhibitor
of soybean LOX has been used as a reference compound (28
and CA (600 V) as the positive control.
Perusal of the IC50’s inhibition values (Table 1) shows that the
most potent inhibitors are the compounds 14 (IC50 33.5 M), 10
(IC50 40.5 M) and 7 (IC50 41.5 M), followed by the conjugates
15 (IC50 65 M), 13 (IC50 81.5 M) and 11 (IC50 87 M). The inhib-
lV)
l
l
l
l
l
l
l
itory effect of the last two mentioned compounds should be mainly
attributed to the presence of the dopamine unit because in contrast
to dopamine (IC50 100
(23% at 0.1 mM) present notable inhibitory values. Accordingly, the
symmetric spermine conjugate 8 containing -DOPA is showing
slightly more inhibitory activity (32% at 0.1 mM) compared to
lM) neither ACI (1% at 0.1 mM) nor L-DOPA
L
L-
DOPA. This shows that the easily oxidizable catechol unit, which
is also an efficient chelator for the Fe3+ ion present in the enzyme
active site, is not the only factor to account for the difference in
activity between the conjugates 8 and 13. Although lipophilicity
is referred as an important physicochemical property for LOX
inhibitors,37 herein the theoretically calculated log P38 values are
too high to be realistic and thus, we do not take them into consid-
eration in the discussion of the results.
The inhibitory activity of the other compounds which do not
contain an easily oxidizable moiety might be attributed mainly
to their ability to attach to the active site of the enzyme and to a
lesser degree to their electron donating ability. The common struc-
tural element of compounds 11 and 13 is the dopamine moiety and
applies to conjugate 13, where conjugation of L-DOPA (65% inhibi-
tion value) and dopamine (43% inhibition value) also resulted to
complete loss of the inhibitory ability towards LPO.
Lower inhibition values are also obtained when conjugating
TRAA with SPM either alone (compound 5) or with ATRA (com-
pound 6) or ACI (compound 7). Also, conjugation of ATRA with
SPM (compound 3) leads to significantly lower inhibition value,
which becomes even lower when SPM is replaced by 1,12-diami-
nododecane (compound 9). On the contrary, conjugation of ACI
(with higher inhibition value than ATRA) with SPM provides a
stronger inhibitor whereas the benzylamide (compound 10) and
the dopaminamide (compound 11) of ACI resulted in very low inhi-
bition values compared to ACI. In addition, the inhibitory activity of
it seems that replacement of ACI by L-DOPA as the acid component
dopamine is totally abolished when conjugated to
ever, when -DOPA is conjugated to SPM the resulting conjugate
8 is slightly more inhibitory than -DOPA itself. Finally, amidation
of TRAA (compound 12) results in lower inhibitory activity than
either the free acid (TRAA) or the corresponding tert-butyl ester
(TRAB).
L
-DOPA. How-
slightly improves inhibition. On the other hand, compound 10 and
11 both have ACI as the acid component (they are ACI amides) and
differ only in the amine component of the conjugate. It therefore
seems that benzylamine is a much better replacement for dopa-
mine. Compound 14 combines characteristics of two molecules
(DCSP and FICA) with high inhibition values which reflect to the
higher value observed for this conjugate. On the other hand com-
pound 15, which also combines characteristics of two molecules
L
L
Eicosanoids are oxygenated metabolites of arachidonic acid
with a broad implication in a diversity of diseases. Upon appropri-

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8211
Table 2
(DCSP and TRAB) with high inhibition values, showed an inhibitory
Cytotoxicity of compounds on RAMEC rat
endothelial cells (24 h incubation)
value which is a little less than the value for the weaker (DCSP) of
the afore mentioned two inhibitors. It is worth noting that the SPM
conjugates with ATRA (compound 3) and ACI (compound 4) pres-
ent almost identical interaction values with LOX which are defi-
nitely much higher than the unconjugated retinoids, indicating
the beneficial nature of the conjugation in these cases. High inter-
action value is observed by the hybrid conjugate TRAA-SPM-ACI
(7), which is higher than those for either the symmetric conjugates
4 and 5 or the asymmetric conjugate TRAA-SPM-ATRA (6).
Furthermore, replacement of the polyamine chain by an ali-
phatic chain, as in conjugate 9, leads to higher interaction values
than the unconjugated retinoid but certainly much lower than
the corresponding conjugate with the polyamine chain (conjugate
3), which incorporates two free secondary amino functions. On the
Compda
IC50
(lV)
b
KukA
DCSP
TRAB
ATRA
ACI
3
3.5
11.3
2.9
18.3
14.6
2.6
4
4.7
9
2.8
15
12.9
a
Full structures of all compounds in this
column are presented in Supplementary data
section (Table 2).
b
Values are means of six different deter-
other hand, conjugation of
slightly improve the inhibition capability of
L
-DOPA with SPM seemed to only
-DOPA. Furthermore,
minations. Viability data are subject to 5%
error.
L
it seems that amides of the acid TRAA (e.g., compound 12) are bet-
ter inhibitors than the free acid (TRAA) but much weaker inhibitors
when compared to esters, for example, the tert-butyl ester TRAB.
15) also resulted in a high decrease (ca. five times) in the anti-
inflammatory activity (46% for TRAB and 42% for DCSP). Therefore,
with the exception of the conjugate ATRA-SPM-ATRA (3), conjuga-
tion of compounds with high anti-inflammatory activities results
to a serious decrease in their anti-inflammatory effect.
Also, the conjugation of L-DOPA with dopamine (conjugate 13) re-
sulted in a stronger inhibitor than the parent molecules. It there-
fore seems that in most cases conjugation of antioxidants have a
beneficial effect on their inhibitory effect on LOX.
2.2.2. Anti-inflammatory activity in vivo
2.2.3. Cytotoxicity studies
In acute preliminary toxicity experiments, the in vivo examined
compounds did not present toxic effects in doses up to 0.1 mmol/
kg body weight. For the anti-inflammatory assay, we used a dose
of 0.01 mmol/kg equimolar to the administered standard drug
indomethacin for the sake of comparison.
For the in vivo screening, the selection of the compounds was
generally based on their good inhibitory activity on LOX and
anti-lipid peroxidation. Thus, the most interesting compounds in
this series, namely compounds 3, 4, 10, 11 and 15 were selected
to be examined in vivo by using the functional model of Carra-
geenan-induced rat paw oedema. Although compound 13 pre-
sented comparable to compound 11 LOX inhibition, it was not
chosen for further studies because (a) it had no anti-LPO activity,
For the cytotoxicity studies, the selection of the compounds was
generally based on their good anti-inflammatory activity, namely
compounds KukA, DCSP, TRAB, ACI, 3 and 4 or their good inhibitory
activity on LOX and anti-lipid peroxidation activity, namely conju-
gate 15. For the sake of comparison, ATRA and conjugate 9 were
also included in the study. The cytocompatibility of these com-
pounds was evaluated by the viability of rat endothelial cells
(RAMEC) cells in the presence of different concentrations of the
compounds (0.5–50
of IC50 values for the examined compounds.
