Synthesis and antioxidant activity of [60]fullerene-flavonoid conjugates

Article abstract of DOI:10.1016/j.tet.2008.10.066

Chalcone, flavone, and arylideneflavanone derivatives bearing one or two 3,5-di-tert-butyl-4-hydroxyphenyl groups were synthesized from 2′,4′-dihydroxyacetophenone and 3,5-di-tert-butyl-4-hydroxybenzaldehyde. These flavonoids were converted into the corre

Full text of DOI:10.1016/j.tet.2008.10.066

Tetrahedron 65 (2009) 253–262
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and antioxidant activity of [60]fullerene–flavonoid conjugates
a
a
a,
a
b
*
´
´
Roger F. Enes , Andreia S.F. Farinha , Augusto C. Tome , Jose A.S. Cavaleiro , Riccardo Amorati ,
Silvia Petrucci b, Gian Franco Pedulli b
,
*
a Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
b
`
Dipartimento di Chimica Organica ‘A. Mangini’, Universita di Bologna, Via S. Giacomo 11, 40126 Bologna, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2008
Received in revised form 21 October 2008
Accepted 21 October 2008
Available online 25 October 2008
Chalcone, flavone, and arylideneflavanone derivatives bearing one or two 3,5-di-tert-butyl-4-hydroxy-
phenyl groups were synthesized from 20,40-dihydroxyacetophenone and 3,5-di-tert-butyl-4-hydroxy-
benzaldehyde. These flavonoids were converted into the corresponding malonates and then reacted with
C60 to yield the title compounds. The O–H bond dissociation enthalpies (BDE) and the chain-breaking
antioxidant activity of the flavonoid derivatives and the corresponding C60 conjugates were evaluated.
These results are consistent with the phenolic moiety being the main responsible for the reaction with
peroxyl radicals, while the C60 part of the molecule acts synergically by trapping alkyl radicals under
reduced O2 partial pressure. These novel C60–flavonoid conjugates are therefore promising leads for
broad-spectrum radical scavengers.
Keywords:
Fullerenes
Antioxidants
Flavonoids
Phenols
Ó 2008 Elsevier Ltd. All rights reserved.
Radicals
EPR spectroscopy
1. Introduction
antiatherogenic, antimicrobial, and anti-inflammatory proper-
ties.12–18 The biological activity and the healthy effects of flavonoids
Oxidative stress plays a significant pathological role in many
human diseases, namely in all inflammatory and ischemic diseases
and also in the neurologic ones such as Alzheimer0s and Parkinson
diseases, multiple sclerosis, and amyotrophic lateral sclerosis.1
Cancer diseases are also associated with oxidative stress; malig-
nant cells in general are more active than normal cells in the
production of superoxide.2 These diseases result from the over-
production of reactive oxygen species (ROS), generated endoge-
nously during the cellular metabolic activities, which lead to
cellular damage including lipid peroxidation, DNA adduct forma-
tion, protein oxidation, and enzyme inactivation, which can all
ultimately lead to cell death.2 The detrimental effect of ROS can be
prevented by antioxidants. It is unanimously accepted that diets
rich in food containing antioxidants (legumes, vegetables, and
fresh fruit) have beneficial effects on human health.3–5 The bene-
ficial effects of natural polyphenolic antioxidants have been
reviewed recently.6–8
have been reviewed recently.19–21
In recent years it has been shown that fullerene derivatives
display a range of biological activities and, specifically, may be used
as protective drugs against diseases related with oxidative stress.22
In fact, fullerenes and their organic derivatives are regarded as
‘radical sponges’ as they are able to trap multiple radicals per
molecule.23,24 For instance, it has been shown that C60 is a more
effective antioxidant than
a-tocopherol (vitamin E) on the pre-
vention of lipid peroxidation induced by superoxide and hydroxyl
radicals.25 In addition, aqueous C60 suspensions not only have no
acute or subacute toxicity in rodents but they also protect their
livers in a dose dependent manner against free radical damage.26
Poly-hydroxylated fullerenes [C60(OH)n] are also excellent antioxi-
dants able to reduce the free radical damage of neuronal tis-
sues.27,28 Carboxyfullerene derivatives have also been successfully
used in vivo as protective drugs against neurodegenerative diseases
related with oxidative stress.29–32 The water-soluble fullerene de-
rivative C60(ONO2)7ꢀ2 attenuates ischemia-reperfusion-induced
lung injury in young Wistar rats due to its antioxidant properties.33
Water-soluble fullerene derivatives are able to protect human skin
keratinocytes from UV irradiation by scavenging ROS.34,35 Recent
studies indicate that dendritic water-soluble C60 derivatives, es-
pecially the anionic ones, exhibit high antioxidant activity against
superoxide anions,36 and fullerene-derivatized amino acids also
show potent antioxidant activities.37,38
Among the natural compounds with high antioxidant activity,
flavonoids, a widely distributed class of phytochemicals, have
a
central role.9–11 Natural flavonoids also show anticancer,
* Corresponding authors. Tel.: þ351 234 370 712; fax: þ351 234 370 084.
E-mail addresses: actome@ua.pt (A.C. Tome´), gianfranco.pedulli@unibo.it
(G.F. Pedulli).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.10.066

254
R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
Recently, we reported the synthesis of new fullerene derivatives
The low yield of the acid-catalyzed reaction prompted us to
carry out a base-catalyzed aldol condensation. However, since un-
der alkaline conditions aldehyde 3 does not behave as an electro-
phile (it predominates as a quinone methide anion),48 it was
necessary to protect the hydroxyl group. Reaction of aldehyde 3
with an excess of methoxyethylmethyl chloride (MEM-Cl)49 in
dried THF, using NaH as base, afforded aldehyde 6 in 87% yield
(Scheme 2). Condensation of aldehyde 6 with acetophenone 2 in
the presence of a 60% NaOH aqueous solution afforded the MEM-
protected chalcone 7 in 35% yield. The cleavage of the MEM
protecting group afforded a mixture of chalcone 4 and the corre-
sponding isomeric flavanone. Because of that, chalcone 7 was used
in the following steps and the cleavage of the MEM protecting
group was left for the final synthetic step. In that way the expected
products were obtained in good yields.
