Synthesis and antioxidative/anti-inflammatory activity of novel fullerene-polyamine conjugates

Article abstract of DOI:10.1016/j.tet.2012.06.066

(E)-4-(Fullerenopyrrolidin-1-yl)-3-methylbut-2-enoic acid and its corresponding succinimidyl ester, readily obtained through Prato-type modification of C₆₀, were used for the selective N-acylation of polyamines. The thus obtained conjugates were evaluated for their antioxidative and anti-inflammatory activity and their cytotoxicity was determined. Members of this family of compounds showed interesting anti-lipid peroxidation, anti-lipoxygenase and anti-inflammatory activity and comparable cytocompatibility to spermidine.

Full text of DOI:10.1016/j.tet.2012.06.066

Tetrahedron 68 (2012) 7041e7049
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and antioxidative/anti-inflammatory activity of novel
fullereneepolyamine conjugates
,
George E. Magoulas a, Thomas Garnelis a, Constantinos M. Athanassopoulos a, Dionissios Papaioannou a
,
*
,
George Mattheolabakis b, Konstantinos Avgoustakis b, Dimitra Hadjipavlou-Litina c
*
a Laboratory of Synthetic Organic Chemistry, Department of Chemistry, University of Patras, 26504 Patras, Greece
b Department of Pharmacy, University of Patras, 26504 Patras, Greece
c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
(E)-4-(Fullerenopyrrolidin-1-yl)-3-methylbut-2-enoic acid and its corresponding succinimidyl ester,
readily obtained through Prato-type modification of C60, were used for the selective N-acylation of
polyamines. The thus obtained conjugates were evaluated for their antioxidative and anti-inflammatory
activity and their cytotoxicity was determined. Members of this family of compounds showed interesting
anti-lipid peroxidation, anti-lipoxygenase and anti-inflammatory activity and comparable cytocompat-
ibility to spermidine.
Received 13 January 2012
Received in revised form 25 May 2012
Accepted 18 June 2012
Available online 26 June 2012
Keywords:
Fullerenes
Ó 2012 Elsevier Ltd. All rights reserved.
Polyamines
Conjugates
Anti-inflammatory activity
Lipoxygenase inhibitors
1. Introduction
delivery systems as recently exemplified with a fullerene conju-
gate with the well-known anticancer drug taxol.7 In this particular
Fullerenes, such as C60 (FUL, 1), constitute a particularly im-
portant family of compounds consisting solely of carbon atoms,
which are arranged in the outer surface of a sphere in the form of
condensed six- and five-membered rings. Their hydrophobic
character as well as their ability to act as free-radical sponges1 and
to induce interesting biochemical effects under the influence of
even low-energy visible light, make fullerenes attractive structural
moieties for various biological applications. The biological evalua-
tion of a plethora of fullerene derivatives, which have been so far
synthesized showed that members of this family exert interesting
pharmacological effects, like anticancer, antiviral and antibacterial
action, antioxidant and neuroprotectory properties, enzyme in-
hibition, interaction with DNA and have been proposed for photo-
dynamic therapy and various gene transfer applications.2e6 Among
polyfunctionalized fullerenes, anionic compounds give higher
protection than cationic ones.
system, an ester bond labile to cellular esterases is used to attach
the drug on a suitably modified C60 molecule through a spacer of
the a,u-dicarboxylic acid type. Modification of C60 is widely used
for the improvement of the solubility of C60 in organic solvents as
well as to provide the handle for anchoring various other structural
units or drugs on C60.8 Modification of C60 is already a well-studied
process and usually takes place through either the Prato reaction or
the DielseAlder reaction or the BingeleHirsch reaction.2
Spermine (SPM) is a natural antioxidant, which is found in all
living organisms. Research shows that SPM has an important
function 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. SPM’s role is that of protecting cells against
free radicals and their destructive effects.9 Persistently high levels
of reactive oxygen species (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. There is an in-
creasing 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.
Another, particularly interesting, application of fullerenes have
been recently exploited with success, namely their use as drug-
* Corresponding authors. Tel.: þ30 2610 962954; fax: þ30 2610 962956 (D.P.);
tel.: þ30 2310 997627; fax: þ30 2310 997679 (D.H.-L.); e-mail addresses: dapa-
paio@chemistry.upatras.gr (D. Papaioannou), hadjipav@pharm.auth.gr (D.
Hadjipavlou-Litina).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.06.066

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G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
Today, there is an increased interest in the combination of two
would be effected by a C60-modified derivative bearing an a,b-un-
pharmacophores on the same scaffold. This procedure leads to
hybrid molecules or conjugates. Hybrid drugs, just as their name
implies, combine two drugs in a single molecule with the goal of
creating a chemical entity, which is more medically effective than
its individual components. These combination drugs can indeed be
more powerful than either of their precursors.10
saturated succinimidyl ester-type side-chain, such as compound 29
(Scheme 1). This compound was readily obtained using glycine
Taking into consideration the ability of fullerenes to act as rad-
ical sponges being capable of effectively quenching ROS, and that
polyamines (PAs), such as SPM and spermidine (SPD), present
antioxidative activity, which is correlated to the number of the
amino groups in the molecule (SPM more efficient than SPD),11 we
thought it was of interest to examine the possibility of preparing
FUL-PA conjugates, as exemplified with the conjugates 2e7, as well
as FUL-PA-drug hybrid conjugates, as exemplified with the conju-
gates 8 and 9 (Fig. 1, fully extended structures of compounds 2e9
Scheme 1. Synthesis of FUL-derived acid 28 and active ester 29. Reaction conditions:
(a) Ph3P]CHCOt2Bu, MeCN, 82 ꢀC, 16 h, 90%; (b) o-NO2eC6H4SO2Cl, Et3N, CHCl3, 0 ꢀC,
3 h, 88%; (c) Ph3P, DIAD, THF, 0 ꢀC for 5 min then 25 ꢀC, 2 h, 96%; (d) PhSH, Na2CO3,
DMF, 25 ꢀC, 12 h, 84%; (e) 6 N NaOH, MeOH, 0e25 ꢀC, 30 min, then 1 N HCl to pH 4.5,
80%; (f) C60, (CH2O)n, PhMe, 90 ꢀC, 3 h, 48%; (g) 50% TFA in CH2Cl2, 0 ꢀC, 1 h, 96%; (h)
HOSu, DIC, THF/1,2-dichlorobenzene (3:1), 0 ꢀC for 30 min then 25 ꢀC for 16 h, 85%.
derivative 26 as key-intermediate. The synthesis of the latter is
outlined in Scheme 1. Thus, nosylation of glycine ethyl ester fol-
lowed by Mitsunobu-type condensation of the thus obtained
N-nosylated derivative 24 with alcohol 23, readily obtained
through the condensation of hydroxyacetone with phosphorane
Ph3P]COt2Bu,19 gave the fully protected compound 25. Sequential
removal of the nosyl protecting group and selective saponification
provided the required N-alkylated glycine derivative 26. Prato re-
action of key-intermediate 26 with FUL (1) provided the anticipated
C60 derivative 27 in 48% yield. Using the same type of reaction,
other types of fullerene-based saturated amino acids and peptides
as well as multifullerene peptides have been synthesized.3,8c
Treatment of ester 27 with 50% trifluoroacetic acid (TFA) in
CH2Cl2 resulted in the removal of the tBu group, thus providing acid
28 in 96% yield. Activation of acid 28, under the action of N,N0-
diisopropylcarbodiimide (DIC) and N-hydroxysuccinimide (HOSu)
in THF/1,2-dichlorobenzene (3:1), finally provided succinimidyl
ester 29 in 85% yield.20 FUL-derived acid 28 and the active ester 29
Fig. 1. Structures of compounds encountered in the present work.
may be seen in the Supplementary data material), and evaluate
their antioxidative/anti-inflammatory activity. The hybrid conju-
gates incorporate drugs currently in use for the treatment of pso-
riasis and other dermatological disorders,12 namely acitretin (10)
and trioxsalen (11), which have been introduced into the PA back-
bone in the form of the isolable succinimidyl esters 1213 and 13,14
respectively. As PAs, the commercially available SPM (16) and SPD
(17), were used as well as the synthetic PAs 19 and 20,15 available in
our laboratory through other research projects. It should be noted
that a functionalized fullerene bearing a tetraamine moiety has
been synthesized and shown to be an efficient gene transfer
agent.16 Also, other types of functionalized C60 have been synthe-
sized and their antioxidative activity towards lipid peroxidation or
their anti-inflammatory activity has been studied.17
were then used for attaching various PAs on C60
.