The retinoids ATRA (IC50 18.3 M) and ACI (IC50 14.6 lM)
lM) and is presented in Table 2 in the form
l
were the least cytotoxic for the RAMEC cells in this series of
compounds. Their corresponding conjugates with SPM, namely
(b) its substructure L-DOPA showed low inhibitory value for LOX
compounds 3 (IC50 2.6 lM) and 4 (IC50 4.7 lM) were however
and (c) the anti-inflammatory activity of dopamine (the other sub-
structure of 13) had been already found to be rather low (only
13%). In addition, compound 11 was chosen for further in vivo test-
ing because it contained the substructure of the retinoid drug ACI,
which had previously shown high anti-inflammatory activity
(63%).10 Unconjugated molecules were also included in the study
for the sake of comparison.
more toxic than the unconjugated molecules. It is interesting
to note that although ACI seems to be more toxic than ATRA,
the reverse holds true for their conjugates. It would be tempting
to hypothesize that the increased toxicity of conjugates com-
pared to the unconjugated retinoids is due to the presence of
the SPM chain. This is not however supported by the IC50 value
(2.8 lM) for the conjugate 9 which contains a conjugating chain
Carrageenan-induced oedema is a non-specific inflammation
resulting from a complex of diverse mediators.39 As shown in Table
1, all conjugates, with the exception of conjugate 3 (40%) which
exhibited a comparable effect to indomethacin (47%), showed
much less potent anti-inflammatory activity (3.6–19%). It is worth
noting that although conjugate 3 was almost four times more ac-
tive than the unconjugated ATRA, the reverse holds for the conju-
gate 4, which is ca. three times less active than ACI. Also, conjugate
3 is ca. two times more active than conjugate 4, although ATRA it-
self is almost six times less inhibitory than ACI. A similar trend has
been observed with the inhibitory activity of these compounds on
the enzymic activity of RNase P, that is although ACI is stronger
inhibitor than ATRA, the reverse is true for their symmetric conju-
gates with SPM.11 Furthermore, amidation of the very active ACI
(compound 10) leads to a large decrease of its activity (ca. twenty
times), which is not as bad (ca. five times less active) when the
amino component is changed to dopamine (compound 11). Finally,
conjugation of the substructures of the DCSP and TRAB (compound
of identical length to that of SPM but lacks the two secondary
amino functions of SPM. It seems that the main cause of in-
creased toxicity is the conjugation per se and not the presence
of SPM. Also, a comparison of the IC50 values of the conjugates
KukA (IC50 3.5 lM) and DCSP (IC50 11.3 lM) shows that the main
cause of increased toxicity should be attributed to the change in
the non-SPM part of the molecules and in particular to the pres-
ence of the free phenolic hydroxyls. Finally, the conjugate 15,
incorporating structural characteristics of conjugate DCSP (IC50
11.3
l
M) and of ester TRAB (IC50 2.9
lM), presented lower toxic-
ity (IC50 12.9
l
M) than either of these compounds. Therefore,
conjugation of antioxidant/anti-inflammatory agents results in
variable cytotoxicity on RAMEC rat endothelial cells depending
primarily on the structure of the unconjugated molecule(s) and
to a much lesser degree to the structure of the conjugating en-
tity. In the cases examined, the presence of the SPM moiety does
not seem to play a particularly important role in increasing
cytotoxicity.

8212
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
the University of Patras. Flash column chromatography was per-
3. Conclusions
formed on Merck silica gel 60 (230–400 mesh) and TLC on Merck
60F254 films (0.2 mm) precoated on aluminium foil. Spots were visu-
alized with UV light at 254 nm, charring agents or ninhydrine. The
eluent systems used were: (A) PhMe/EtOAc (9:1), (B) PhMe/EtOAc
(8:2), (C) PhMe/EtOAc (6:4), (D) PhMe/EtOAc (1:1), (E) EtOAc, (F)
CHCl3/MeOH (98:2), (G) CHCl3/MeOH (97:3), (H) CHCl3/MeOH
(9:1), (I) CHCl3/MeOH/concd NH3 (7:3:0.3).
All the chemicals used were of analytical grade and commer-
cially available from Aldrich or Fluka. ACI and SPM were purchased
from CHEMOS GmbH and ACROS ORGANICS, respectively. Com-
pounds such as 16 (TRAA),14 FICA,10 18,11 21,11 DMCA22 and 2222
were available in our laboratory from previous research projects.
Finally, conjugates 3,11 4,11 514 and 714 were synthesized, for the
purpose of the present study, according to literature protocols.
In general, conjugation of molecules with an interest as antiox-
idants and/or anti-inflammatory agents leads to compounds which
generally present, with a few exceptions, higher inhibition on LOX
and less potent anti-inflammatory activity. Regarding their ability
to interact with AAPH, the highest activity was observed for conju-
gates 4 and 15 which both incorporate moieties (ACI, TRAB, DCSP)
characterized by high interaction values with AAPH. Both com-
pounds had low interaction values with DPPH whereas on the con-
trary the highly interacting with DPPH conjugate 13 exhibited zero
interaction with AAPH, thus showing that lipid peroxidation activ-
ity is not always accompanied by DPPH radical-scavenging ability
and vice-versa as it has been also previously referred.10,40 This
can be attributed to the different chemical reactions involved in
the two assays.29 In the DPPH assay, a reduction of the long-lived
N-centred radical mainly takes place through ET from the antioxi-
dant. Particularly effective such antioxidants are the phenoxide an-
ions from phenolic compounds, such as the catechol unit
4.2. N1-(All-trans-retinoyl)-N12-[3-(trioxsalen-40-
yl)acryloyl]spermine (6)
To an ice-cold suspension of 16 (0.08 g, 0.27 mmol), Boc3-SPM
(0.14 g, 0.28 mmol) and TEA (0.09 mL, 0.66 mmol) in DMF
(0.7 mL), HBTU (0.11 g, 0.30 mmol) was added and the resulting
solution was stirred for 45 min at ambient temperature. Then, the
reaction mixture was diluted with CHCl3 and washed with a 5%
aqueous solution of NaHCO3 and H2O. The organic layer was dried
over Na2SO4 and evaporated to dryness to leave an oily residue,
which upon FCC purification, using the eluant system E gave the
anticipated fully protected spermine analog (0.09 g, 74%) as a yellow
oil. Thiswasthentreatedwithanice-coldsolutionof50%TFA/CH2Cl2
(0.5 mL) and stirred at this temperature for 2 h. Volatile components
were removed under vacuo and the residue was triturated with Et2O
and left refrigerated overnight to give compound 17 as the corre-
sponding tris-trifluoroacetate salt.14 This compound was used as
such into the next experiment as follows.