having flavonoid moieties of synthetic and natural origin.39–41 We
also reported the synthesis and antioxidant activity of fullerene
derivatives bearing 3,5-di-tert-butyl-4-hydroxyphenyl groups,42
i.e., compounds structurally related with BHT,43 a phenolic anti-
oxidant largely used in the food industry.44,45 It has been demon-
strated that C60 alone is not a powerful chain-breaking antioxidant
as believed, as it does not efficiently trap peroxyl radicals that are
involved in the propagation step of the peroxidative chain re-
action.42 Instead, flavonoids bearing a 3,5-di-tert-butyl-4-hydroxy-
phenyl moiety are powerful scavengers of peroxyl radicals. They
exhibit high antioxidant protective activity against LDL perox-
idation induced by metal ions or peroxyl radical and do not exhibit
prooxidant activity.46 It is thus expected that the addition of a C60
moiety to flavonoids incorporating groups structurally related to
BHT could, eventually, show increased antioxidant and radical
The oxidative cyclization of chalcone 7 with dimethylsulfoxide
in the presence of a catalytic amount of iodine47 afforded directly
the unprotected flavone 8 (Scheme 3). The cleavage of the MEM
group is probably due to the formation of HI during the cyclization
step.
scavenging activities. In this paper, we report the synthesis of C60
–
flavonoid conjugates incorporating one or two 3,5-di-tert-butyl-4-
hydroxyphenyl groups and the evaluation of their antioxidant
activities by measuring the O–H bond dissociation enthalpies and
the rate constants for their reaction with peroxyl radicals.
2.2. Synthesis of the C60–flavonoid conjugates
2. Results and discussion
The C60–flavonoid conjugates were synthesized by the cyclo-
propanation of C60 with the malonates obtained in the reaction of
3-chloro-3-oxopropanoate with the 3-hydroxypropyl derivatives 5,
7, and 8. The malonates, obtained in excellent yields (80–98%), were
coupled to C60 following a variation50 of the Bingel methodology.51
The reaction of C60 with the malonates, in the presence of CBr4 and
DBU, afforded the expected C60–flavonoid conjugates in moderate
yields (21–48%) (Schemes 4–6). Finally, compound 11 was obtained
in an almost quantitative yield (97%) by acid catalyzed cleavage of
the protecting MEM group (Scheme 4).
2.1. Synthesis of the flavonoids
A conventional route to flavonoids (chalcones, flavanones, and
flavones) involves the aldol condensation of a 20-dihydroxy-
acetophenone with a benzaldehyde. These reactions lead to 20-
dihydroxychalcones which can be converted into flavanones, by
cycloisomerization, or into flavones, by oxidative cyclization.47 To
prepare flavonoids adequately functionalized to be linked to C60 we
used 2,040-dihydroxyacetophenone (1) and 3,5-di-tert-butyl-4-
hydroxybenzaldehyde (3), both commercially available, as starting
materials. Our idea was to functionalize acetophenone 1 with
a hydroxyalkyl group in order to use it, in a later stage, to prepare
malonate derivatives which could be linked to C60 by cyclo-
propanation (see Schemes 4–6). Selective alkylation of the 40-hy-
droxyl group of acetophenone 1 with 3-iodopropan-1-ol leads to 2
in 82% yield (Scheme 1). Acid-catalyzed condensation of this
compound with aldehyde 3 affords a 3:1 mixture of the chalcone 4
and the arylideneflavanone 5, resulting from the cyclization of 4 to
the corresponding flavanone and subsequent reaction with another
molecule of aldehyde 3.48
2.3. Structural characterization of the synthesized
compounds
All compounds were characterized by 1H and 13C NMR spec-
troscopy and mass spectrometry. The 1H NMR spectra of all syn-
thesized flavonoids showa singlet at
to the tert-butyl groups. In the case of arylideneflavanone de-
rivatives, two singlets at 1.28–1.29 ppm and 1.36 ppm are ob-
served. The spectra of the chalcone derivatives are characterized
d 1.48–1.51 ppm corresponding
d
by the presence of two doublets at
d 7.37–7.44 ppm and at d
HO
HO
I
+
K2CO3
acetone
OH
O
82%
1
2''
5'
HO
O
OH
6'
4'
3'
1''
3''
+
OHC
2'
OH
O
2
3
HCl
EtOH, rt
OH
2''
5'
2''
8
5
3
OH
HO
O
4
5
HO
O
O
O
2
2
6'
4'
3'
7
6
1''
3''
1''
α
3''
+
3
2'
OH
4
β
6
OH
O
H
5 (10%)
4 (30%)
Scheme 1.

R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
255
NaH,
OH
O
O
THF, rt
Cl
O
O
+
O
87%
OHC
(MEM-Cl)
OHC
3
6
NaOH 60%
(40 equiv.)
MeOH
35%
HO
O
OMEM
2
+
6
OH
O
7
Scheme 2.
3'
4' OH
5'
2'
2''
8
5
DMSO, I2
reflux, 1 h
61%
HO
O
OMEM
2
O
HO
O
6'
7
6
1''
3''
3
4
OH
O
8
O
7
Scheme 3.
7.82–7.91 ppm corresponding to the resonances of protons H-
a
and
H-
b
, respectively. The coupling constant of 15.4 Hz confirms a trans
O
O
O
O
configuration for the C–C double bond. The singlet at ca. 13.5 ppm,
due to the resonance of the 20-OH proton, is also distinctive of all
chalcone derivatives. The singlet corresponding to the 4-OH proton
i
OMEM
7
H3CO
H3CO
O
O
O
89%
appears typically at
d
5.0–5.5 ppm; this signal is absent in the 1H
NMRspectra of thecompoundsbearing theMEMgroup. The 1HNMR
spectra of the flavone derivatives show a characteristic singlet at
OH
O
ii
48%
O
9
d
6.67–6.70 ppm corresponding to the resonance of proton H-3. The
1H NMR spectra of the arylideneflavanone derivatives 5, 14, and 15
OR
show the expected singlets corresponding to the resonances of
protons H-2 and C]CHAr at
d 6.50–6.51 and 8.01–8.05 ppm, re-
spectively. The chemical shifts of the C]CHAr protons (dw8 ppm)
OH
O
indicate that these compounds have an E-configuration.52,53
10, R = MEM
11, R = H
iii (97%)
The resonance of the ester methyl group of malonate derivatives
appears as a singlet at
d
3.73–3.74 ppm while in the C60–flavonoid
Scheme 4. (i) ClCOCH2COOMe, NEt3, CH2Cl2, 0 ꢁC to rt; (ii) C60, CBr4, DBU, toluene, rt;
(iii) HCl 10% in methanol, 70 ꢁC.
OH
O
O
i
80%
O
5
O
O
H3CO
OH
OH
O
O
i
8
O
O
O
O
H3CO
98%
OH
O
H
14
ii
21%
O
12
ii
43%
OH
O
O
O
O
O
O
O
O
O
O
H3CO
H3CO
OH
O
H
13
15
Scheme 5. (i) ClCOCH2COOMe, NEt3, CH2Cl2, 0 ꢁC to rt; (ii) C60, CBr4, DBU, toluene, rt.