For the synthesis of asymmetric conjugates, such as 2e5 we
envisaged that, with the exception of PA derivative 20, suitably
protected PAs should be used. Such derivatives are, for example, the
N1,N4,N9-tri(tert-butoxycarbonyl)spermine (30),21 for anchoring
one FUL unit on the SPM skeleton, and the N1-trityl (32a)- or N8-
trityl(32b)spermidine,22 for anchoring one FUL unit on the SPD
skeleton. Thus, condensation of SPM derivative 30 with active ester
29 in the presence of Et3N produced the fully protected monoamide
31 (Scheme 2) in 82% yield. This, upon TFA-mediated deprotection,
gave the SPM monoamide 2, as the corresponding tristrifluorocetate
salt, in 94% yield. Similarly, condensation of active ester 29 with, for
example, N1-tritylspermidine (32a) gave amide 33 in 91% yield upon
selective acylation, as expected, of the primary amino function.
Routine detritylation with 30% TFA/CH2Cl2 gave the monoacylated
SPD conjugate 3, as the corresponding bistrifluoroacete salt, in 95%
yield. Unexceptionally, PA derivative 20 was also coupled with ac-
tive ester 29 to provide the anticipated amide 4 in 78% yield. The
suitability of monoacylated PA derivatives 2 or 3 to provide hybrid
conjugates with suitably derivatized antipsoriatic or other types of
drugs was exemplified through the preparation of compounds 8 and
9, using as starting material the FUL-SPM conjugate 2. Indeed,
compound 2 was selectively acylated at the primary amino function
with active esters 12 and 13 in the presence of iPr2NEt, to produce
the desired hybrid SPM conjugates 8 and 9 in 56% and 52% yield,
2. Results
2.1. Synthesis
We have shown in a series of publications that PAs can be di-
rectly and selectively acylated at their primary amino functions in
the presence of secondary ones by using isolable a,b-unsaturated
succinimidyl esters, such as compounds 1213 and 13,14 as well as
succinimidyl esters of cinnamic acids.18 We therefore envisaged
that the direct selective functionalization of PAs, such as SPM,

G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
7043
respectively. For the synthesis of the N4-acylated SPD conjugate 5,
2.2. Biological testing
the FUL-derived acid 28 was condensed with N1,N8-ditritylspermi-
dine (34)23 in the presence of iPr2NEt in DMF using
bromopyrrolidino-phosphonium hexafluorophosphate (PyBrOP) as
the coupling agent. That way, the fully protected conjugate 35 was
obtained in 58% yield, which upon TFA-mediated deprotection gave
the anticipated conjugate 5, as the corresponding bistrifluoroacete
salt, in 95% yield.
2.2.1. Antioxidative activity in vitro. The antioxidant properties of
water-soluble fullerene derivatives mainly tris-malonic acid ful-
lerene derivatives and dendrofullerenes have been studied in
detail.6 In the present investigation, the conjugates 2e9 as well as
the parent molecule 1 and the FUL-linker adducts 27 and 28 were
studied with regard to their antioxidant ability, using the antioxi-
dant assay, which involves the interaction of antioxidants with the
water-soluble azo compound 2,2-azobis(2-amidinopropane) dihy-
drochloride (AAPH), and for their ability to inhibit soybean lip-
oxygenase (LOX) by the UV absorbance based enzyme assay.25 The
5-lipoxygenase (5-LOX) catalyzes the first two steps in the me-
tabolism of arachidonic acid to leukotrienes with a marked relation
to the severity of cardiovascular diseases, asthma and cancer.
Inhibitors of LOX have attracted attention initially as potential
agents for the treatment of inflammatory and allergic diseases,
asthma, certain types of cancer and cardiovascular diseases.26,27
The data for the antioxidant properties of the synthesized
compounds and of selected unconjugated molecules are tabulated
in Table 1. For the sake of comparison, corresponding data for the
compounds 10,28 14e15,29 16e1730,31 and 21e22,30 obtained
through previous studies, are also included in this table.
Table 1
% Inhibition of lipid peroxidation (AAPH %); in vitro inhibition of soybean lip-
oxygenase (LOX) (IC50); inhibition % of Carrageenan-induced rat paw oedema (ICPE
%)
Scheme 2. Synthesis of the FUL-PA conjugates 2e5 and 8e9. Reaction conditions: (a)
Et3N, CH2Cl2, 25 ꢀC, 30e45 min, 82% (for 31), 91% (for 33) and 78% (for 4); (b) 50% TFA
Compounda
AAPH % @
IC50 mM or %
LOX inh. @
ICPE %
i
in CH2Cl2, 25 ꢀC, 2 h, 94% (for 2) and 95% (for 5); (c) Pr2NEt, CH2Cl2/DMF (1:1), 25 ꢀC,
M (ꢁSD)b
0.01 mmol/kg
4 h, 56% (for 8) and 52% (for 9); (d) 30% TFA in CH2Cl2, 25 ꢀC, 2 h, 95%; (e) iPr2NEt,
100
m
(ꢁSD)b
PyBrOP, DMF, 25 ꢀC, 1 h, 58%.
100
m
M/50
m
M
(ꢁSD)b
5ꢁ0.1
1
2
3
4
5
6
7
8
9
10
14
15
16
17
9.6ꢁ0.3
100ꢁ5
86ꢁ1.7
0.09ꢁ0.0
86ꢁ1.9
0.06ꢁ0.0
82ꢁ2.1
98ꢁ1.7
0.4ꢁ0.0
85ꢁ1.6
73ꢁ5.3
80ꢁ6.2
na
30.5*ꢁ1.8
26**ꢁ1.2
42*ꢁ1.0
21*ꢁ0.8
nt
nt
nt
nt
77.5
78.5
m
m
Mꢁ3.4
Mꢁ3.1
The applicability of active ester 29 for the preparation of sym-
metric FUL-PA conjugates, such as 6 and 7 was initially tested with
its reaction with SPM. The reaction mixture was however complex
and we therefore decided to use the corresponding N4,N9-ditrityl-
spermine (18) in the coupling reaction.
50
m
Mꢁ2.2
42/0ꢁ1.1
67
na
80
50
m
Mꢁ2.2
m
m
Mꢁ3.5
Mꢁ1.4
The latter was obtained in 27% total yield from SPM through
sequential selective N1,N12-bistrifluoroacetylation,24 N4,N9-ditrity-
lation and finally removal of the trifluoroacetyl protecting groups
with saponification.23 Coupling of active ester 29 with compound
18 in the presence of Et3N in CH2Cl2 produced after 2 h at 25 ꢀC the
anticipated symmetric conjugate 36 and the monoacylated SPM
derivative 37 in 35 and 15% yields, respectively (Scheme 3).
From compound 36, the symmetric conjugate 6 was obtained as
the corresponding bistrifluoroacete salt in 92% yield, through rou-
tine deprotection with 50% TFA in CH2Cl2. Similarly, condensation
of active ester 29 with PA 19, also in the presence Et3N, produced
after 3 h at 25 ꢀC the anticipated symmetric conjugate 7 (34% yield)
together with the corresponding monoacylated PA derivative 38 in
20% yield.