To an ice-cold solution of 17 (0.17 g, 0.2 mmol) and 18 (0.05 g,
0.13 mmol) in DMF/CHCl3 (1:1, 0.7 mL), DIEA (0.11 mL, 0.65 mmol)
was added dropwise. The resulting solution was stirred at this tem-
perature for 10 min and then left to attain ambient temperature
were it kept for further 5 h. Then, the reaction mixture was diluted
withCHCl3 and washed once withan 5% aqueoussolution of NaHCO3
and twice with H2O. The organic layer was dried over Na2SO4 and
evaporated to dryness to leave an oily residue. Conjugate 6 (0.09 g,
61%) was obtained pure, after FCC purification using solvent system
I for elution, as an orange foam. Rf (I): 0.34; IR (thin film, CHCl3, cm
ꢁ1): 1718, 1684, 1676, 1654, 1648, 1636, 1600, 1577, 1400, 1240,
1120, 1101; ESI-MS: m/z 787.6 [M+Na], 765.6 [M+H], 441.4
[(M+H)ꢁC20H22N2O4], 409.3 [(M+H)ꢁC23H36N2O]; RP-HPLC:
tR = 18.08 min; 1H NMR (DMSO-d6, d): 8.31 (s, 1H, NH), 8.26 (t, 1H,
J = 5.6 Hz, NHCO), 7.99 (t, 1H, J = 5.6 Hz, NHCO), 7.81 (s, 1H, H-5),
7.48 (d, 1H, J = 16.0 Hz, H-15), 7.09 (dd, 1H, J = 15.2 and 11.6 Hz, H-
22), 6.66 (s, 1H, H-2), 6.55 (d, 1H, J = 16.0 Hz, H-16), 6.29 (d, 2H,
J = 15.2 Hz, H-21 and H-25), 6.12 (d, 1H, J = 11.6 Hz, H-23), 6.10 (d,
1H, J = 16.0 Hz, H-26), 5.89 (s, 1H, H-19), 3.33–3.24 (two overlapping
q, 4H, J = 6.4 Hz, H-10 and H-100), 3.13 and 3.12 (two overlapping t,
4H, J = 6.8 Hz, H-30 and H-80), 2.62–2.55 (m, 4H, H-40 and H-70),
2.58 (s, 3H, H-12), 2.53 (s, 3H, H-13), 2.43 (s, 3H, H-14), 2.32 (s, 3H,
H-33), 1.96 (s, 3H, H-34), 1.80 (s, 1H, NH), 1.72 (s, 3H, H-35), 1.65
(quint., 2H, J = 6.8 Hz, H-30), 1.55 (quint., 4H, J = 6.8 Hz, H-20 and
H-90), 1.48–1.39 (m, 8H, H-50, H-60, H-29 and H-31), 0.93 (s, 6H, H-
36 and H-37); 13C NMR (DMSO-d6, d): 167.6, 166.7, 161.3, 159.1,
156.3, 154.9, 153.4, 149.5, 148.2, 138.4, 138.3, 136.2, 136.0, 134.0,
130.0, 129.5, 128.9, 128.3, 123.0, 122.9, 122.1, 121.1, 116.7, 113.2,
112.9, 112.7, 110.2, 109.6, 49.3, 49.2, 47.4, 47.3, 43.7, 38.3, 37.8,
29.8 (two C), 28.8, 27.6, 21.5, 19.5, 17.5, 14.1, 13.8, 13.0, 12.0, 8.5.
containing compounds DHCA, KukA, L-DOPA, dopamine and the
conjugates 8, 11 and 13 as well as the reference compound NDGA.
On the other hand in the AAPH assay, the highly reactive alkyl per-
oxyl radical is neutralized mainly by HAT from the antioxidant.
Compounds incorporating phenol moieties in the present set of
compounds seem not to be particularly effective HAT agents. Obvi-
ously, compounds acting as particularly effective HAT agents are
the conjugates DCSP, 4 and 15. The ability of conjugates to interact
with either DPPH or AAPH varies and depends on the structure and
the reducing ability of the unconjugated molecules. The incorpora-
tion of compounds like L-DOPA or dopamine, containing the highly
oxidizable catechol unit, in conjugates provides compounds with
increased interaction values against DPPH and inhibitory activity
on LOX but generally very low inhibition of LPO and low anti-
inflammatory potency. With the exception of conjugates DCSP
and 15 which presented cytotoxicity on cells comparable to that
of the well-known retinoids ATRA and ACI, the other conjugates
KukA, 3, 4 and 9 and the ester TRAB which were tested exhibited
increased cytoxicity, indicating that the safety of use of these com-
pounds for biomedical applications may be compromised at high
doses.
4. Experimental section
4.1. Materials and methods
Melting points were determined with a Buchi SMP-20 apparatus
and are uncorrected. IR spectra were recorded for KBr pellets on a
Perkin–Elmer 16PC FT-IR spectrophotometer. 1H NMR spectra were
obtained at 400.13 MHz and 13C NMR spectra at 100.62 MHz on a
Bruker DPX spectrometer; TMS was used as reference. ESI-MS spec-
tra were recorded on a Waters micromass ZQ spectrometer
equipped with a quadropole analyzer, using MeOH as solvent. Opti-
cal rotationswere measured at25 °C with a Schmidt–Haensch Polar-
tronic D polarimeter with a 0.5 dm cell. Analytical RP-HPLC were
performed on a Waters system (2695 Alliance). Elution of the com-
pounds was determined from the absorbance at 254 nm (Waters
2996 Diode array detector). Compound purity was assessed using
a Lichrosphere™ RP 8e C8 column (5
l
m, 125 ꢂ 4 mm) and a linear
gradient of 10–60% acetonitrile (containing 0.08% TFA) in water
(containing 0.08% TFA) for compounds 6, 8 and 9 or a linear gradient
of 10–40% acetonitrile (containing 0.08% TFA) in water (containing
0.08% TFA) for compound 13 over 30 min at a flow rate of 1 mL/
min. Microanalyses were performed on a Carlo Erba EA 1108 CHNS
elemental analyzer in the Laboratory of Instrumental Analysis of

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8213
4.3. Synthesis of N1,N12-bis(3,4-dihydroxy-
phenylalanyl)spermine (8)
L-
1H, J = 1.6 Hz, H-9), 6.40 (dd, 1H, J = 1.6 and 8.0 Hz, H-5), 3.80
(dt, 1H, J = 6.4 and 8.8 Hz, H-2), 3.68 (d, 1H, J = 6.4 Hz, NH), 2.76
(dd, 1H, J = 8.8 and 13.2 Hz, H-3a), 2.16 (dd, 1H, J = 8.8 and
13.2 Hz, H-3b); 13C NMR (DMSO-d6, d): 171.3, 146.6, 146.0 (three
C), 145.9, 145.6, 133.6, 132.3, 128.9 (six C), 128.1 (six C), 127.1,
126.8 (three C), 125.8, 124.7, 120.8, 120.3, 119.5, 117.2, 71.7,
57.7, 40.2 (buried under the solvent).