Scheme 6. (i) ClCOCH2COOMe, NEt3, CH2Cl2, 0 ꢁC to rt; (ii) C60, CBr4, DBU, toluene, rt.

256
R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
O
O
OH
OH
HO
O
H3CO
O
O
β
α
OH
O
OH
O
C60-Chalcone
Chalcone
OH
OH
O
O
HO
O
O
O
O
O
H3CO
β
O
O
Flavone
C60-Flavone
OH
OH
O
O
b
γ
O
O
HO
O
O
O
H3CO
α
a
OH
OH
O
O
H
H
Flavanone
C60-Flavanone
Figure 1. Flavonoids and C60–flavonoid conjugates used in the antioxidant activity studies.
derivatives it appears at
of the C60–flavonoid conjugates show the expected signals
corresponding to the methano bridge at
w54 ppm and to the sp3
d
3.95–4.08 ppm. The 13C NMR spectra
for spectral parameters). In the case of flavanone derivatives, only
the radical from the phenolic moiety a (see Fig. 1) was detectable.
When photolyzing solutions of C60–chalcone, only a singlet
(g¼2.0033; peak-to-peak line widthz0.7 G), whose intensity
d
carbons of C60 at
d 69.1–72.5 ppm. The mass spectra of the C60–
flavonoid conjugates show the [MþH]þ ion, confirming the pro-
posed structures.
2.4. Antioxidant activity of the C60–flavonoid conjugates
The antioxidant activity of the flavonoid derivatives and of the
corresponding C60 conjugates, shown in Figure 1, was evaluated for
each compound by determining two physico-chemical parameters
that provide a good estimate of this property that is the O–H bond
dissociation enthalpies (BDE) and the rate constant for their re-
action with peroxyl radicals. The BDE values were measured by
using the electron paramagnetic resonance (EPR) radical equili-
bration technique,54 as described below.
2.4.1. EPR spectra
Phenoxyl radicals were produced at room temperature inside
the cavity of an EPR spectrometer by reacting the compounds
shown in Figure 1 with alkoxyl radicals generated photolytically
from di-tert-butyl peroxide in deoxygenated benzene solution. The
measured g factors were typical of phenoxyl radicals (see Table 1
Table 1
EPR spectral parameters for the phenoxyl radicals obtained by abstraction of the OH
hydrogen atom from the title compounds
Molecule
a(2Hm)/G
a(other)/G
g
Chalcone
C60–chalcone
Flavone
1.82
da
3.01 (1Ha); 6.02 (1Hb)
d
2.0043
d
1.95
1.95
1.81
1.83
3.63 (1Hb)
3.63 (1Hb)
2.70 (1Ha); 5.43 (1Hg)
2.70 (1Ha); 5.52 (1Hg)
2.0045
2.0045
2.0044
2.0044
C60–flavone
Flavanoneb
C
60–flavanoneb
Figure 2. Experimental (upper) and computer simulated EPR spectra observed at
room temperature when photolyzing a deoxygenated benzene solution of flavanone
and BHT in a concentration ratio of 5.2:1.
a
See text.
Referred to ring a.
b

R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
257
Table 2
whose intensity increased under irradiation at the expenses of the
phenoxyl radicals.
O–H BDE values of the title compounds measured at room temperature in benzene
containing 10% di-tert-butyl peroxide, rate constants kinh for their reaction with
peroxyl radicals in cumene at 30 ꢁC, and number of radicals n trapped by each
antioxidant molecule
The results, reported in Table 2, show that the flavonoid com-
pounds are characterized by
a BDE(O–H) value of about
1 kcal molꢂ1 larger than that of BHT, and that these values are not
Molecule
BDE(O–H)a/kcal molꢂ1
kinhb/103
M
ꢂ1 sꢂ1
nb
significantly influenced by the covalent link to C60
.
Chalcone
C60–chalcone
Flavone
81.0
d
13
13
2.0
2.0
1.8
1.8
3.7
3.8
2.0e
2.4.3. Autoxidation studies
80.6
d
80.9c
80.8c
79.9d
8.1
8.9
9.3
10
The chain-breaking antioxidant activity of the title compounds
was evaluated by studying the inhibition of the thermally initiated
autoxidation of cumene or styrene (RH) in chlorobenzene, (Eqs. 3–
8). This reaction, initiated by the thermal decomposition of azobis-
(isobutyronitrile) (AIBN) at 30 ꢁC, was followed by monitoring the
oxygen consumption (see Fig. 3) with an automatically recording
gas-absorption apparatus, built in our laboratory and described
previously, which uses as detector a commercial differential
pressure transducer.58 The solutions of cumene or styrene were
either air-saturated, or bubbled with a gas mixture containing N2
and 1% oxygen; BHT was used as reference chain-breaking
inhibitor.
C60–flavone
Flavanone
C60–flavanone
BHT
11e
Errorꢀ0.2 kcal molꢂ1
.
a
b
Error: ꢀ10%.
c
Referred to the ring a.
Ref. 56.
Ref. 42.
d
e
increased during irradiation, was observed. This line was also vis-
ible when photolyzing C60–flavone solutions, together with the
spectrum of the corresponding phenoxyl radical. Since the g factor
and the line width do not correspond to those typical of the addi-
Ri
Initiator / Rꢃ
(3)
(4)
42,55
tion of alkoxyl radicals to C60
,
we tentatively attribute this
singlet to a persistent adduct to the fullerene moiety of tri-
chloromethyl radicals, which might be formed by H-atom ab-
straction from chloroform, employed in little amount (10%) to
enhance the solubility of C60–chalcone and C60–flavone.
Rꢃ þ O2/ROOꢃ
kp
ROOꢃ þ RH / ROOH þ ROꢃ
(5)
(6)
2.4.2. BDE(O–H)
The determination of the O–H bond dissociation enthalpies was
done by measuring, by means of EPR spectroscopy, the equilibrium
constant K1 for the hydrogen atom transfer reaction between two
phenols and the corresponding phenoxyl radicals (Eq. 1) generated
under continuous photolysis (as an example, see Fig. 2). The BDEs
for the species ArOH were calculated, with the assumption that the
entropic term can be neglected,56 by means of Eq. 2 from K1 and the
known BDE value of a reference species Ar0OH, which in the present
case was 2,6-di-tert-butyl-4-methylphenol (BHT) whose recently
2kt
ROOꢃ þ ROOꢃ / non-radical products
kinh
ROOꢃ þ ArOH / ROOH þ ArOꢃ
(7)
(8)
ROOꢃ þ ArOꢃ/non-radical products
In the presence of the investigated compounds, the oxidation of
cumene was strongly retarded (induction period), the oxygen up-
take being given by Eq. 9, where kp is the rate constant for the
propagation step (0.32 Mꢂ1 sꢂ1),42 kinh the inhibition rate constant
revised BDE(O–H) value is 79.9 kcal molꢂ1 56,57
.