9.2*ꢁ0.5
63**ꢁ3.4
nt
1/naꢁ10ꢂ3
9/naꢁ0.8
9.4
m
Mꢁ3.1
46**ꢁ1.8
na/na
na/na
nt
na
nt
21
22
27
28
97ꢁ3.8
33ꢁ2.2
53ꢁ0.8
55ꢁ1.9
85
95
na
m
m
Mꢁ6.7
19*ꢁ1.2
Mꢁ2.2
nt
nt
43/0ꢁ2.1
36.5*ꢁ0.7
NDGA
Trolox
Indomethacin
28
m
Mꢁ1.3
nt
nt
63ꢁ1.2
47**ꢁ3.1
a
Full structures of all compounds in this column are presented in the
Supplementary data section (Table S1).
b
Values are meansꢁSD of three or four different determinations; na: no activity
under the reported experimental conditions; nt: not tested; means within each
column differ significantly (p <0.05); 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. Each value represents the mean obtained from 6 to 15 animals in two
independent experiments. In all cases, significant difference from control: *p <0.1,
**p <0.01 (Student’s t test).
2.2.2. Anti-inflammatory activity in vivo. Recently, the possibility to
use of C60 as anti-inflammatory agent has been reported.32 For the
in vivo anti-inflammatory screening we used the functional model
of Carrageenan-induced rat paw oedema. Carrageenan-induced
oedema is a non-specific inflammation resulting from a complex
of diverse mediators.33 The selection of FUL-PA conjugates for
screening was generally based on their good inhibitory activity on
Scheme 3. Synthesis of the symmetric FUL-PA conjugates 6 and 7. Reaction conditions:
(a) Et3N, CH2Cl2, 25 ꢀC, 2e3 h, 34% (for 7) and 35% (for 36); (b) 50% TFA in CH2Cl2, 25 ꢀC,
1 h, 92%.

7044
G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
LOX. For the sake of comparison, compounds 1 and 28 were also
included in the study. The data for the anti-inflammatory potential
of the tested compounds are also tabulated in Table 1.
peroxidation, although SPM itself presents no activity in this sys-
tem. A comparison of the inhibitory values of conjugates 2 (100%),
3 (86%) and 5 (86%) reveals that the number (three better than two)
and not the nature (primary or secondary) of the free amino
functions are particularly important for inhibition. On the other
hand, attachment of a second C60 moiety or a trioxsalen-derived
moiety on conjugate 2 leads to conjugates 6 and 9 with a dra-
matic fall of the inhibitory potency (0.06 and 0.4%, respectively). On
the contrary, the inhibitory activity of 2 is essentially retained upon
attaching an acitretin moiety (conjugate 8, 98%). Worth noting,
however, is that the corresponding symmetric conjugates 22 and 21
had previously shown 33% and 97% inhibitory potencies,
respectively. Interestingly, when in the inactive conjugate 6 the two
secondary amino functions are replaced by tetrazole rings the
resulting compound 7 exhibits a high inhibitory value (82%).
However, extremely low inhibition is observed with the structure 4,
which looks like half of the structure of conjugate 7.
2.2.3. Cytotoxicity. It has been reported that lipophilic substitution
on N atoms of PAs leads to increased toxicity of the natural poly-
amines (SPM more than SPD)34 whereas, on the other hand, cat-
ionic fullerenes are associated with cytotoxicity.4 We therefore
evaluated the cytocompatibility of the fullerene-polyamine conju-
gates 2e6, 8 and 9, as well as of the FUL-derived acid 28, through
the viability of RAMEC rat endothelial cells in the presence of dif-
ferent concentrations of these compounds (1e50 mM), using the
MTT test essentially as described previously.35 For the sake of
comparison, SPD was also included in the study. The results are
given graphically in Fig. 2 (see also Table S2 in the Supplementary
data section).
Regarding the inhibitory activity of the synthesized conjugates
on soybean LOX, perusal of the IC50 inhibition values (Table 1)
shows that the most potent, and equipotent, inhibitors are the
conjugates 4 and 9 (IC50 50
67 M), 2 (IC50 77.5 M), 3 (IC50 78.5
PAs SPM and SPD, ester 27 and the conjugate 7 were found inactive.
Interestingly, the linear SPD conjugate 3 (87% at 100 M, IC50
78.5 M) was twice as potent inhibitor as its branched analogue,
namely SPD conjugate 5 (42% at 100 M). On the other hand, C60
had very low inhibitory activity (5% at 100 M) whereas acid 28
presented an inhibitory value of 43% (at 100 M). It is therefore
mM), followed by the conjugates 6 (IC50
m
m
m
M) and 8 (IC50 80 M). The
m
m
m
m
m
m
evident that attaching the linker on C60 leads to a significant
improvement of the inhibitory potency on LOX, which is not
however improved when the resulting adduct is attached at the
inner N atom of the SPD chain (branched conjugate 5, 42% at
Fig. 2. Cytotoxicity of FUL-PA conjugates on RAMEC cells (24 h incubation).
100 mM). On the contrary, when the linker is attached on the outer
3. Discussion
N atom of SPD (linear conjugate 3) the inhibitory activity is dou-
bled. Also, it is interesting to note that the longer PA chain of SPM
(conjugate 2) leads to a very small improvement in the inhibitory
activity. In any case, attachment of the FUL-linker adduct on a PA
chain leads to great improvement in the inhibitory potency of PAs
on LOX. Furthermore, attachment of a second moiety of acid 28 on
the PA (SPM) chain seems to offer further improvement of the in-
A very simple, efficient and versatile methodology has been
developed for the conjugation of fullerenes with PAs. The Prato
reaction was employed for the modification of C60 with an
a,b-unsaturated carboxylic acid-type linker and the resulting
compound 27 was readily converted to the corresponding isolable
succinimidyl ester 28. The latter compound is useful for the selec-
tive acylation of primary amino functions in the presence of sec-
ondary ones in PA molecules, whereas the former is useful for the
direct acylation of secondary amino functions of selectively pro-
tected PAs in the presence of an appropriate coupling agent.
The versatility of the method was demonstrated with prepara-
tion of PAs bearing one or two C60 units per PA skeleton (conjugates
2 and 6), as well as selectively monofunctionalized PAs at either the
primary or the secondary amino functions (conjugates 3 and 4). In
addition, the FUL-PA conjugates can be used to carry pharmaceu-
tically important molecules, exemplified herein with hybrid con-
jugates 8 and 9. Finally, replacement of C60 by other fullerenes or
carbon nanotubes can be envisaged, which will further augment
the versatility of the present methodology. The synthesized FUL-PA
conjugates in the context of the present work are suitable for
structureebiological activity relationship studies. In our anti-
oxidative activity studies, AAPH was used as a free radical initiator
to follow oxidative changes of linoleic acid to conjugated diene
hydroperoxide. The FUL-linker adducts, namely acid 28 (55%) and
tert-butyl ester 27 (53%) presented almost equipotent inhibitory
potencies, a little lower than trolox (Table 1) but much higher than
the parent fullerene C60 (9.6%). Therefore, the linker seems to in-
crease significantly the low inhibitory efficiency of C60. Further-
more, a comparison of the structures of acid 28 and conjugate 2 and
their corresponding inhibition values reveals that the presence of
the SPM chain causes significant increase of inhibition of lipid
hibitory potency against LOX (conjugate 6, IC50 67
tency is further improved when the second acid 28 moiety is
replaced by the trioxsalen-derived acid 14 (conjugate 9, IC50 50
but not with the acitretin (10) moiety (conjugate 8, IC50 80
This can be taken to mean that the primary amino function of 2 can
be replaced by an amido function whose carbonyl group comes
from a lipophilic acid, which is neither too long (acitretin) nor too
bulky (acid 14). In any case, amidation of either trioxsalen-derived
acid 14 or acitretin (10) with the PA conjugate 2 greatly improves
their inhibitory activity on LOX. Furthermore, a comparison of the
mM). This po-
mM)
mM).
inhibition of the symmetric conjugates 21 (75% at 100
85 M) and 22 (57% at 100 M, IC50 95 M) with the corresponding
asymmetric conjugates 8 (IC50 80 M) and 9 (IC50 50 M) shows
mM, IC50
m
m
m
m
m
that attachment on the SPM chain of a second molecule of either
acitretin (10) or acid 14 is less effective on inhibiting LOX than
attaching the FUL-derived acid 28, in particular in the case of
acid 14.