4.3.1. Synthesis of N-trityl-L-DOPA (19)
To a suspension of -DOPA (0.59 g, 3 mmol) in anhydrous CHCl3
L
(9 mL), Me3SiCl (1.71 mL, 13.5 mmol) was added and the reaction
mixturewasrefluxedfor20 min. TEA(1.87 mL, 13.5 mmol)wasthen
added with cooling and then reflux was continued for further
45 min. The resulting solution was cooled to 0 °C and treated drop-
wise with a solution of i-PrOH (0.34 mL, 4.5 mmol) in CH2Cl2
(1 mL), followedby thesequentialaddition ofTEA(0.42 mL, 3 mmol)
and TrtCl (0.83 g, 3 mmol). The thus obtained reaction mixture was
stirredfor 1 h at ambient temperature, CH3OH (7 mL) was added, left
at ambient temperature for 30 min and then evaporated to dryness.
The residue was treated with a 5% aqueous citric acid and extracted
twice with Et2O. The organic layer was washed with 1 N NaOH and
water and the aqueous phases were combined, cooled to 0 °C and
then acidified with 5% aqueous citric acid solution. The organic
material was extracted twice with EtOAc and the combined organic
4.3.3. Preparation of conjugate 8
To a solution of N4,N9-bistritylspermine (0.14 g, 0.21 mmol) and
active ester 20 (0.22 g, 0.4 mmol) in CH2Cl2 (0.5 mL), DIEA
(0.09 mL, 0.5 mmol) was added dropwise. The resulting solution
was stirred at ambient temperature for 2 h. The reaction mixture
was then diluted with CH2Cl2, washed once with a 5% aqueous
solution of NaHCO3 and twice with H2O, dried over Na2SO4 and
evaporated to dryness. The anticipated bisamide (0.23 g, 72%)
was obtained pure, after FCC purification, using the solvent system
F for elution, as pale brown foam. It had Rf (F): 0.19; IR (KBr, cmꢁ1):
3408, 1654, 1638, 1508, 1174, 774, 746, 706; ESI-MS: m/z 1530.1
[M+H], 1287.5 [(M+H)ꢁTrt], 243.6 [Trt].
The bisamide (0.2 g, 0.14 mmol) was treated with an ice-cold
solution of 20% TFA/CH2Cl2 (1 mL) and kept at this temperature
for 2 h. Then, volatile components were removed under vacuo
and the residue was triturated with Et2O and left refrigerated over-
night. The resulting tetra-trifluoroacetate salt was converted to its
corresponding tetrahydrochloride salt upon treatment with a
1.5 M solution of HCl in EtOH, followed by evaporation and tritur-
ation with Et2O. Conjugate 8 (0.065 g, 83%) was obtained, after fil-
layers were washed with H2O and brine. Pure Trt-L-DOPA (19)
(1.07 g, 81%) was thus obtained as a foam and had: Rf (H): 0.20; MS
(ESI, 30 eV): m/z 440.41 [M+H], 243.41 [Trt]; The latter was con-
verted to the corresponding crystalline diethylammonium salt
according to the following procedure: A solution of Trt-L-DOPA in
EtOAc (20 mL) was treated with Et2NH (0.32 mL, 3 mmol) and left
to crystallize at ambient temperature for overnight. The precipitate
was filtered to give Trt-
tion from acetone, as a light brown solid. This salt had mp 159 °C;
+14.2 (c 1.0, MeOH); Rf (H): 0.37; IR (KBr, cmꢁ1): 3490, 3418,
L-DOPA.DEA (0.86 g, 80%), after recrystalliza-
tration under vacuo, as a white solid. It had ½a D25
ꢃ
+24.3 (c 0.7,
MeOH); IR (KBr, cmꢁ1): 3566, 3548, 3480, 3414, 3231, 1636,
1616, 620; ESI-MS: m/z 561.3 [M+H]; RP-HPLC: tR = 13.65 min;
1H NMR (DMSO-d6, d): 9.09 (br s, 4H, NH2þ), 8.92 (s, 4H, OH),
8.70 (t, 2H, J = 5.6 Hz, NHCO), 8.32 (br s, 6H, NH3þ), 6.70 (d, 2H,
J = 8.0 Hz, H-6), 6.64 (d, 2H, J = 1.6 Hz, H-9), 6.49 (dd, 2H, J = 1.6
and 8.0 Hz, H-5), 4.19–4.12 (m, 4H, H-10), 3.82 (t, 2H, J = 7.2 Hz,
H-2), 2.85–2.83 (m, 8H, H-30 and H-40), 2.81–2.75 (m, 4H, H-3),
1.82–1.70 (m, 8H, H-20 and H-50); 13C NMR (DMSO-d6, d): 168.6
(two C), 145.6 (two C), 144.9 (two C), 126.1 (two C), 120.8 (two
C), 117.4 (two C), 116.2 (two C), 54.4 (two C), 46.5 (two C), 44.9
(two C), 39.2 (two C, burried under the solvent), 36.9 (two C),
36.4 (two C), 25.9 (two C).
½ ꢃ
a 2D5
3056, 2980, 2924, 1616, 1526, 1378, 1260, 774, 744, 706; ESI-MS:
m/z 440.5 [M+H], 243.5 [Trt]; 1H NMR (DMSO-d6, d): 7.41–7.35,
7.26–7.21 and 7.18–7.12 (three m, 15H, Trt), 6.56 (d, 1H, J = 2 Hz,
H-6), 6.51 (d, 1H, J = 8.0 Hz, H-9), 6.32 (dd, 1H, J = 2.0 and 8.0 Hz,
H-5), 3.11 (t, 1H, J = 5.2 Hz, H-2), 2.69 (q, 4H, J = 7.2 Hz, (CH3CH2)2N),
2.40 (dd, 1H, J = 4.8 and 13.2 Hz, H-3b), 1.99 (dd, 1H, J = 4.8 and
13.2 Hz, H-3a), 1.06 (t, 6H, J = 7.2 Hz, (CH3CH2)2N); 13C NMR
(DMSO-d6, d): 177.0, 147.3, 144.9, 143.7, 130.5, 129.2, 128.0, 126.5,
121.2, 118.1, 115.2, 71.5, 59.1, 41.9 (2C), 12.6.