K1
ArOH þ Ar0Oꢃ # ArOꢃ þ Ar0OH
(1)
(2)
À
Á
of the antioxidant and
value of kinh was obtained from the slope of the linear plot of
). The stoichiometric factor (that is the
s
the length of the induction period.59 The
BDEðArO—HÞ ¼ BDE Ar0O—H ꢂ RTlnðK1Þ
This approach could not be applied in the cases of C60–chalcone
and C60–flavone due to the presence of the singlet line at g¼2.0033,
ꢂD
[O2] versus ln(1ꢂt/
s
number of peroxyl radicals trapped by each antioxidant molecule),
(a)
(b)
3.0
a
b
c
d
c
a
b
d
e
2.5
2.0
1.5
1.0
0.5
0.0
e
0
2000
4000
time/s
6000
8000
0
2000
4000
time/s
6000
8000
Figure 3. Oxygen consumption traces observed during AIBN (0.05 M) initiated cumene autoxidation (6.2 M) in chlorobenzene solution at 30 ꢁC, in the absence (a) and in the
presence of 6.3ꢄ10ꢂ6 M of the investigated compounds. Panel a: (b) C60–flavone; (c) BHT; (d) C60–chalcone; (e) C60–flavanone. Panel b: (b) flavone; (c) BHT; (d) chalcone; (e) flavanone.

258
R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
C60–flavone possesses an antioxidant activity smaller than BHT, but
larger than C60, in agreement with the results obtained in cumene.
The autoxidation of styrene was also performed at low oxygen
concentration, by bubbling in the reaction vessel, before starting
the reaction, a N2/O2 mixture containing 1% O2 (Fig. 5b). In these
conditions, C60 behaves as a better inhibitor than under air, pre-
sumably because at low O2 concentration the C60 moiety is able to
compete with O2 for Rꢃ, thus interrupting some radical chains.61
Interestingly, at low O2 partial pressure, C60–flavone is more effec-
tive in reducing the oxidation rate not only than C60, but also than
BHT, differently to what observed under air. This result can be ra-
tionalized by admitting that the fullerene moiety behaves as a good
trap toward alkyl radicals, and that this action is synergic with the
ROOꢃ scavenging by the flavonoid part of the molecule. This is
consistent with the reported reactivity of alkyl radicals toward C60
(1.4ꢄ107 Mꢂ1 sꢂ1 for benzyl radical),62 larger than that with BHT
(5ꢄ104 Mꢂ1 sꢂ1 for neophyl radical).63
76
77
78
79
BDE(O-H) / kcal/mol
80
81
82
83
84
3. Conclusions
Figure 4. Plot of Log(kinh) versus the BDE(O–H) values of the investigated compounds
(empty circles). Similar plots are also reported for 2,6-di-tert-butyl (C) and 2,6-di-
methyl (6) 4-substituted phenols.56,60
Flavonoid derivatives bearing one or two 3,5-di-tert-butyl-4-
hydroxyphenyl groups were synthesized and linked covalently to
fullerene C60 via cyclopropanation reactions. The chain-breaking
antioxidant activity of these compounds was evaluated by mea-
suring the O–H bond dissociation enthalpies (BDE) and the rate
constant for the reaction with peroxyl radicals (kinh). From the
comparison of the BDE and kinh values of flavonoids with those of
the corresponding C60 conjugates, it is evident that the ROOꢃ trap-
ping ability of the studied compounds in air-saturated solutions is
due to the phenolic moiety, similar to that of the synthetic inhibitor
BHT. On the other hand, under reduced O2 partial pressure, the
investigated compounds showed better antioxidant activity than
C60 and BHT alone, because in these conditions the ability of the
fullerene moiety to trap alkyl radical acts synergically with the
peroxyl radical scavenging of the phenolic part of the molecule.
This property may become important in the case of the autoxida-
tion of unsaturated lipids, since at the relatively low oxygen partial
pressure present in most living cells, alkyl radicals are not quanti-
tatively converted to peroxyl radicals (Eq. 4).64 Furthermore, the
use of C60–flavonoid conjugates may have other biological benefits
relatively to the flavonoids alone: a better lipophilicity of the con-
jugates, the spherical shape of C60, which could adapt to active sites
of enzymes,22d and the fact that C60 may act as a ‘passport’ for the
intracellular delivery of drugs65 and as a slow-release system.66
These biological properties induced by the fullerene moiety make
the use of C60–antioxidant conjugates particularly interesting in
medical research.
n, was determined from the length of
which n¼2.0.59
s
with respect to BHT, for
(9)
kp½RHꢅ
ꢂ
D
½O2ꢅt ¼
lnð1 ꢂ t= Þ
s
kinh
The results obtained in the case of cumene, reported in Table 2
and Figure 3, show that all the investigated compounds possess
a kinh value comparable to that of BHT and a stoichiometric co-
efficient of about 2, with the exception of flavanone and C60–fla-
vanone, which trap ca. 4 peroxyl radicals due to the presence of two
phenolic moieties. In the case of C60–flavonoids, at the end of the
induction period the oxygen consumption is still slightly retarded
with respect to the uninhibited reaction (see Fig. 3a), suggesting
that the fullerene moiety possesses some antioxidant activity.42
Figure 4 shows that the BDE(O–H) and kinh values obtained in the
present work are in reasonable agreement with those reported in
the literature for 2,6-di-tert-butylphenols.60
Instead, when using styrene as oxidizable substrate, only a slight
reduction of the oxygen consumption rate was observed, this being
in line with the kp value of styrene (41 Mꢂ1 sꢂ1 60 much higher than
)
that of cumene. Figure 5a, reporting the relative reduction of the
reaction rates as a function of inhibitor concentration, shows that
(b)
(a)
1.1
20.9% O2
1% O2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
1
1
3
2
3
2
0
1
2
3
105 [AH] / M
4
5
6
0
1
2
3
105 [AH] / M
4
5
6
Figure 5. Relative decrease of the rate of oxygen consumption during the autoxidation of styrene initiated by AIBN at 30 ꢁC in chlorobenzene in the presence of increasing amounts
of: (1) C60, (2) C60–flavone, (3) BHT; in air (a) and 1% O2 (b).