Finally, the extreme difference in inhibitory potency between
the conjugate 6 (IC50 67 mM) and the conjugate 7 (not active) needs
to be discussed. This difference should be attributed to two factors,
namely the replacement of the basic secondary amino functions
(protonated at physiological pH) by the tetrazole rings and the
replacement of the conformationally non-rigid secondary amino
functionalities by the tetrazole ring, which is considered as cis-
amide bond surrogate. On the contrary, the much simpler conju-
gate 4 (compared to conjugate 7) was shown to be the most potent

G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
7045
(together with conjugate 9) inhibitor of LOX, approaching the ac-
tivity of the reference compound NDGA (IC50 28 M). Conjugate 4,
NMR spectra at 100.62 MHz on a Bruker DPX spectrometer. Tetra-
methylsilane (TMS) was used as internal standard. Chemical shifts
m
incorporating an N-benzylated tetrazole ring, is an analogue of the
conjugate of C60 with the simpler PA 1,3-diaminopropane (a com-
mon substructure in SPM and SPD) and can be considered as a lead-
compound for the development of more active LOX inhibitors.
The most interesting conjugates, 2e4 and 9 were selected for
anti-inflammatory studies. In addition and for the sake of com-
parison, FUL (1) and the FUL-derived acid 28 were also examined.
As shown in Table 1, the most potent inhibitor was conjugate 3 with
an inhibitory potency (42%) comparable to indomethacin (47%).
Conjugate 3, in which the PA chain is shorter (SPD in place of SPM)
than in conjugate 2, was a more potent inhibitor than conjugate 2
(26%). It should be noted that C60 itself presents interesting anti-
inflammatory activity (30.5%), which is slightly improved (36.5%)
upon attaching the linker (FUL-derived acid 28). The anti-
inflammatory activity is further improved upon attaching the SPD
chain (conjugate 3) but significantly lowered when the SPD chain is
replaced by the more hydrophilic SPM chain (conjugate 2).
are reported in d units, parts per million (ppm) downfield from
TMS. Electro-spray ionization (ESI) mass spectra were recorded on
a Micromass-Platform LC spectrometer using MeOH as solvent. The
MALDI high-resolution MS (HRMS) experiments were carried out
in an Applied Biosystems MALDI-TOF/TOF A/B 4700 Proteomics
Analyzer instrument using dithranol as matrix. In the case of ESI-
HRMS the experiments were carried out in an Applied Biosystems
Q-STAR instrument (Qq-TOF). Elemental analyses were determined
on a Carlo Erba EA 1108 CHNS elemental analyzer in the In-
strumental Analysis Centre of the University of Patras and the re-
sults are within ꢁ0.4% of the calculated values. Flash column
chromatography (FCC) was performed on Merck silica gel 60
(230e400 mesh) and TLC on 60 Merck 60 F254 films (0.2 mm)
precoated on aluminium foil. Spots were visualized with UV light at
254 nm and the ninhydrin agent. All solvents were dried and/or
purified according to standard procedures prior to use. Anhydrous
Na2SO4 was used for drying organic solvents and subsequently
solvents were routinely removed at ca. 40 ꢀC under reduced pres-
sure (water aspirator). All reagents employed in the present work
were purchased from commercial suppliers and used without fur-
ther purification. All reactions were routinely carried out under
an argon atmosphere with the exception of TFA-mediated
deprotections.
As concerns the cytotoxicity of the C60-PA conjugates and at the
higher concentration examined (50 mM), all compounds showed
decreased viability in the range 52e72%. Compared to SPD (68%) all
compounds, with the exception of the SPD conjugate 5 (71.7% sur-
vival), were more cytotoxic than SPD. The highest cytotoxicity was
observed for the FUL-derived acid 28 (52.8%). Compared to the SPM
conjugate 2 (63%), the SPD conjugates 3 (66%) and 5 (71.7%) wereless
cytotoxic. The results from the cytotoxicity studies taking together
with the results from the anti-inflammatory, antioxidative and LOX
inhibition studies indicate that conjugate 3 is a compound deserving
further development as an antioxidant/anti-inflammatory agent.
Purity determination. Analytical reversed phase high-
performance liquid chromatography was performed on a Waters
Alliance 2695 HPLC system equipped with an autosampler, a high-
pressure pump and a photodiode array detector. The purity of the
final compounds was determined using UV detection (
The chromatographic method employed the following: column,
m); mobile phase A, 0.08%
l¼254 nm).
4. Conclusions
Supercosil LC-8 (25.0 cmꢃ4.6 mm, 5
m
TFA in water; mobile phase B, acetonitrile with 0.08% TFA; flow rate,
1.0 mL/min; elution profile, gradient elution from 30e60 to 100% B
over 30 min According to these analyses, purities for all tested
compounds were >96.5% (see Supplementary data).
A newlinker, namelysuccinimidyl (E)-3-methyl-4-(pyrrolidin-1-
yl)but-2-enoate, for the selective attachment of FUL moieties on the
primaryamino functions of PAs has been developed and shown to be
useful in the preparation of FUL-PA and of FUL-PA-antipsoriatic
agent conjugates. Biological evaluation of the antioxidative/anti-
inflammatory potential of the synthesized conjugates revealed
that conjugation of C60 with SPM or SPD resulted in large improve-
ment of both the anti-LOX and the anti-lipid peroxidation activity of
the unconjugated molecules whereas an improvement in the anti-
inflammatory potency of C60 was observed only with the conju-
gate 3 (42%), incorporating the less hydrophilic PA chain (SPD),
which was comparable to the reference compound indomethacin
(47%). Regarding their cytotoxicity, the majority of the synthesized
compounds, and in particular the FUL-SPD conjugates 3 and 5 were
5.2. Fullerene derivatization related compounds
5.2.1. (E)-tert-Butyl 4-hydroxy-3-methylbut-2-enoate (23). A solu-
tion of hydroxyacetone (1.37 mL, 20 mmol) and stabilized phos-
phorane Ph3P]CHCOt2Bu (9.79 g, 26 mmol) in MeCN (20 mL) was
refluxed for 3 h. The reaction mixture was evaporated to dryness,
triturated with Et2O and cooled overnight. The precipitated Ph3P]
O and excess of phosphorane were filtered off and discarded.
Evaporation of the filtrate followed by FCC purification gave pure
ester 23. Yield: 3.1 g (90%), yellow oil; Rf (PhMe/EtOAc 9:1): 0.13; IR
(neat, cmꢂ1): 3426, 1708, 1698, 1659; MS (ESI, 30 eV): m/z 195.21
of comparable toxicity, even at the highest concentration (50 mM)
examined, to the naturally present in the cells SPD and therefore
they could be safely used for possible biomedical application. Ap-
plication of the present methodology to the synthesis of further
examples of fullerene-PA conjugates and of carbon nanotube-PA
conjugates, the biological evaluation of the synthesized com-
pounds in various other cellular targets, and investigation of the
mechanisms through which the most potent compounds in this
series exert their inhibitory effect on LOX and inflammation are
currently in progress.
(MþNa); 1H NMR (CDCl3,
): 5.89 (1H, d, J¼1.2 Hz), 4.11 (2H, d,
d
J¼4.8 Hz), 2.06 (3H, br s), 1.99 (1H, t, J¼4.8 Hz),1.49 (9H, s); 13C NMR
(CDCl3, d): 166.4, 155.4, 113.0, 79.9, 67.1, 28.4 (three C), 15.4.
5.2.2. Ethyl 2-(2-nitrophenylsulfonamido)acetate (24). To an ice-
cold suspension of glycine ethyl ester hydrochloride (1.4 g,
10 mmol) and 2-nitrobenzenesulfonyl chloride (2.44 g, 11 mmol) in
CHCl3 (15 mL), Et3N (3.14 mL, 22.5 mmol) was added dropwise and
the reaction mixture was kept at 0 ꢀC for further 3 h. Volatile
components were evaporated under vacuo, the residue was taken
up in CHCl3 and washed sequentially twice with a 5% aq solution of
citric acid and twice with water. The organic layer was dried over
Na2SO4 and evaporated to dryness to leave an oily residue, which
upon trituration with Et2O and overnight refrigeration afforded the
titled compound in pure form. Yield: 2.45 g (85%), white solid; mp
127e129 ꢀC; Rf (PhMe/EtOAc 8:2): 0.26; IR (KBr, cmꢂ1): 3332, 1736,
1594, 1546; MS (ESI, 30 eV): m/z 327.27 (MþK), 311.27 (MþNa),
5. Experimental section
5.1. General
Melting points were determined with a Buchi SMP-20 apparatus
and are uncorrected. IR spectra were recorded as KBr pellets, unless
otherwise stated, on a PerkineElmer 16PC FT-IR spectrophotome-
ter. Routine 1H NMR spectra were recorded at 400.13 MHz and 13C
289.25 (MþH); 1H NMR (CDCl3,
d): 8.10e8.01 (1H, m), 7.95e7.90

7046
G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
(1H, m), 7.75e7.72 (2H, m), 6.04 (1H, unresolved t), 4.06 (2H, q,
J¼7.2 Hz), 4.00 (2H, d, J¼6.0 Hz), 1.15 (3H, t, J¼7.2 Hz); 13C NMR
d): 164.0, 154.1, 152.4, 146.8, 145.8, 145.6, 145.5, 145.2, 145.0, 144.8,
144.1, 142.6, 142.2, 141.8, 141.6, 141.5, 139.8, 135.9, 119.6, 78.4, 67.4,
62.7 (two C), 29.9 (three C), 14.4. Anal. Calcd for C71H19NO2: C,
92.90; H, 2.09; N, 1.53. Found: C, 92.95; H, 1.74; N, 1.92.