4.3.2. Synthesis of benzotriazolyl N-Trt-
L-DOPA (20)
4.4. Synthesis of N1,N12-bis(all-trans-retinoyl)-1,12-
Trt- -DOPA.Et2NH (0.51 g, 1 mmol), was partitioned between
L
aminododecane (9)
EtOAc and an ice-cold 5% aqueous citric acid solution. The organic
layer was separated and the aqueous re-extracted with EtOAc. The
combined organic layers were washed once with H2O, dried and
To a suspension of 1,12-diaminododecane (0.026 g, 0.13 mmol)
in TEA (0.09 mL, 0.66 mmol) and CHCl3 (1 mL), succinimidyl ester
18 (0.12 g, 0.3 mmol) was added. The resulting suspension was
heated at 40 °C for 2 h. The reaction mixture was diluted in CHCl3
and washed twice with H2O, dried over Na2SO4 and evaporated to
dryness to leave a residue. Pure 9 (0.09 g, 90%) was obtain, after
FCC purificationusing initiallythesolvent systemD and thenthe sol-
vent system B as eluents, as an orange foam and had Rf (B): 0.28; IR
(KBr, cmꢁ1): 3412, 1718, 1654, 1638; ESI-MS: m/z 803.1 [M+K],
787.4 [M+Na], 765.3 [M+H]; RP-HPLC: tR = 17.50 min 1H NMR
(DMSO-d6 d): 7.69 (unresolved t, 2H, NHCO), 6.81 (dd, 2H, J = 12
and 16 Hz, H-5), 6.22 (d, 2H, J = 16 Hz, H-4), 6.18 (d, 2H, J = 16 Hz,
H-8), 6.12 (2H, d, J = 12 Hz, H-6), 6.08 (d, 2H, J = 16 Hz, H-9), 5.77
(s, 2H, H-2), 2.61–2.42 (m, 4H, H-10), 2.19 (s, 6H, H-16), 1.95 (unre-
solv. t, 4H, H-12), 1.89 (s, 6H, H-17), 1.62 (s, 6H, H-18), 1.53 (t, 4H,
J = 6.0 Hz, H-14), 1.41–1.38 (m, 8H, H-13 and H-20), 1.25 (br s, 16H,
H-30, H-40, H-50 and H-60), 0.96 (s, 12H, H-19 and H-20); 13C NMR
(DMSO-d6, d): 161.6 (two C), 159.9 (two C), 141.9 (two C), 137.6
(two C), 137.0 (two C), 133.9 (two C), 133.8 (two C), 130.5 (two C),
129.9 (two C), 129.1 (two C), 110.6 (two C), 42.1 (two C), 39.8 (two
C, burried under solvent), 34.2 (two C), 33.8 (two C), 33.1 (two C),
evaporated to dryness to leave Trt-L-DOPA as a brown foam. This
was then dissolved in THF (3 mL), cooled to 0 °C and treated
sequentially with HOBt (0.20 g, 1.5 mmol) and DCC (0.23 g,
1.1 mmol). The resulting suspension was stirred at this tempera-
ture for 30 min and then left to attain ambient temperature, where
it was stirred further for 2 h to complete the reaction. Then, a few
drops of AcOH and H2O were added and the mixture was stirred
additional for 30 min to destroy excess DCC. DCU was then filtered
off and the filtrate was evaporated to dryness to leave an oily res-
idue. This was then dissolved in EtOAc, washed with an ice-cold 5%
aqueous solution of NaHCO3 and H2O. The organic layer was dried
over Na2SO4 and evaporated to dryness. Pure active ester 20
(0.47 g, 85%) was finally obtained after FCC purification, using the
eluant system B, as light brown foam. ½a D25
ꢁ8.48 (c 0.7, CHCl3);
ꢃ
Rf (B): 0.36; IR (KBr, cmꢁ1): 3332, 1820, 1768, 1600, 1446, 1282,
774, 744, 706; ESI-MS: m/z 595.4 [M+K], 579.6 [M+Na], 557.2
[M+H], 243.5 [Trt]; 1H NMR (DMSO-d6, d): 8.92 and 8.91 (two s,
2H, OH), 8.10 (d, 1H, J = 8.0 Hz, H-10), 7.54–7.47 (m, 7H, Trt and
H-11), 7.38–7.31 (m, 6H, Trt), 7.27–7.17 (m, 4H, Trt and H-12),
6.86 (d, 1H, J = 8.0 Hz, H-13), 6.69 (d, 1H, J = 8.0 Hz, H-6), 6.44 (d,

8214
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
29.6 (two C), 29.5 (four C), 28.9 (four C), 27.0 (two C), 21.7 (two C),
19.2 (two C), 14.6 (two C), 13.0 (two C).
sequentially with a 5% aqueous solution of citric acid, H2O, a 5%
aqueous solution of NaHCO3 and H2O, dried over Na2SO4 and evap-
orated to afford pure 12 (0.036 g, 78%) as a white solid. Compound
12 had mp 224–226 °C; Rf (C): 0.13; IR (KBr, cmꢁ1): 3282, 1736,
1656, 1614, 1602, 1104; ESI-MS: m/z 729.1 [2V+Na], 707.2
[2M+H], 354.3 [M+H], 281.3 [(M+H)ꢁPhCH2NH2]; 1H NMR
(DMSO-d6, d): 8.14 (t, 1H, J = 5.6 Hz, NHCO), 7.77 (s, 1H, H-5),
7.47 (d, 1H, J = 16.0 Hz, H-15), 6.70 (d, 1H, J = 16.0 Hz, H-16), 6.35
(s, 1H, H-2), 3.23 (q, 2H, J = 6.4 Hz, H-18), 2.58 (s, 3H, H-12), 2.52
(s, 3H, H-13), 2.43 (s, 3H, H-14), 1.49 (quint., 2H, J = 6.8 Hz, H-
19), 1.33 (sextet, 2H, J = 7.2 Hz, H-20), 0.91 (t, 3H, J = 7.2 Hz, H-
21); 13C NMR (DMSO-d6, d) 165.5, 160.3, 159.3, 154.3, 154.2,
149.1, 128.4, 122.6, 122.2, 116.6, 113.4, 113.0, 112.6, 108.8, 38.8,
38.7, 32.0, 20.1, 19.3, 14.2, 12.9, 8.7; Fnal. Calcd for C21H23NO4:
C, 71.37; H, 6.56; N, 3.96. Found: C, 71.58; H, 6.24; N, 4.09.
4.5. Syntheses of acitretin and trioxsalen amides
4.5.1. Synthesis of acitretin benzylamide (10)
To a solution of 21 (0.05 g, 0.12 mmol) and DIEA (0.03 mL,
0.18 mmol) in CH2Cl2 (0.2 mL), benzylamine (0.014 mL, 0.13 mmol)
was added dropwise. After 15 min, a yellow solid precipitated out.