R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
259
4. Experimental section
4.1. General
30.2 and 31.7 [C(CH3)3], 34.3 and 34.4 [C(CH3)3], 59.9, 65.6, 79.2,
102.2, 109.8, 116.2, 124.9, 125.9, 128.0, 128.8, 129.3, 129.5, 135.8,
136.1, 139.6, 153.9, 155.4, 161.0, 165.1, 181.3 (C]O). MS (MALDI-
TOF): m/z: 643 (MþH)þ. HRMS-ESI m/z calcd for C41H55O6 (MþH)þ
643.3993, found 643.4001.
1H and 13C NMR spectra were recorded on Bruker Avance 300
and 500 spectrometers at 300 or 500 MHz and 75 or 125 MHz,
respectively. CDCl3 was used as solvent and TMS as internal refer-
4.4. 3,5-Di-tert-butyl-4-[(2-methoxyethoxy)meth-
ence. Chemical shifts (
d
) are quoted in parts per million relative to
oxy]benzaldehyde, 6
TMS and the coupling constants (J) are expressed in hertz (Hz).
Mass spectra were recorded on a 4800 MALDI TOF/TOF Analyser
(Applied Biosystems). Flash chromatography was carried out with
silica gel 0.032–0.063 mm. Melting points were measured in
a Bu¨chi Melting Point B-540 apparatus.
NaH (197 mg, 4 equiv) was added to a solution of 3,5-di-tert-
butyl-4-hydroxybenzaldehyde (3) (500 mg, 2.05 mmol) in dry THF
(20 mL) and the mixture was stirred for 15 min at room temperature
under a nitrogen atmosphere. MEM-Cl (0.70 mL, 3 equiv) was added
to the reaction mixture and it was stirred for 3 h. The reaction
mixture was poured into ice and neutralized to pH 6–7 with a 10%
HCl aqueous solution. The resulting mixture was extracted with
chloroform (3ꢄ30 mL) and the organic phase was concentrated. The
aldehyde 6 was purified by preparative TLC using a mixture of
chloroform/petroleum ether (6:4) as the eluent. Compound 6
(575 mg, 87% yield) was obtained as an oil. 1H NMR (300.13 MHz,
4.2. Synthesis of flavonoid derivatives
4.2.1. 20-Hydroxy-40-(3-hydroxypropoxy)acetophenone, 2
20,40-Dihydroxyacetophenone (1) (600 mg) and K2CO3 (1.6 g,
3 equiv) were stirred in acetone (15 mL) for 15 min, at room tem-
perature. To this mixture was added 3-iodopropan-1-ol (0.45 mL,
1.2 equiv). The reaction mixture was heated at reflux for 14 h. The
reaction mixture was filtered and the solid residue was washed
with acetone. Most part of the solvent was evaporated and the
residue was added to water and acidified to pH 4–5 with a 10% HCl
aqueous solution. The resulting mixture was extracted with ethyl
acetate (3ꢄ30 mL). The organic phase was concentrated and the
acetophenone 2 was purified by column chromatography using
a mixture of chloroform/acetone (8.5:1.5) as the eluent. Aceto-
phenone 2 was crystallized from ethyl ether/petroleum ether, in
the freezer, yielding 673 mg (82%) of white solid with mp 46–47 ꢁC.
CDCl3):
OCH2CH2O), 3.98–4.01 (m, 2H, OCH2CH2O), 5.03 (s, 2H, OCH2O), 7.80
(s, 2H, Ar–H), 9.92(s,1H, CHO).13C NMR (125.77 MHz, CDCl3):
d
¼1.48 (s, 18H, t-Bu), 3.43 (s, 3H, OCH3), 3.64–3.67 (m, 2H,
d¼31.8
[C(CH3)3], 35.9 [C(CH3)3], 59.1, 69.3, 99.8, 128.5, 131.5, 145.7, 160.2,
192.1. MS (EIþ): m/z (%): 322 (Mꢃþ,10), 247 (100). HRMS-ESI m/z calcd
for C19H31O4 (MþH)þ 323.2217, found 323.2208.
4.5. 3,5-Di-tert-butyl-20-hydroxy-40-(3-hydroxypropoxy)-4-
[(2-methoxyethoxy)methoxy] chalcone, 7
1H NMR (500.13 MHz, CDCl3):
d
¼2.06 (quintet, J¼6.0 Hz, 2H, H-200),
NaOHaqueoussolution(60%, 40 equiv)wasaddedtoanicecooled
solution of acetophenone 2 (420 mg, 2.0 mmol) and aldehyde 6
(587 mg, 1.2 equiv) in methanol (10 mL). The reaction mixture was
stirred for 15 min at room temperature and then it was heated at
reflux for 8 h using an oil bath. The reaction mixture was poured into
ice, acidified to pH 3–4 with a 10% HCl aqueous solution, and
extracted with chloroform (3ꢄ30 mL). Compound 7 was purified by
preparative TLC using a mixture of petroleum ether/ethyl acetate
(1:1) as the eluent. Chalcone 7 was crystallized from ethyl ether/
petroleum ether yielding yellow crystals (305 mg, 35%) with mp 99–
2.55 (s, 3H, CH3), 3.86 (t, J¼6.0 Hz, 2H, H-300), 4.15 (t, J¼6.0 Hz, 2H,
H-100), 6.42 (d, J¼2.5 Hz, 1H, H-30), 6.44 (dd, J¼8.7, 2.5 Hz, 1H, H-50),
7.62 (d, J¼8.7 Hz, 1H, H-60). 13C NMR (75.47 MHz, CDCl3):
¼26.2,
d
31.7, 59.7, 65.5, 101.4, 107.8, 113.9, 132.3, 165.1, 165.3, 202.6 (C]O).
MS (EIþ): m/z (%): 210 (Mꢃþ, 41), 195 (33), 137 (100). HRMS-ESI m/z
calcd for C11H15O4 (MþH)þ 211.0965, found 211.0965.
4.3. 3,5-Di-tert-butyl-20,4-dihydroxy-40-(3-hydroxypropoxy)-
chalcone (4) and arylideneflavanone 5
100 ꢁC. 1H NMR (300.13 MHz, CDCl3):
d
¼1.48 (s, 18H, t-Bu), 2.08
These compounds were prepared as described in the literature48
using acetophenone 2 (273 mg, 1.3 mmol) and aldehyde 3 (316 mg,
1.3 mmol). Compounds 4 and 5 were separated by crystallization
from a chloroform/methanol mixture.