(CDCl3, d): 168.5, 147.8, 134.0, 133.7, 132.9, 130.6, 125.7, 61.9, 45.0,
13.9. Anal. Calcd for C10H12N2O6S: C, 41.66; H, 4.20; N, 9.72; S, 11.12.
Found: C, 41.89; H, 4.11; N, 9.60; S, 11.25.
5.2.6. Fullerene-derived acid 28. To a suspension of ester 27 (0.1 g,
0.11 mmol) in CH2Cl2 (4.0 mL), TFA (4.0 mL) was added dropwise
and the resulting mixture was left to stir at ambient temperature
for 1 h. Volatile components were evaporated and the residue was
triturated with hexane, the solvent was decanted and the residue
was finally dried under vacuo to give acid 28. Yield: 0.1 g (96%),
brown solid; Rf (PhMe/AcOEt 1:1): 0.52; IR (KBr, cmꢂ1): 3450, 1684,
1638; HRMS (m/z): [MþH]þ calcd for C67H12NO2, 862.0868; found,
5.2.3. tert-Butyl (E)-4-(N-(2-ethoxy-2-oxoethyl)-2-nitrophenyl-sul-
fonamido)-3-methylbut-2-enoate (25). To an ice-cold solution of 24
(2.16 g, 7.5 mmol), 23 (1.68 g, 9.75 mmol) and triphenyl phosphine
(TPP) (2.56 g, 9.75 mmol) in THF (22 mL), diisopropyl azodi-
carboxylate (DIAD) (1.92 mL, 9.75 mmol) was added. The reaction
mixture left at this temperature for 5 min and then left to attain
ambient temperature for further 2 h. Then, it was evaporated to
dryness, triturated with Et2O and cooled overnight. The pre-
cipitated Ph3P]O was filtered off and discarded. Evaporation of the
filtrate followed by FCC purification gave pure diester 25. Yield:
3.19 g (96%), yellow oil; Rf (PhMe/EtOAc 8:2): 0.36; IR (neat, cmꢂ1):
1746, 1712, 1659, 1545, 1369; MS (ESI, 30 eV): m/z 481.28 (MþK),
465.29 (MþNa), 443.31 (MþH), 387.27 (MHeCH2]CMe2), 369.27
862.0897; 1H NMR (CS2,
3.81 (2H, s), 2.51 (3H, s); 13C NMR (CS2,
d
): 7.20 (1H, s), 6.33 (1H, s), 4.43 (4H, s),
): 172.7, 153.4, 150.4, 148.4,
d
147.9, 145.8, 145.3, 144.8, 143.8, 143.5, 142.5, 141.5, 141.0, 140.2,
139.5, 136.4, 77.3, 67.1 (two C), 14.6. Anal. Calcd for C67H11NO2: C,
93.38; H, 1.29; N, 1.63. Found: C, 93.40; H, 1.39; N, 1.37.
[MꢂOCMe3]; 1H NMR (CDCl3,
d
): 8.08e8.02 (1H, m), 7.73e7.68 (2H,
5.2.7. Fullerene-derived ‘active’ ester 29. To an ice-cold solution of
acid 28 (0.1 g, 0.1 mmol) in THF (0.6 mL) and 1,2-dichlorobenzene
(0.2 mL) were added HOSu (0.047 g, 0.41 mmol) and DIC (0.039 g,
0.31 mmol). The reaction mixture was left at 0 ꢀC for 30 min and
then at ambient temperature overnight. The reaction was diluted
with CHCl3 and then washed once with a 5% aq solution NaHCO3
and twice with water, dried over Na2SO4 and evaporated to dry-
ness. Pure active ester 29 was obtained after purification with FCC.
Yield: 0.083 g (85%), dark brown solid; Rf (PhMe/AcOEt 95:5): 0.26;
IR (KBr, cmꢂ1): 1734, 1684, 1654; HRMS (m/z): [MþH]þ calcd for
m), 7.64e7.61 (1H, m), 5.69 (1H, br s), 4.12 (2H, s), 4.10 (2H, s), 4.07
(2H, q, J¼7.2 Hz), 2.04 (3H, d, J¼0.8 Hz), 1.46 (9H, s), 1.18 (3H, t,
J¼7.2 Hz); 13C NMR (CDCl3,
d): 168.3, 165.2, 149.3, 147.8, 133.7, 133.3,
131.8, 130.9, 124.2, 120.6, 80.5, 61.5, 55.5, 47.4, 28.2 (three C), 16.0,
14.0.
5.2.4. Synthesis of (E)-2-((4-(tert-butoxy)-2-methyl-4-oxobut-2-en-
1-yl)amino)acetic acid (26). To a solution of 25 (2.88 g, 6.5 mmol)
and thiophenol (0.83 mL, 8.13 mmol) in DMF (80 mL) was added
Na2CO3 (2.76 g, 26 mmol) and the resulting suspension was stirred
vigorously for 12 h. The reaction mixture was evaporated under
highly reduced pressure and the oily residue was diluted with
CH2Cl2 and washed several times with water and brine. The organic
layer was dried over Na2SO4, and evaporated to dryness. The de-
sired intermediate was obtained pure after FCC purification. Yield:
1.4 g (84%), yellow oil; Rf (PhMe/EtOAc 8:2): 0.15; IR (neat, cmꢂ1):
1736, 1636, 1558; MS (ESI, 30 eV): m/z 515.49 (2MþH), 296.29
(MþK), 280.21 (MþNa) 258.25 (MþH), 202.15 (MHeCH2]CMe2);
C71H15N2O4, 959.1032; found, 959.1091; 1H NMR (CDCl3,
d
): 6.66
(1H, s), 4.48 (4H, s), 3.88 (2H, s), 2.90 (4H, br s), 2.51 (3H, s); 13C
NMR (CDCl3, ): 169.6, 162.2 (two C), 154.4, 149.3, 147.3, 146.3, 146.1,
d
146.0, 145.7, 145.5, 145.3, 143.1, 142.7, 142.2, 142.1, 141.9, 137.4, 136.2,
70.4, 67.6 (two C), 25.4 (two C), 18.4. Anal. Calcd for C71H14N2O4: C,
88.93; H, 1.47; N, 2.92. Found: C, 89.01; H, 1.68; N, 2.61.
5.3. General procedure for the synthesis of compounds 4, 31
and 33
1H NMR (CDCl3,
d
): 5.81 (1H, s), 4.20 (2H, q, J¼7.2 Hz), 3.38 (2H, s),
3.26 (2H, s), 2.11 (3H, s), 1.76 (1H, br s), 1.48 (9H, s), 1.28 (3H, t,
To a solution of amine 20 (0.018 g, 0.084 mmol) or 30 (0.042 g,
0.084 mmol) or 32a (0.033 g, 0.084 mmol) and Et3N (23 mL,
J¼7.2 Hz); 13C NMR (CDCl3,
d): 172.3, 166.2, 154.7, 117.6, 79.7, 60.2,
56.6, 50.0, 28.2 (three C), 17.0, 14.2.
0.168 mmol) in CH2Cl2 (0.16 mL), ‘active’ ester 29 (0.040 g,
0.042 mmol) was added. The resulting mixture was stirred at am-
bient temperature until the completion of the reaction (monitored
by TLC). Then, it was diluted with CH2Cl2, washed once with a 5% aq
solution NaHCO3 and twice with water. The organic layer was dried
over Na2SO4 and evaporated to dryness to afford pure 4, 31, and 33,
respectively, after FCC purification.