Additional CH2Cl2 was added to dissolve the precipitate and thus ob-
tained solution was washed sequentially with a 5% aqueous solution
of citric acid, H2O, a 5% aqueous solution of NaHCO3 and H2O, dried
over Na2SO4 and evaporated to dryness to afford pure 10 (0.034 g,
68%) as yellow solid. It had mp 165–169 °C; Rf (A): 0.28; IR (KBr,
cmꢁ1): 3320, 1630, 1616, 1586, 1536, 1120, 966; ESI-MS: m/z
438.2 [M+Na], 416.3 [V+H], 309.4 [(M+H)ꢁPhCH2NH2]; 1H NMR
(DMSO-d6, d): 8.45 (t, 1H, J = 5.8 Hz, NHCO), 7.32 (t, 2H, J = 7.2 Hz,
H-24), 7.26 (d, 2H, J = 7.2 Hz, H-23), 7.23 (t, 1H, J = 7.2 Hz, H-25),
6.96 (dd, 1H, J = 11.6 and 15.6 Hz, H-5), 6.69 (s, 1H, H-12), 6.67 (d,
1H, J = 15.6 Hz, H-4), 6.34 (d, 1H, J = 15.8 Hz, H-8), 6.31 (d, 1H,
J = 11.6 Hz, H-6), 6.27 (d, 1H, J = 15.8 Hz, H-9), 5.91 (s, 1H, H-2),
4.31 (d, 2H, J = 5.8 Hz, H-21), 3.75 (s, 3H, OCH3), 2.31 (s, 3H, H-20),
2.25 (s, 3H, H-16), 2.18 (s, 3H, H-18), 2.06 (s, 6H, H-17 and H-19);
13C NMR (DMSO-d6, d): 165.5, 156.1, 147.0, 140.1, 138.2, 138.1,
137.0, 135.6, 134.0, 131.2, 129.8, 129.6, 128.7 (2C), 128.0, 127.8
(2C), 127.2, 123.3, 122.0, 110.6, 55.8, 42.4, 21.6, 17.7, 13.6, 13.1,
12.2; Fnal. Calcd for C28H33NO2: C, 80.93; H, 8.00; N, 3.37. Found:
C, 80.58; H, 8.22; N, 3.59.
4.6. Synthesis of N-[2-(3,4-dihydroxyphenyl)ethyl]-3,4-
dihydroxy-L-phenylalaninamide (13)
To an ice-cold suspension of dopamine hydrochloride (0.04 g,
0.22 mmol) in CHCl3/DMF (6:1, 0.23 mL), DIEA (0.08 mL, 0.46 mmol)
was added and the resulting suspension was stirred for further
10 min at that temperature. Then, compound 20 (0.1 g, 0.18 mmol)
was added and the reaction mixture was progressively turned into a
clear solution. Stirring was continued for 30 min, at ambient tem-
perature, to complete the reaction. The reaction mixture was then
diluted with CHCl3 and washed sequentially with a cooled 5% aque-
ous solution of citric acid, H2O, a cooled 5% aqueous solution of NaH-
CO3 and H2O, dried over Na2SO4 and evaporated to dryness to leave
an oily residue. Pure N-tritylated 13 (0.055 g, 53%) was obtained as a
white foam, after FCC purification using as eluent the solvent system
H. This compound had Rf (H): 0.29; IR (thin film, CHCl3, cmꢁ1): 3334,
1638, 1602, 1446, 1284, 780, 748, 706; ESI-MS: m/z 596.9 [M+Na],
243.2 [Trt].
Protected 13 (0.05 g, 0.087 mmol) was then diluted in an ice-cold
solution of 30% TFA/CH2Cl2 (3 mL) and was stirred at that tempera-
ture for 30 min. Then, solvents were evaporated, the residue was
triturated with Et2O and refrigerated overnight. Pure 13 (0.033 g,
85%) was thus obtained, following filtration under vacuo, as the cor-
responding trifluoroacetate salt. The latter was converted to its
hydrochloride salt (a white solid) upon treatment with a 1.5 M solu-
4.5.2. Acitretin 2-(3,4-dihydroxyphenyl)ethylamide (11)
To an ice-cold suspension of dopamine hydrochloride (0.04 g,
0.21 mmol) in CHCl3/DMF 5:1 (0.18 mL), DIEA (0.08 mL,
0.46 mmol) was added and the resulting suspension was stirred
for further 10 min at this temperature. Then, succinimidyl ester
21 (0.05 g, 0.12 mmol) was added and the resulting reaction mix-
ture was stirred at 0 °C for 5 min and at room temperature for fur-
ther 2 h. Progressively, the reaction mixture was turned into a
solution. After completion of the reaction, the mixture was diluted
with CHCl3, washed sequentially with a 5% aqueous solution of
NaHCO3, H2O, a 5% aqueous solution of citric acid and H2O, dried
over Na2SO4 and evaporated to dryness to give a residue. Pure 11
(0.047 g, 85%) was obtained as yellow solid, after FCC purification
using as eluant the solvent system H. It had mp 152–155 °C; Rf
(H): 0.36; IR (KBr, cmꢁ1): 3472, 3414, 1636, 1616, 1120; ESI-MS:
m/z 484.3 [V+Na], 462.3 [M+H], 309.3 [(M+H)ꢁ(HO)2Ph(CH2)2
NH2]; 1H NMR (DMSO-d6, d): 8.55 (br s, 2H, OH), 7.84 (t, 1H,
J = 5.2 Hz, NHCO), 6.93 (dd, 1H, J = 11.6 and 14.8 Hz, H-5), 6.68 (s,
1H, H-12), 6.66 (d, 1H, J = 16.0 Hz, H-8), 6.64 (d, 1H, J = 8.4 Hz, H-
25), 6.59 (d, 1H, J = 1.6 Hz, H-28), 6.45 (dd, 1H, J = 1.6 and 8.4 Hz,
H-24), 6.32 (d, 1H, J = 11.2 Hz, H-6), 6.31 (d, 1H, J = 14.8 Hz, H-4),
6.26 (d, 1H, J = 16.0 Hz, H-9), 5.83 (s, 1H, H-2), 3.76 (s, 3H, OCH3),
3.25 (H-21, burried under H2O), 2.56 (t, 2H, J = 7.6 Hz, H-22), 2.28
(s, 3H, H-20), 2.25 (s, 3H, H-16), 2.18 (s, 3H, H-18), 2.07 (s, 3H,
H-17) 2.06 (s, 3H, H-19); 13C NMR (DMSO-d6, d): 166.5, 156.0,
146.4, 145.5, 144.0, 138.2, 138.0, 137.1, 135.6, 133.9, 131.2,
130.8, 129.8, 129.3, 127.9, 123.6, 122.0, 119.6, 116.4, 115.9,
110.6, 55.8, 41.0, 35.1, 21.6, 17.6, 13.6, 13.0, 12.2; Fnal. Calcd for
tion of HCl in EtOH, which had ½a D25
ꢃ
+16.2 (c 0.5, MeOH); Rf (I): 0.17;
IR (KBr, cmꢁ1): 3408, 1674, 1638, 1616, 1524, 1446, 1286, 1198; ESI-
MS: m/z 665.3 [2M+H], 333.2 [M+H]; RP-HPLC: tR = 4.43 min 1H
NMR (DMSO-d6, d): 8.88, 8.85, 8.76 and 8.71 (four s, 4H, OH), 8.36
(t, 1H, J = 5.6 Hz, NHCO), 8.02 (br s, 3H, NH3þ), 6.66 (d, 1H,
J = 8.0 Hz, H-6), 6.63 (d, 1H, J = 8.4 Hz, H-16), 6.61 (d, 1H, J = 2.0 Hz,
H-9), 6.57 (d, 1H, J = 2.0 Hz, H-13), 6.45 (dd, 1H, J = 2.0 and 8.0 Hz,
H-5), 6.41 (dd, 1H, J = 2.0 and 8.4 Hz, H-17), 3.25 (H-10, burried un-
der H2O), 3.13 (sextet, 1H, J = 6.0 Hz, H-2), 2.82 (dd, 1H, J = 6.0 and
13.6 Hz, H-3a), 2.70 (dd, 1H, J = 6.0 and 13.6 Hz, H-3b), 2.48 (t, 2H,
J = 7.6 Hz, H-11); 13C NMR (DMSO-d6, d): 168.3, 145.7, 145.6,
145.0, 144.1, 130.2, 126.0, 120.7, 119.7, 117.2, 116.4, 116.0, 115.9,
54.4, 41.1, 37.1, 34.7.