(quintet, J¼6.0 Hz, 2H, H-200), 3.43 (s, 3H, OCH3), 3.65–3.68 (m, 2H, O–
CH2 CH2–O), 3.85–3.90 (m, 2H, H-300), 3.99–4.02 (m, 2H, O–CH2CH2–
O), 4.18 (t, J¼6.0 Hz, 2H, H-100), 5.02 (s, 2H, O–CH2–O), 6.49–6.52 (m,
2H, H-30 andH-50), 7.44 (d, J¼15.4 Hz,1H, H-
a), 7.55 (s, 2H, H-2 and H-
6), 7.84(d, J¼9.6 Hz,1H,H-60), 7.86(d, J¼15.4 Hz,1H,H-
b),13.52(s,1H,
4.3.1. Chalcone 4
20-OH). 13C NMR (75.47 MHz, CDCl3):
d
¼31.7 (C-200), 31.9 [C(CH3)3],
Yield 135 mg (30%), mp 140–141 ꢁC. 1H NMR (300.13 MHz,
35.8 [C(CH3)3], 59.1, 59.8, 65.6, 69.1, 71.6, 99.7,101.6,107.8,114.1,118.7,
127.2, 129.5,131.2, 145.2, 157.1, 165.1, 166.4,166.5,166.9,191.9 (C]O).
MS (EIþ): m/z (%): 514 (Mꢃþ, 9), 438 (24),137 (23), 89 (100). Elemental
analysis (%) calcd for C30H42O7: C 70.01, H 8.23; found: C 70.15, H 8.09.
CDCl3):
d
¼1.49 (s, 18H, t-Bu), 2.08 (quintet, J¼6.0 Hz, 2H, H-200), 3.88
(t, J¼6.0 Hz, 2H, H-300), 4.18 (t, J¼6.0 Hz, 2H, H-100), 5.02 (s, 1H, 4-
OH), 6.48–6.51 (m, 2H, H-30 and H-50), 7.41 (d, J¼15.4 Hz, 1H, H-
a),
7.50 (s, 2H, H-2 and H-6), 7.82–7.91 (m, 2H, H-60 and H-
b), 13.63 (s,
1H, 20-OH). 13C NMR (75.47 MHz, CDCl3):
d
¼30.1 (C-200), 31.7
4.6. 30,50-Di-tert-butyl-40-hydroxy-7-(3-hydroxypropoxy)-
flavone, 8
[C(CH3)3], 34.3 [C(CH3)3], 59.9, 65.6, 101.6, 107.7, 114.2, 116.8, 126.1,
131.1,136.5,146.0,156.7,165.1,166.5,191.9 (C]O). MS (MALDI-TOF):
m/z: 427 (MþH)þ. HRMS-ESI m/z calcd for C26H35O5 (MþH)þ
427.2479, found 427.2488.
A solution of chalcone 7 (60 mg, 0.12 mmol) and iodine (1.2 mg,
0.04 equiv) in DMSO (2 mL) was heated at reflux for 1 h, under a ni-
trogen atmosphere. The reaction mixture was poured into water, and
the iodine was reduced with an aqueous solution of 5% Na2S2O3
(20 mL). The resulting solution was extracted with ethyl acetate
(3ꢄ25 mL), the organic phase was concentrated and purified by
preparative TLC (silica gel) using a mixture of chloroform/ethyl ace-
tate (8:2) as the eluent. Compound 8 was crystallized from CHCl3/
MeOH giving white crystals (30 mg, 61% yield) with mp 176–177 ꢁC.
4.3.2. Arylideneflavanone 5
Yield 45 mg (10%), mp 180–181 ꢁC. 1H NMR (300.13 MHz,
CDCl3):
d
¼1.28 (s, 18H, t-Bu), 1.36 (s, 18H, t-Bu), 2.02 (quintet,
J¼6.0 Hz, 2H, H-200), 3.81–3.86 (m, 2H, H-300), 4.10 (t, J¼6.0 Hz, 2H,
H-100), 5.21 (s, 1H, OH), 5.47 (s, 1H, OH), 6.35 (d, J¼2.4 Hz, 1H, H-8),
6.51 (s, 1H, H-2), 6.52 (dd, J¼8.9, 2.4 Hz, 1H, H-6), 7.07 (s, 2H, H-20
and H-60), 7.27 (s, 2H, H-20 and H-60), 7.90 (d, J¼8.9 Hz, 1H, H-5),
1H NMR (300.13 MHz, CDCl3):
d
¼1.51 (s, 18H, t-Bu), 2.13 (quintet,
8.05 (s, 1H, C]CH–Ar). 13C NMR (75.47 MHz, CDCl3):
d
¼30.0 (C-200),
J¼6.0 Hz, 2H, H-200), 3.89–3.94 (m, 2H, H-300), 4.26 (t, J¼6.0 Hz, 2H,

260
R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
H-100), 5.66 (s, 1H, OH), 6.70 (s, 1H, H-3), 6.96–7.00 (m, 2H, H-6 and
(30 mg, 0.040 mmol) and C60 (1.5 equiv) in anhydrous toluene
(50 mL) were added CBr4 (1 equiv) and DBU (2 equiv). The reaction
mixture was stirred for 18 h at room temperature, under a nitrogen
atmosphere, and then it was concentrated and purified by flash
chromatography (silica gel) using a (9:1) or (98:2) mixture of tol-
uene/ethyl acetate as the eluent. Compounds 10, 13, and 15 were
crystallized from CHCl3/MeOH.
H-8), 7.73 (s, 2H, H-20 and H-60), 8.12 (d, J¼9.5, 1H, H-5). 13C NMR
(75.47 MHz, CDCl3):
d
¼30.1 [C(CH3)3], 31.8 (C-200), 34.5 [C(CH3)3],
59.7, 65.9, 101.1, 106.0, 114.3, 117.8, 122.8, 123.5, 127.0, 136.5, 157.0,
157.9,163.2,164.4,178.0 (C]O). MS (MALDI-TOF): m/z: 425 (MþH)þ.
MS (EIþ): m/z (%): 424 (Mꢃþ, 100), 409 (61), 137 (17). Elemental
analysis(%) calcd for C26H32O5: C 73.56, H 7.60; found: C 73.77, H8.00.
4.7. Synthesis of malonate derivatives
4.8.1. C60–chalcone conjugate 10
Black solid (39 mg, 48% yield), mp >300 ꢁC.1HNMR (300.13 MHz,
General procedure. A solution of methyl 3-chloro-3-oxopropa-
noate (2 equiv) in anhydrous CH2Cl2 (2 mL) was added dropwise to
an ice cooled solution of chalcone 7 (100 mg, 0.19 mmol), flavone 8
(58 mg; 0.12 mmol), or arylideneflavanone 5 (40 mg, 0.085 mmol)
and anhydrous NEt3 (4 equiv) in anhydrous CH2Cl2 (10 mL). The ice
bath was removed and the reaction mixture was stirred at room
temperature for 3 h. It was then washed with an aqueous solution
of 0.5% HCl (2ꢄ15 mL) and with a saturated aqueous solution of
NaHCO3 (2ꢄ15 mL). The organic phase was dried (Na2SO4) and the
solvent was evaporated under vacuum. Malonates 9, 12, and 14
were purified by preparative TLC (silica gel) using a 95:5 mixture of
CHCl3/AcOEt as the eluent.