To an ice-cold solution of this intermediate (1.34 g, 5.2 mmol) in
MeOH (8.0 mL) an aqueous solution 6 N NaOH (2.5 mL) was added
and the reaction mixture was left to stir for 30 min. The solvent was
evaporated under vacuo and the residue was diluted with a mini-
mum amount of water. The anticipated acid 26 was obtained pure
after careful acidification to pH 4.5e5.0 with an aqueous solution of
1 N HCl, overnight refrigeration and filtration of the thus obtained
precipitate. Yield: 0.95 g (80%), white solid; mp 133e135 ꢀC; IR
(KBr, cmꢂ1): 3460, 3070e2680, 1704, 1650, 1598; HRMS-ESI (m/z):
[MþH]þ calcd for C11H20NO4, 230.1392; found, 230.1408; 1H NMR
Compound 4. Reaction time: 45 min; yield: 0.034 g (78%), brown
solid; Rf (PhMe/EtOAc 1:1): 0.33; IR (KBr, cmꢂ1): 3360, 1670, 1624;
HRMS (m/z): [MþH]þ calcd for C77H23N6O, 1047.1933; found,
1047.2010; 1H NMR (CDCl3/CS2,
d): 7.73 (1H, unresolved t), 7.29 (3H,
(DMSO-d6,
d
): 5.77 (1H, s), 3.30 (2H, s), 3.15 (2H, s), 2.02 (3H, s), 1.42
unresolved m), 7.21 (2H, unresolved m), 6.26 (1H, s), 5.52 (2H, s),
4.70 (4H, s), 3.97 (2H, s), 3.65 (2H, unresolved t), 3.57 (2H, un-
(9H, s); 13C NMR (DMSO-d6,
d): 171.2, 165.3, 153.8, 117.4, 79.2, 54.8,
49.4, 27.8 (three C), 16.6.
resolved t), 2.46 (3H, s); 13C NMR (CDCl3/CS2,
d): 168.8, 154.6, 153.6,
148.5, 146.5, 146.3, 145.2, 144.9, 144.7, 144.5, 143.8, 143.4, 142.3,
141.9, 141.6, 141.4, 141.3, 141.2, 140.1, 139.4, 136.6, 135.4, 133.1, 129.2,
129.0, 127.7, 125.9, 69.6, 66.8 (two C), 50.4, 42.4, 24.6, 17.6.
5.2.5. Fullerene-derived ester 27. A solution of C60 (0.2 g,
0.27 mmol), paraformaldehyde (0.042 g, 1.35 mmol) and 26
(0.045 g, 0.19 mmol) in PhMe (90 mL) was refluxed for 3 h. The
reaction mixture was evaporated to a minimum volume and sub-
jected to FCC using as eluant the solvent system PhMe/hexane 1:1
to recover unreacted C60 and then PhMe/hexane 9:1 to afford pure
ester 27. Yield: 0.12 g (48%), brown solid; Rf (PhMe/hexane 9:1):
0.54; IR (KBr, cmꢂ1): 1710, 1650; HRMS (m/z): [MþH]þ calcd for
Compound 31. Reaction time: 30 min; yield: 0.046 g (82%) brown
solid; Rf (PhMe/EtOAc 1:1): 0.28; IR (KBr, cmꢂ1): 3448, 1670, 1636;
HRMS (m/z): [MþH]þ calcd for C92H60N5O7, 1346.4493; found,
1346.4528; 1H NMR (CDCl3,
d): 6.93 (1H, br s), 6.20 (1H, s), 5.18 (1H,
br s), 4.48 (4H, s), 3.81 (2H, s), 3.32e3.00 (12H, m), 2.29 (3H, s),
1.70e1.55 (8H, m), 1.39 and 1.37 (27H, two s); 13C NMR (CDCl3,
d):
C71H20NO2, 918.1494; found, 918.1467; 1H NMR (CS2,
d
): 6.08 (1H,
166.1 (four C), 153.5, 147.3, 146.3, 146.2, 145.6, 145.4, 144.5, 143.2,
142.7, 142.1, 142.0, 140.3, 137.8, 136.1, 129.0, 128.2, 125.3, 79.8 (two
s), 4.39 (4H, s), 3.70 (2H, s), 2.44 (3H, s), 1.49 (9H, s); 13C NMR (CS2,

G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
7047
C), 79.6, 69.7, 62.0 (two C), 46.8, 45.7, 44.4, 37.4, 37.0, 29.7, 28.5
(nine C), 27.6, 24.6, 24.2, 23.8, 14.0.
(twelve C), 127.1 (twelve C), 127.0, 126.9, 126.4, 125.6 (twelve C),
69.4 (two C), 66.6 (two C), 63.0 (four C), 46.8 (two C), 45.5 (two C),
36.9 (two C), 25.7 (two C), 22.4 (two C), 16.6 (two C).
Compound 37. Yield: 0.019 g (15%), brown solid; Rf (CHCl3/
MeOH/concd NH3 9:1:0.1): 0.23; HRMS (m/z): [MþH]þ calcd for
C115H64N5O, 1530.5111; found, 1530.5197.
Compound 33. Reaction time: 30 min; yield: 0.048 g (91%),
brown solid; Rf (CHCl3/MeOH 95:5): 0.19; IR (KBr, cmꢂ1): 3396,
1662,1618, 748, 704; HRMS-ESI (m/z): [MþH]þ calcd for C93H43N4O,
1231.3437; found, 1231.3821; 1H NMR (CDCl3, ): 1H NMR (CDCl3,
d
d): 7.45e7.41, 7.32e7.25 and 7.23e7.17 (15H, three m), 6.48 (1H,
unresolved t), 6.23 (1H, s), 4.41 (4H, s), 3.70 (2H, s), 3.39e3.34 (4H,
m), 2.87 (2H, t, J¼6.4 Hz), 2.79 (2H, t, J¼6.0 Hz), 2.50 (3H, s), 2.49
(1H, s), 2.34 (2H, quintet, J¼6.0 Hz), 1.76e1.66 (4H, m); 13C NMR
5.6. General procedure for the deprotection of compounds 31,
33, 35 and 36
(CDCl3,
d
): 162.6, 154.8, 147.2, 146.2 (three C), 146.1, 145.6, 145.4,
To an ice-cold suspension of compound 31 (0.04 g, 0.03 mmol)
or 33 (0.04 g, 0.032 mmol) or 35 (0.03 g, 0.02 mmol) or 36 (0.03 g,
145.3, 144.5, 143.0, 142.6, 142.2, 142.1, 141.9, 140.1, 136.2, 128.6 (six
C), 128.1 (six C), 128.0, 127.4, 126.6 (three C), 70.4, 67.3, 63.3 (two C),
50.3, 48.0, 44.0, 42.0, 32.6, 29.5, 27.5, 17.4.
0.015 mmol) in CH2Cl2 (0.1 mL) TFA (0.1 mL or 40
compound 33) was added and the resulting solution was stirred at
ꢀC for 2 h. The reaction mixture was evaporated to dryness and
mL in the case of
0
5.4. Procedure for the synthesis of compound 35
triturated with Et2O to afford the corresponding trifluoroacetate
salts 2, 3, 5 and 6 as brown solids.