4.7. Preparation of conjugates 14 and 15
C
29H35NO4: C, 75.46; H, 7.64; N, 3.03. Found: C, 75.62; H, 7.44;
4.7.1. (E)-N-(5-Hydroxy-3-oxopentyl)-3,4-dimethoxycinnamide
(23)
N, 2.92.
To an ice-cold solution of 22(0.31 g, 1 mmol) and 2-(2-aminoeth-
oxy)ethanol (0.10 mL, 1 mmol) in DMF (1.7 mL), TEA (0.28 mL) was
added dropwise. The resulting solution was stirred at that tempera-
ture for 10 min and then left to attain ambient temperature where it
was kept for further 45 min. After completion of the reaction, the
mixture was diluted with CHCl3 and washed once with a 5% aqueous
solution of NaHCO3 and twice with H2O. The organic layer was dried
4.5.3. N-Butyl-3-(trioxsalen-40-yl)acrylamide (12)
To an ice-cold suspension of 16 (0.04 g, 0.13 mmol), 1-butyl-
amine (0.014 mL, 0.14 mmol) and DIEA (0.05 mL, 0.26 mmol) in
DMF (0.25 mL), HBTU (0.07 g, 0.19 mmol) was added. The resulting
solution was stirred at ambient temperature for 30 min to com-
plete the reaction. The mixture was diluted with CHCl3 washed

D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
8215
over Na2SO4 and evaporated to dryness to leave an oily residue. Pure
23 (0.26 g, 88%) was obtained, as a colourless oil after FCC purifica-
tion and using solvent system H as eluant. Compound 23 had Rf
(H): 0.37; IR (thin film, CHCl3, cmꢁ1): 3356, 3288, 1660, 1598,
1554, 1514, 1260, 1138; ESI-MS: m/z 318.4 [M+Na], 296.4 [M+H],
191.5 [(M+H)ꢁHO(CH2)2O(CH2)2NH2]; 1H NMR (CDCl3, d): 7.56 (d,
1H, J = 16.0 Hz, H-3), 7.05 (d, 1H, J = 8.0 Hz, H-6), 7.01 (s, 1H, H-9),
6.81 (d, 1H, J = 8.0 Hz, H-5), 6.51 (br s, 1H, NHCO), 6.32 (d, 1H,
J = 16.0 Hz, H-2), 3.88 (s, 6H, OCH3), 3.76 (t, 2H, J = 4.0 Hz, H-12),
3.67–3.56 (m, 6H, H-10, H-11 and H-13), 2.43 (br s, 1H, OH); 13C
NMR (CDCl3, d): 161.8, 145.8, 144.3, 136.3, 123.1, 117.2, 113.7,
106.3, 105.0, 67.5, 65.2, 57.0, 51.2, 51.1, 34.8.
and indomethacin were obtained from Sigma Chemical, Co. (St.
Louis, MO, USA) and Carrageenan, type K, was commercially avail-
able. For the in vivo experiments, male and female Fischer-344 rats
(180–240 g) were used. For the in vitro tests a Lambda 20 (Perkin–
Elmer) UV–vis double beam spectrophotometer was used. Each
in vitro experiment was performed at least in triplicate and the
standard deviation of absorbance was less than 10% of the mean.
4.9. In vitro assays
4.9.1. Determination of the reducing activity (RA) of the stable
radical DPPH8,9
To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol
an equal volume of the compounds dissolved in DMSO was added.
The mixture was shaken vigorously and allowed to stand for
20 min or 60 min; absorbance at 517 nm was determined spectro-
photometrically and the percentage of activity was calculated. All
tests were undertaken on three or four replicates and the results
were averaged (Table 1).
4.7.2. General procedure for coupling 23 with the carboxylic
acids FICA and TRAA
To an ice-cold suspension of the carboxylic acids FICA or TRAA
(0.3 mmol), 23 (0.09 g, 0.3 mmol) and TPP (0.09 g, 0.36 mmol) in
THF (1 mL), DIAD (0.07 mL, 0.36 mmol) was added dropwise and
the resulting solution was stirred at ambient temperature to com-
plete the reaction. The reaction mixtures were directly chromato-
graphed (FCC) to afford pure conjugates 14 and 15 respectively.
4.9.2. Soybean LOX inhibition study in vitro8,9
The tested compounds dissolved in DMSO were incubated at rt
with sodium linoleate (0.1 mL) and 0.2 mL of enzyme solution
(1/9 ꢂ 10ꢁ4 w/v in saline). The conversion of sodium linoleate to
13-hydroperoxylinoleic acid at 234 nm was recorded and com-
pared with the appropriate standard inhibitor.
4.7.2.1. (E,E)-N-[11-Aza-14-(3,4-dimethoxyphenyl)-5,8-dioxa-4,12-
dioxotetradeca-2,13-dienoyl] indole-3-carboxanilide (14). Reac-
tion time: 40 min; Elution system D; Yield: 0.12 g (64%), yellow solid;
mp 115–117 °C; Rf (D): 0.25; IR (KBr, cmꢁ1): 3474, 3412, 3280, 1734,
1720, 1654, 1616, 1600, 1542, 1438, 1184, 1106, 754, 720, 694; ESI-
MS: m/z 634.1 [V+Na], 612.0 [M+H]; 1H NMR (DMSO-d6, d): 10.14 (s,
1H, PhNHCO), 8.93 (s, 1H, H-18), 8.45–8.40 (m, 1H, H-29), 8.31–8.26
(m, 1H, H-26), 8.07 (t, 1H, J = 5.6 Hz, NHCO), 7.90 (d, 1H, J = 16.0 Hz,
H-15), 7.76 (unresolv. dd, 2H, H-22), 7.67–7.64 (m, 2H, H-27 and H-
28), 7.57–7.54 (m, 3H, H-23 and H-24), 7.32 (d, 1H, J = 16.0 Hz, H-3),
7.10 (d, 1H, J = 1.6 Hz, H-9), 7.08 (d, 1H, J = 16.0 Hz, H-16), 7.04 (dd,
1H, J = 1.6 and 8.4 Hz, H-5), 6.88 (d, 1H, J = 8.4 Hz, H-6), 6.54 (d, 1H,
J = 16.0 Hz, H-2), 4.42–4.36 (m, 2H, H-12), 3.75 and 3.72 (two s, 6H,
OCH3), 3.76–3.73 (m, 2H, H-13), 3.56 (t, 2H, J = 5.6 Hz, H-11), 3.50
(2H, H-11, buried under water); 13C NMR (DMSO-d6, d): 165.9, 165.1,
163.4, 162.1, 150.5, 149.3, 139.2, 135.8, 134.5, 133.8, 133.7, 132.7,
132.5, 129.2 (two C), 129.1, 126.2, 125.5, 124.8, 122.3, 121.7, 120.7
(two C), 120.3, 116.9, 116.6, 112.1, 110.5, 69.6, 68.3, 65.0, 55.9, 55.8,
39.1; Fnal. Calcd for C34H33N3O8: C, 66.77; H, 5.44; N, 6.87. Found: C,
66.56; H, 5.23; N, 7.03.