CDCl3):
d
¼1.48 (s, 18H, t-Bu), 2.37 (quintet, J¼5.9 Hz, 2H, H-200), 3.41
(s, 3H, OCH3), 3.65–3.68 (m, 2H, OCH2CH2O), 3.99–4.02 (m, 2H,
OCH2CH2O), 4.07 (s, 3H, CO2CH3), 4.19 (t, J¼5.9 Hz, 2H, H-300), 4.73 (t,
J¼5.9 Hz, 2H, H-100), 5.03 (s, 1H, OCH2O), 6.43 (d, J¼2.5 Hz,1H, H-30),
6.52 (dd, J¼9.0, 2.5 Hz,1H, H-50), 7.39 (d, J¼15.4 Hz,1H, H-
a), 7.80 (d,
J¼9.0 Hz, 1H, H-60), 7.84 (d, J¼15.4 Hz, 1H, H-
b
), 13.47 (s, 1H, 20-OH).
13C NMR (125.77 MHz, CDCl3):
d
¼28.1 (C-200), 31.9 [C(CH3)3], 35.8
[C(CH3)3], 54.1 (methano bridge), 59.1, 63.6, 64.0, 69.1 (C60-sp3), 71.4,
71.6 (C60-sp3), 99.7,101.6,107.9,114.4,118.7,127.2,129.5,131.4,138.4,
139.4,140.9,141.7,141.8,142.2,142.9,143.0,143.7,143.9,144.4,144.5,
144.60, 144.62, 144.65, 144.77, 144.81, 144.86, 145.09, 145.14, 145.18,
145.18, 145.23, 145.27, 145.4, 157.1, 163.6, 164.1, 165.0, 166.6 (2malo-
nate C]O),191.9 (chalcone C]O). HRMS-ESI m/z calcd for C94H45O10
(MþH)þ 1333.3007, found 1333.2978.
4.7.1. Malonate 9
Obtained as an yellow oil (106 mg, 89% yield). 1H NMR
(300.13 MHz, CDCl3):
d
¼1.48 (s, 18H, t-Bu), 2.18 (quintet, J¼6.1 Hz,
4.8.2. C60–chalcone conjugate 11
2H, H-200), 3.41 (s, 2H, OC–CH2–CO), 3.43 (s, 3H, OCH3), 3.63–3.68
(m, 2H, OCH2CH2O), 3.74 (s, 3H, CO2CH3), 3.99–4.02 (m, 2H,
OCH2CH2O), 4.11 (t, J¼6.1 Hz, 2H, H-300), 4.37 (t, J¼6.1 Hz, 2H, H-100),
5.03 (s, 2H, OCH2O), 6.46 (d, J¼2.5 Hz, 1H, H-30), 6.50 (dd, J¼8.9,
A solution of 10% HCl in methanol (2 mL) was added to a solution
of compound 10 (19 mg, 0.014 mmol) in chloroform (3 mL). The
mixture was stirred at 70 ꢁC for 3 h, it was neutralized with an
aqueous solution of Na2CO3 and then compound 11 was extracted
with chloroform (2ꢄ25 mL). The organic layer was dried (Na2SO4)
and concentrated. Compound 11 was crystallized from CHCl3/MeOH
affording a black solid (17 mg, 97% yield) with mp >300 ꢁC. 1H NMR
2.5 Hz, 1H, H-50), 7.44 (d, J¼15.4 Hz, 1H, H-
), 7.84 (d, J¼8.9 Hz, 1H,
a
H-60), 7.86 (d, J¼15.4 Hz, 1H, H-
b
), 13.53 (s, 1H, 20-OH). 13C NMR
(75.47 MHz, CDCl3):
d
¼28.1 (C-200), 31.8 [C(CH3)3], 35.8 [C(CH3)3],
41.2, 52.5, 59.1, 62.0, 64.4, 69.1, 71.6, 99.6, 101.4, 107.9, 114.1, 118.6,
127.2, 129.5, 131.2, 145.2, 157.1, 165.1, 166.5 (2malonate C]O), 191.8
(chalcone C]O). MS (MALDI-TOF): m/z: 615 (MþH)þ.
(300.13 MHz, CDCl3):
d
¼1.49 (s, 18H, t-Bu), 2.33–2.41 (m, 2H, H-200),
4.07 (s, 3H, CO2CH3), 4.19 (t, J¼5.8 Hz, 2H, H-300), 4.73 (t, J¼5.8 Hz, 2H,
H-100), 5.61 (s,1H, 4-OH), 6.42 (d, J¼2.5 Hz,1H, H-30), 6.51 (dd, J¼9.0,
2.5 Hz,1H, H-50), 7.37 (d, J¼15.4 Hz,1H, H-
), 7.81 (d, J¼9.0 Hz,1H, H-
a
4.7.2. Malonate 12
60), 7.86 (d, J¼15.4 Hz, 1H, H-
b
), 13.59 (s, 1H, 20-OH). 13C NMR
¼28.1 (C-200), 30.2 [C(CH3)3], 34.4 [C(CH3)3],
Obtained as
a
colorless oil (55 mg, 98% yield). 1H NMR
(125.77 MHz, CDCl3):
d
(300.13 MHz, CDCl3):
d¼1.50 (s, 18H, t-Bu), 2.23 (quintet, J¼6.1 Hz,
54.1 (methano bridge), 63.6, 64.0, 71.4 (C60-sp3), 101.6, 107.8, 114.4,
116.8, 126.1, 126.3, 136.5, 138.5, 141.0, 141.7, 141.8, 142.16, 142.18,
142.90, 142.97, 143.04, 143.7, 143.9, 144.45, 144.52, 144.63, 144.67,
144.80, 144.83, 144.87, 145.10, 145.15, 145.20, 145.23, 146.1, 156.7,
163.6, 164.1, 164.9, 166.5 (2malonate C]O), 192.0 (chalcone C]O).
MS (MALDI-TOF): m/z: 1245 (MþH)þ, 720 (C6ꢃþ0). HRMS-ESI m/z calcd
for C90H37O8 (MþH)þ 1245.2483, found 1245.2447.