To a suspension of acid 28 (0.04 g, 0.041 mmol), SPD derivative
Compound 2. Yield: 0.039 g (94%); IR (KBr, cmꢂ1): 3396,
3030e2700, 1684, 1654, 1202, 1132; HRMS (m/z): [MþH]þ calcd for
34 (0.05 g, 0.08 mmol) and PyBrOP (0.028 g, 0.06 mmol) in DMF
i
(0.15 mL) was added Pr2NEt (35
mL, 0.2 mmol). The reaction mix-
C77H36N5O, 1046.2920, found 1046.2974; 1H NMR (DMSO-d6,
d):
ture was left to stir at ambient temperature for 1 h and then was
diluted with CHCl3, and washed once with a 5% aq solution NaHCO3
and twice with water. The organic layer was dried over Na2SO4 and
evaporated to dryness. The anticipated product was obtained pure
after FCC purification. Yield: 0.035 g (58%), brown solid; Rf (PhMe/
EtOAc 9:1): 0.25; IR (KBr, cmꢂ1): 3288, 1642, 1619, 770, 748, 706;
HRMS-ESI (m/z): [MþH]þ calcd for C112H57N4O, 1473.4532; found,
8.71 (2H, br s), 8.56 (2H, br s), 8.27 (1H, t, J¼6.0 Hz), 7.88 (3H, br s),
6.22 (1H, s), 4.48 (4H, s), 3.76 (2H, s), 3.23 (2H, q, J¼6.0 Hz),
3.05e2.85 (10H, m), 2.39 (3H, s), 1.86 (2H, quintet, J¼8.0 Hz), 1.78
(2H, quintet, J¼8.0 Hz), 1.68e1.60 (4H, unresolved m); 13C NMR
(DMSO-d6, d): 168.3, 155.8, 147.2, 146.6, 146.2, 146.0, 145.8, 145.3,
145.2, 144.5, 143.2, 143.1, 142.6, 142.3, 142.0, 141.9, 141.8, 139.8,
136.2, 71.3 (two C), 67.3, 46.7, 44.4, 36.7, 31.1, 27.1, 24.3, 23.2, 23.1,
17.4. The two missing PA backbone C atoms are buried under the
solvent.
1473.4105; 1H NMR (CDCl3,
d): 7.45e7.30, 7.21e7.17 and 7.16e7.05
(30H, three m), 6.47 (1H, 2 s), 4.31 (4H, 2 s), 3.60 (2H, 2 s), 3.42 and
3.38 (2H, two t, J¼6.4 Hz), 3.25 and 3.21 (2H, two t, J¼6.4 Hz), 2.28
(3H, s), 2.17e2.08 (4H, m), 1.80e1.68 (2H, m), 1.56e1.37 (4H, m); 13C
Compound 3. Yield: 0.037 g (95%); IR (KBr, cmꢂ1): 3398,
3030e2700, 1678, 1654, 1198, 1135; HRMS (m/z): [MþH]þ calcd for
NMR (CDCl3,
d
): 167.7, 154.8, 147.3, 146.3 (six C), 146.1, 146.0, 145.8,
C74H29N4O, 989.2341, found 989.2381; 1H NMR (DMSO-d6,
d): 8.47
145.7,145.6,145.5, 145.3, 144.6,143.1, 142.7, 142.2, 142.1, 141.9,140.2,
137.9, 136.2, 129.0 (twelve C), 128.6, 128.5, 128.2 (twelve C), 127.8,
125.3 (six C), 70.7 (two C), 67.7, 61.3 (two C), 47.0, 45.6, 30.8, 29.7,
28.3, 27.2, 25.9, 17.8.
(2H, br s), 8.13 (1H, t, J¼6.0 Hz), 7.80 (3H, br s), 6.20 (1H, s), 4.47 (4H,
s), 3.74 (2H, s), 3.17 (2H, q, J¼6.0 Hz), 3.01e2.84 (6H, m), 2.39 (3H, s),
1.86 (2H, quintet, J¼6.4 Hz), 1.61 (2H, quintet, J¼6.4 Hz), 1.52 (2H,
quintet, J¼6.4 Hz); 13C NMR (DMSO-d6,
d): 167.9, 155.8, 147.2, 146.6,
146.2, 146.0, 145.8, 145.3, 145.2, 143.0, 142.6, 142.3, 142.0, 141.9,
140.0, 136.2, 71.3 (two C), 67.3, 47.1, 44.4, 38.1, 36.7, 26.9, 24.2, 23.6,
17.3.
5.5. General procedure for the synthesis of compounds 7 and
36
Compound 5. Yield: 0.023 g (95%); IR (KBr, cmꢂ1): 3394,
3030e2800, 1652, 1630, 1200, 1137; HRMS (m/z): [MþH]þ calcd for
To a solution of PA 19 (0.045 g, 0.084 mmol) or 18 (0.058 g,
0.084 mmol) and Et3N (29
mL, 0.21 mmol) in DMF (0.1 mL), active
C74H29N4O, 989.2341, found 989.2346; 1H NMR (DMSO-d6,
d): 7.82
ester 29 (0.040 g, 0.042 mmol) was added. The resulting mixture
was left to stir at ambient temperature until the completion of the
reaction (monitored by TLC). Then, it was diluted with CHCl3,
washed once with a 5% aq solution NaHCO3 and twice with water.
The organic layer was dried over Na2SO4 and evaporated to dryness
to afford pure 7 and 38 and 36 and 37, respectively, after FCC
purification.
(6H, br s), 6.59 and 6.54 (1H, two s), 4.48 (4H, s), 3.79 (2H, s),
2.85e2.79 (6H, unresolved m), 2.18 and 2.17 (3H, two s), 1.80 (2H,
m), 1.62e1.46 (6H, m); 13C NMR (DMSO-d6,
d): 168.0 and 167.2 (1C),
155.8, 147.2, 147.0, 146.6, 146.2, 146.0, 145.8, 145.3, 145.2, 144.5,
143.3, 143.1, 142.6, 142.3, 142.0, 141.9, 140.0, 136.2, 71.1 (two C), 67.1,
47.9, 44.1, 37.3, 35.7, 26.1, 24.8, 23.4, 17.7.
Compound 6. Yield: 0.029 g (92%); IR (KBr, cmꢂ1): 3248,
3030e2700, 1654, 1560, 1168; HRMS (m/z): [MþH]þ calcd for
Compound 7. Reaction time: 2 h; yield: 0.029 g (34%), brown
solid; Rf (CHCl3/MeOH/concd NH3 95:5:0.5): 0.48; IR (KBr, cmꢂ1):
3460, 1684, 1654; ESI-MS (m/z): 1984.32 (MþNHþ4 ), 1966.51 (M);
HRMS (m/z): [MþH]þ calcd for C144H39N12O2, 1967.3319; found,
1889.3678 [(MþHþ)ꢂ6Nþ6H].
C144H45N6O2, 1889.3604, found 1889.3552; 1H NMR (DMSO-d6,
d):
8.80 (4H, br s), 8.25 (2H, t, J¼6.0 Hz), 6.22 (2H, s), 4.50 (8H, s),
3.76 (4H, s), 3.27 (4H, q, J¼6.0 Hz), 2.96e2.84 (8H, unresolved m),
2.39 (6H, s), 1.86 (4H, quintet, J¼6.0 Hz), 1.69e1.61 (4H, un-
Compound 38. Yield: 0.019 g (20%), brown solid; Rf (CHCl3/
MeOH/concd NH3 95:5:0.5): 0.27; HRMS (m/z): [MþH]þ calcd for
C77H30N11O, 1124.2635; found, 1124.2645.
resolved m); 13C NMR (DMSO-d6,
d): 169.5 (two C), 157.0 (two C),
148.4, 147.8, 147.4, 147.2, 146.9, 146.5, 146.4, 144.4, 144.3, 143.8,
143.5, 143.2, 143.1, 143.0, 141.2, 137.4, 72.5 (four C), 68.5 (two C),
46.7 (two C), 45.1 (two C), 36.9 (two C), 25.5 (two C), 23.0 (two C),
18.6 (two C).
Compound 36. Reaction time: 3 h; yield: 0.035 g (35%), brown
solid; Rf (CHCl3/MeOH 99:1): 0.25; IR (KBr, cmꢂ1): 3420, 1626, 742,
706; HRMS (m/z): [MþH]þ calcd for C182H73N6O2, 2373.5795;
found, 2373.5423; 1H NMR (CDCl3,
d): 8.14 (2H, s), 7.51e7.49,
5.7. General procedure for the synthesis of compounds 8 and
9
7.30e7.27 and 7.19e7.08 (30H, three m), 6.42 (2H, s), 4.52 (8H, s),
3.89 (4H, s), 3.62 (4H, t, J¼7.2 Hz), 3.16e3.02 (8H, m), 2.60 (6H, s),
1.74 (4H, quintet, J¼7.2 Hz), 1.49e1.42 (4H, m); 13C NMR (CDCl3,
d
):
To a suspension of SPM derivative 2 (0.04 g, 0.025 mmol) in
CH2Cl2/DMF (1:1, 0.1 mL) were added successively active ester 12
161.6 (two C), 153.8 (two C), 146.2, 145.2, 145.1, 144.6, 144.4, 144.3,
144.1, 143.5, 143.1, 142.0, 141.6, 141.2, 141.1, 140.9, 139.1, 135.2, 127.6
i
(0.009 g, 0.021 mmol) or 13 (0.008 g, 0.021 mmol) and Pr2NEt

7048
G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
(17
m
L, 0.1 mmol). The resulting mixture was stirred at ambient
5.10. In vivo assays: inhibition of the Carrageenan-induced
oedema28
temperature for overnight. Then, it was diluted with CHCl3 and
washed once with a 5% aq solution NaHCO3 and twice with water.