4.9.3. Inhibition of linoleic acid lipid peroxidation8,9
Production of conjugated diene hydroperoxide by oxidation of
linoleic acid in an aqueous dispersion is monitored at 234 nm.
AAPH is used as a free radical initiator. This assay can be used to
follow oxidative changes and to understand the contribution of
each tested compound.
Azo compounds generating free radicals through spontaneous
thermal decomposition are useful for in vitro studies of free radical
production. The water-soluble azo compound AAPH has been
extensively used as a clean and controllable source of thermally
produced alkylperoxyl free radicals. Ten microlitres of the 16 mM
linoleic acid sodium salt solution was added to the UV cuvette con-
taining 0.93 mL of 0.05 M phosphate buffer, pH 7.4 prethermostat-
ed at 37 °C. The oxidation reaction was initiated at 37 °C under air
by the addition of 50
lL of 40 mM AAPH solution. Oxidation was
carried out in the presence of aliquots (10
lL) in the assay without
4.7.2.2. 5-(3,4-Dimethoxycinnamoylamido)-3-oxapentyl 3-(tri-
oxsalen-40-yl)acrylate (15). Reaction time: 45 min; Elution sys-
tem G; Yield: 0.12 g (68%), white solid; mp 105–110 °C; Rf (G):
0.27; IR (KBr, cmꢁ1): 3274, 3216, 1734, 1710, 1636, 1598, 1510,
1260, 1236, 1104, 722, 696; ESI-MS: m/z 598.1 [V+Na], 576.1
[M+H]; 1H NMR (DMSO-d6, d): 8.01 (t, 1H, J = 5.6 Hz, NHCO), 7.86
(s, 1H, H-26), 7.73 (d, 1H, J = 16.0 Hz, H-16), 7.24 (d, 1H,
J = 16.0 Hz, H-3), 6.94 (s, 1H, H-9), 6.93 (d, 1H, J = 8.0 Hz, H-6), 6.82
(d, 1H, J = 8.0 Hz, H-5), 6.63 (d, 1H, J = 16.0 Hz, H-15), 6.47 (d, 1H,
J = 16.0 Hz, H-2), 6.35 (br s, 1H, H-23), 4.36–4.32 (m, 2H, H-12),
3.74 (s, 3H, OCH3), 3.75–3.72 (m, 2H, H-13), 3.68 (s, 3H, OCH3),
3.56 (t, 2H, J = 5.6 Hz, H-11), 3.50 (2H, H-10, buried under water),
2.62 (s, 3H, H-28), 2.54 (s, 3H, H-29), 2.45 (s, 3H, H-30); 13C NMR
(DMSO-d6, d): 166.9, 165.8, 161.3, 160.3, 156.6, 154.6, 154.3,
150.3, 149.3, 149.1, 139.1, 134.8, 133.6, 132.7, 132.5 (two C),
129.9, 122.0, 121.7, 117.6, 114.1, 112.5, 111.8, 110.0, 68.4 (two C),
63.9, 55.9, 55.7, 19.4, 13.0, 8.6; Fnal. Calcd for C32H33NO9: C,
66.77; H, 5.78; N, 2.43. Found: C, 66.98; H, 5.62; N, 2.31.
antioxidant, lipid oxidation was measured in the presence of the
same level of DMSO. The rate of oxidation at 37 °C was monitored
by recording the increase in absorption at 234 nm caused by con-
jugated diene hydroperoxides.
4.9.4. Evaluation of the cytocompatibility of conjugates
RAMEC rat endothelial cells were isolated as described previ-
ously.41 The cells were routinely subcultured in Dulbecco’s MEM
full growth medium (Biochrom AG, Germany), containing 10% foe-
tal calf serum, 200 mM
L-glutamine, 2.5 lg/mL Amphotericin B,
100 g/mL streptomycin, 100 U/mL penicillin and 0.003% endothe-
l
lial cell growth supplement in a humidified atmosphere containing
5% CO2 at 37 °C.
The cytocompatibility of the compounds was evaluated by the
viability of RAMEC cells in the presence of different concentrations
of the compounds (0.5–50
seeded in 24-well plates at a density of 20,000 cells per well in
500 L Dulbecco’s MEM full growth medium containing 10% foetal
calf serum, 200 mM -glutamine, 2.5 g/mL Amphotericin B,
100 g/mL streptomycin, 100 U/mL penicillin and 0.003% endothe-
l
M) using the MTT test.42 The cells were
l
L
l
4.8. General biological assays
l
lial cell growth supplement. Twenty-four hours after plating, dif-
DPPH, NDGA and CA were purchased from the Aldrich Chemical
Co. Milwaukee, WI, (USA). Soybean LOX, linoleic acid sodium salt
ferent amounts of the compounds were added in the wells. After
24 h of incubation at 37 °C, 50 lL of MTT solution (5 mg/mL in

8216
D. Hadjipavlou-Litina et al. / Bioorg. Med. Chem. 18 (2010) 8204–8217
phosphate buffered saline, pH 7.4) were added into each well and
Acknowledgements
the plates were incubated at 37 °C for 2 h. The medium was with-
drawn and 200 lL of DMSO was added in each well and agitated
We thank the European Social Fund (ESF), Operational Program
for Educational and Vocational Training II (EPEAEK II) and particu-
larly the Program IRAKLEITOS as well as BIOMEDICA for funding
the synthetic part of this work. We also wish to thank Dr. C. Hansch
and Biobyte Corp. 201 West 4th Str., Suite 204, Claremont CA,
California 91711, USA for free access to the C-QSAR program.
thoroughly to dissolve the formazan crystals. The solution was
transferred to 96-well plates and immediately read on a microplate
reader, at a wavelength of 540 nm. The experiments were per-
formed in hexaplicate. Cell viability was calculated from the ratio
between the absorbance provided by the cells treated with the dif-
ferent compounds and the absorbance provided by the non-treated
cells (control) and the 50% growth inhibition values (IC50) were cal-
culated. Viability data are subject to 5% error.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.10.012. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
4.10. In vivo assays
All screening tests were conducted on Fisher-344 inbred rats of
ca. 150–200 g body weight; each group was composed of six ani-
mals. Compounds were dispersed in water with few drops of
Tween 80 and ground in a mortar before use (or diluted in water
as salts) and administered ip. Controls received the liquid vehicle.
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