2H, H-200), 3.42 (s, 2H, OC–CH2–CO), 3.73 (s, 3H, CO2CH3), 4.19 (t,
J¼6.1 Hz, 2H, H-300), 4.41 (t, J¼6.1 Hz, 2H, H-100), 5.66 (s, 1H, OH),
6.70 (s, 1H, H-3), 6.96–7.00 (m, 2H, H-6 and H-8), 7.74 (s, 2H, H-20
and H-60), 8.12 (d, J¼8.8 Hz, 1H, H-5). 13C NMR (75.47 MHz, CDCl3):
d
¼28.3 (C-200), 30.1 [C(CH3)3], 31.8 [C(CH3)3], 34.5, 41.2, 59.1, 62.0,
64.7, 101.2, 106.0, 114.1, 117.9, 122.8, 123.5, 127.0, 136.5, 157.0, 157.9,
163.0, 164.4 (2malonate C]O), 177.9 (flavone C]O). HRMS-ESI m/z
calcd for C30H37O8 (MþH)þ 525.2483, found 525.2496.
4.8.3. C60–flavone conjugate 13
Black solid (25 mg, 43%yield), mp>300 ꢁC.1HNMR (300.13 MHz,
4.7.3. Malonate 14
CDCl3):
d
¼1.49 (s, 18H, t-Bu), 2.42 (quintet, J¼5.9 Hz, 2H, H-200), 4.08
Colorless oil (53 mg, 80% yield). 1H NMR (300.13 MHz, CDCl3):
(s, 3H, CO2CH3), 4.27 (t, J¼5.9 Hz, 2H, H-300), 4.77 (t, J¼5.9 Hz, 2H, H-
100), 5.63 (s,1H, OH), 6.67 (s,1H, H-3), 6.89 (d, J¼2.4 Hz,1H, H-8), 7.00
(dd, J¼8.8, 2.4 Hz, 1H, H-6), 7.62 (s, 1H, H-20 and H-60), 8.11 (d,
d
¼1.28 (s, 18H, t-Bu), 1.36 (s, 18H, t-Bu), 2.12 (quintet, J¼6.1 Hz, 2H,
H-200), 3.38 (s, 2H, OC–CH2–CO), 3.68 (s, 3H, CO2CH3), 4.03 (t,
J¼6.1 Hz, 2H, H-300), 4.33 (t, J¼6.1 Hz, 2H, H-100), 5.22 (s, 1H, OH),
5.48 (s, 1H, OH), 6.33 (d, J¼2.4 Hz, 1H, H-8), 6.51 (dd, J¼8.8, 2.4 Hz,
1H, H-6), 6.52 (s, 1H, H-2), 7.06 (s, 2H, H-20 and H-60), 7.27 (s, 2H, H-
20 and H-60), 7.89 (d, J¼8.8 Hz, 1H, H-5), 8.05 (s, 1H, C]CH–Ar). 13C
J¼8.8 Hz,1H, H-5). 13C NMR (75.47 MHz, CDCl3):
d
¼28.1 (C-200), 30.2
[C(CH3)3], 34.5 [C(CH3)3], 52.0 (methano bridge), 54.1, 63.4, 64.2, 71.3
(C60-sp3), 101.1, 106.2, 114.3, 118.1, 122.8, 123.6, 127.1, 136.5, 138.3,
139.4, 140.85, 140.87, 141.6, 141.8, 142.1, 142.82, 142.88, 142.90,
142.95, 143.03, 143.6, 143.8, 144.2, 144.4, 144.55, 144.59, 144.60,
144.63, 144.71, 144.84, 145.0, 145.07, 145.12, 145.20, 157.0, 157.9,
163.0,163.5,164.0,164.5 (2malonate C]O),177.8 (flavone C]O). MS
(MALDI-TOF): m/z: 1243 (MþH)þ. HRMS-ESI m/z calcd for C90H35O8
(MþH)þ 1243.2326, found 1243.2321.
NMR (75.47 MHz, CDCl3):
d
¼28.2 (C-200), 30.0 and 30.2 [C(CH3)3],
34.3 and 34.4 [C(CH3)3], 41.3, 52.5, 62.1, 64.3, 79.2, 102.3, 109.7,
116.3, 124.9, 125.9, 128.0, 128.9, 129.3, 129.5, 135.9, 136.1, 139.6,
153.9, 155.4, 160.9, 165.0, 166.4, 166.9, 181.3 (C]O).
4.8. Synthesis of C60 derivatives
4.8.4. C60–flavanone conjugate 15
General procedure. To
a
solution of malonate
9
(31 mg,
Black solid (12 mg, 21% yield), mp >300 ꢁC. 1H NMR
0.061 mmol), malonate 12 (22 mg, 0.046 mmol), or malonate 14
(300.13 MHz, CDCl3):
d¼1.29 (s, 18H, t-Bu), 1.36 (s, 18H, t-Bu), 2.18–

R.F. Enes et al. / Tetrahedron 65 (2009) 253–262
261
2.36 (m, 2H, H-200), 3.95 (s, 3H, CO2CH3), 4.10–4.14 (m, 2H, H-300),
4.65–4.72 (m, 2H, H-100), 5.21 (s, 1H, OH), 5.63 (s, 1H, OH), 6.32 (d,
J¼2.3 Hz, 1H, H-8), 6.50 (s, 1H, H-2), 6.54 (dd, J¼8.8, 2.3 Hz, 1H, H-
6), 7.04 (s, 2H, H-20 and H-60), 7.26 (s, 2H, H-20 and H-60), 7.89 (d,
J¼8.8 Hz, 1H, H-5), 8.01 (s, 1H, C]CH–Ar). 13C NMR (75.47 MHz,
2004. R.F.E. also thanks FCT for the grant SFRH/BPD/24213/2005.
Financial support from MIUR (Rome), contract 2004038243 (GFP,
RA), is gratefully acknowledged.
References and notes
CDCl3):
d
¼28.1 (C-200), 30.0 and 30.2 [C(CH3)3], 34.3 and 34.4
[C(CH3)3], 54.0 (methano bridge), 63.4, 63.6, 63.9, 72.5 (C60-sp3),
79.2, 102.1, 109.8, 116.4, 124.9, 125.9, 128.0, 128.9, 129.4, 129.5, 135.9,
136.1,138.6,139.3,139.8,140.9,141.7,141.8,142.2,143.0,143.8,143.9,
144.5, 144.6, 144.7, 144.8, 144.9, 145.10, 145.14, 145.19, 145.23, 153.9,
155.4, 161.0, 163.5, 165.0 (malonate C]O), 178.5 (flavanone C]O).
MS (MALDI-TOF): m/z: 1461 (MþH)þ. HRMS-ESI m/z calcd for
C105H57O9 (MþH)þ 1461.3997, found 1461.3966.
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