The organic layer was dried over Na2SO4 and evaporated to dryness.
The residue was subjected to FCC to afford pure 8 or 9 as brown
solids.
In acute toxicity experiments, the in vivo examined compounds
did not present toxic effects in doses up to 0.1 mmol/kg body
weight. Oedema was induced in the right hind paw of Fisher 344
rats (150e200 g) by the intradermal injection of 0.1 mL 2% Carra-
geenan in water. Both sexes were used. Females pregnant were
excluded. Each group was composed of 6e15 animals. The animals,
which have been bred in our laboratory, were housed under stan-
dard conditions and received a diet of commercial food pellets and
water ad libitum during the maintenance but they were entirely
fasted during the experiment period. Our studies were in accor-
dance with recognised guidelines on animal experimentation. The
tested conjugates 0.01 mmol/kg body weight, were diluted in water
with few drops of Tween 80 and ground in a mortar before use and
they were given intraperitoneally simultaneously with the Carra-
geenan injection. The rats were euthanized 3.5 h after Carrageenan
administration. The difference between the weight of the injected
and uninjected paws was calculated for each animal. The change in
paw weight was compared with that in control animals (treated
with water) and expressed as a percent inhibition of the oedema
(%ICPE values, Table 1). Indomethacin was tested as a reference
compound in 0.01 mmol/kg. Values % ICPE is the mean from two
different experiments with a standard error of the mean less than
10%.
Compound 8. Yield: 0.016 g (56%); Rf (CHCl3/MeOH/concd NH3
8:2:0.2): 0.34; IR (KBr, cmꢂ1): 3402, 1654, 1636, 1118; ESI-MS (m/z):
1355.58 (MþH); HRMS (m/z): [MþH]þ calcd for C98H60N5O3,
1354.4696; found, 1354.4591; 1H NMR (DMSO-d6,
d): 8.27 (1H, t,
J¼5.6 Hz), 8.15 (1H, t, J¼5.6 Hz), 6.96 (1H, dd, J¼11.6 and 15.2 Hz),
6.68 (1H, s), 6.66 (1H, d, J¼16 Hz), 6.33 (1H, d, d, J¼15.2 Hz), 6.25
(1H, d, J¼16 Hz), 6.23 (1H, s), 6.22 (1H, d, J¼11.6 Hz), 5.82 (1H, s),
4.46 (4H, s), 3.75 (5H, s), 3.25e3.18 (4H, m), 2.61e2.54 (2H, m),
2.53e2.50 (6H, m), 2.36 (3H, s), 2.29 (6H, s), 2.24 (3H, s), 2.05 (6H,
s), 1.82e1.74 (4H, m), 1.65e1.59 (4H, m).
Compound 9. Yield: 0.014 g (52%); Rf (CHCl3/MeOH/concd NH3
8:2:0.2): 0.35; IR (KBr, cmꢂ1): 3382, 1736, 1656, 1614, 1602, 1104;
HRMS-ESI (m/z): [MþH]þ calcd for C94H48N5O5, 1326.3655; found,
1326.3750; 1H NMR (DMSO-d6/CF3CO2D¼9:1,
d): 8.72e8.49 (4H,
two m), 8.45 (2H, t, J¼6.0 Hz), 7.91 (1H, s), 7.55 (1H, d, J¼16.0 Hz),
6.80 (1H, d, J¼16.0 Hz), 6.38 (1H, s), 6.18 (1H, s), 5.02 (4H, s), 4.22
(2H, s), 3.32 (2H, t, J¼6.0 Hz), 3.01e2.90 (10H, unresolved m), 2.68
(3H, s), 2.60 (3H, s), 2.57 (3H, s), 2.48 (3H, s), 1.86 (4H, quintet,
J¼6.0 Hz), 1.68e1.56 (4H, unresolved m).
5.8. General biological assays
5.11. Evaluation of the cytocompatibility of conjugates
NDGA and AAPH were purchased from Aldrich Chemical Co.
Milwaukee, WI, (USA). Soybean LOX, linoleic acid sodium salt and
indomethacin were obtained from Sigma Chemical, Co. (St. Louis,
MO, USA) and Carrageenan, type K, was commercially available. For
the in vivo experiments, male and female Fischer-344 rats
(180e240 g) were used. For the in vitro tests a Lambda 20 (Per-
kineElmer) UVevis 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.
RAMEC rat endothelial cells were isolated as described pre-
viously.36 The cells were routinely subcultured in Dulbecco’s MEM
full growth medium (Biochrom AG, Germany), containing 10%
foetal calf serum, 200 mM
L-glutamine, 2.5 mg/mL Amphotericin B,
100 g/mL streptomycin, 100 U/mL penicillin and 0.003% endo-
m
thelial cell growth supplement in a humidified atmosphere con-
taining 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 (1e50
MTT test.35 The cells were 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
mM) using the
m
5.9. In vitro assays
L
m
m
For the stock solutions of the tested compounds, DMSO was
used as a solvent. The solutions were homogeneous and clear when
they were used. In some cases ultrasound stirring was helpful.
penicillin and 0.003% endothelial cell growth supplement. Twenty-
four hours after plating, different amounts of the compounds were
added in the wells. After 24 h of incubation at 37 ꢀC, 50
mL of MTT
solution (5 mg/mL in phosphate buffered saline, pH 7.4) were
5.9.1. Inhibition of linoleic acid lipid peroxidation.28 Ten microlitres
of a 16 mM linoleic acid sodium salt solution was added to the UV
cuvette containing 0.93 mL of 0.05 M phosphate buffer, pH 7.4,
prethermostated at 37 ꢀC. The oxidation reaction was initiated at
added into each well and the plates were incubated at 37 ꢀC for 2 h.
The medium was withdrawn and 200 mL of DMSO was added in
each well and agitated thoroughly to dissolve the formazan crys-
tals. The solution was transferred to 96-well plates and immedi-
ately read on a microplate reader, at a wavelength of 540 nm. The
experiments were performed in hexaplicate. Cell viability was
calculated from the ratio between the absorbance provided by the
cells treated with the different compounds and the absorbance
provided by the non-treated cells (control). Viability data are
subject to ꢁ5% error.
37 ꢀC under air by the addition of 50
Oxidation was carried out in the presence of aliquots (10
mL of 40 mM AAPH solution.
mL from
the stock solution 10 mM in DMSO) in the assay without antioxi-
dant. 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 conju-
gated diene hydroperoxides and compared with the appropriate
standard inhibitor (trolox).
Acknowledgements
5.9.2. Soybean LOX inhibition study in vitro.28 The tested com-
pounds dissolved in DMSO (after sonication) were incubated at RT
with sodium linoleate (0.1 mL) and 0.2 mL of enzyme solution
(1/9ꢃ10ꢂ4 w/v in saline). The conversion of sodium linoleate to 13-
hydroperoxylinoleic acid at 234 nm was recorded and compared
with the appropriate standard inhibitor NDGA.
We thank Prof. Giovanni and Prof. Anna Napoli from the De-
partment of Chemistry of the University of Calabria (Italy) for
providing the HRMS analyses. Also, we thank Prof. Th. Tsegenidis,
Department of Chemistry, University of Patras, for his valuable help
on HPLC analyses.

G.E. Magoulas et al. / Tetrahedron 68 (2012) 7041e7049
7049
Markopoulou, O.; Kontogiorgis, C.; Hadjipavlou-Litina, D. J. J. Med. Chem. 2007, 50,
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