Obtaining new flavanones exhibiting antifungal activities by methyltrioxorhenium-catalyzed epoxidation-methanolysis of flavones

Article abstract of DOI:10.1016/j.tet.2008.05.101

New 3-hydroxy-2-methoxyflavanones have been obtained through epoxidation-methanolysis of the corresponding flavone with urea-hydrogen peroxide (UHP)/methyltrioxorhenium (CH₃ReO₃, MTO) catalytic system in methanol as nucleophilic solv

Full text of DOI:10.1016/j.tet.2008.05.101

Tetrahedron 64 (2008) 7561–7566
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Obtaining new flavanones exhibiting antifungal activities by
methyltrioxorhenium-catalyzed epoxidation–methanolysis of flavones
,
a
a
b
Roberta Bernini a , Enrico Mincione , Gianfranco Provenzano , Giancarlo Fabrizi ,
*
Sabrina Tempesta c, Marcella Pasqualetti c
a
`
Dipartimento di Agrobiologia e Agrochimica, Universita degli Studi della Tuscia, Via S. Camillo De Lellis snc, 01100 Viterbo, Italy
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita degli Studi di Roma La Sapienza, P.le A. Moro 5, 00185 Roma, Italy
Dipartimento di Ecologia e Sviluppo Economico Sostenibile, Universita degli Studi della Tuscia, Via S. Giovanni Decollato 1, 01100 Viterbo, Italy
b
c
`
`
a r t i c l e i n f o
a b s t r a c t
Article history:
New 3-hydroxy-2-methoxyflavanones have been obtained through epoxidation–methanolysis of the
corresponding flavone with urea–hydrogen peroxide (UHP)/methyltrioxorhenium (CH3ReO3, MTO) cat-
alytic system in methanol as nucleophilic solvent. After acetylation of the reaction mixtures, the cor-
responding cis- and trans-3-acetoxy-2-methoxyflavanones have been isolated and characterized by
spectroscopic analyses. Their antifungal activity has been tested in vitro against three fungal strains of
common saprotrophic soil and seed fungi, such as Trichoderma koningii, Fusarium solani and Cladospo-
rium herbarum, potentially pathogenic for humans. Some aspects of the structure–activity relationship
of the most active compounds have been evaluated. The mycelial growth of T. koningii and C. herbarum
has been totally inhibited from cis-3-acetoxy-2,6-dimethoxyflavanone 7c and cis-3-acetoxy-2,7-
dimethoxyflavanone 13c at the lowest concentration (0.5ꢀ10ꢁ4 M).
Received 4 March 2008
Received in revised form 5 May 2008
Accepted 22 May 2008
Available online 27 May 2008
Keywords:
Urea–hydrogen peroxide/
methyltrioxorhenium-catalyzed
epoxidation–methanolysis
New flavanones
Ó 2008 Elsevier Ltd. All rights reserved.
Antifungal activity
1. Introduction
pyrane) rings. Among them, flavones and flavanones have an oxo
group in C4; flavones have also a double bond in C2–C3 (Fig.1, b and
Flavonoids are polyphenolic secondary metabolites ubiquitous
in edible plant foods and beverages. In fact, they naturally occur in
fruits, vegetables, nuts, seeds, tea and wine, and are an integral part
of the human diet.1 In the plant kingdom, they are involved in
important biological processes such as nitrogen fixation, photo-
sensitization, energy transfer, plant growth, control of respiration
and photosynthesis.2 Figure 1 shows the generic chemical structure
of flavonoids (a) based on the flavan nucleus and the numbering
system used to distinguish the carbon positions around the mole-
cule. The three phenolic rings are referred to as the A, B and C (or
c). These compounds as well as their mixtures possess high anti-
fungal potential against different species usually occurring on
grains,3 seeds and pods.4
With the aim of synthesizing new flavanones, we have consid-
ered to functionalize the double bond of flavones by an oxidative
addition pathway. It is known that the C2–C3 double bond of these
compounds is unreactive towards classical oxidants such as H2O2/
8
OHꢁ,5 KMnO4,6 NiO2,7 SeO2
and Tl(OAc)3.9 Only freshly pre-
pared acetone solutions of dimethyldioxirane10 or methyl(tri-
fluoromethyl)dioxirane11 are able to convert flavones into the
corresponding epoxides. However, dioxiranes are difficult to handle
being gaseous, explosive and having a poor solubility. Furthermore,
to the best of our knowledge, no general catalytic oxidation pro-
cedures for the oxidative attack of the double bond of flavones have
been reported. In the last years, methyltrioxorhenium (CH3ReO3,
MTO)12 together with hydrogen peroxide or urea–hydrogen per-
oxide 13 has become an important catalyst for a variety of synthetic
transformations such as the epoxidation of alkenes,14 conjugated
dienes,15 oxidation of alkynes,16 aromatic derivatives,17 sulfur
compounds,18 anilines and amines,19 phosphines,20 C–H and Si–H
3'
4'
5'
2'
8
B
6'
O
O
O
O
O
C
9
2
7
6
1'
A
3
10
5
4
b
a
c
Figure 1. The generic structure of flavonoids.
21
bonds and the Bayer–Villiger rearrangement.22 The reactive in-
termediates for these oxidations are monoperoxo [CH3Re(O)2O2]
* Corresponding author. Tel.: þ39 761 357452; fax: þ39 761 357230.
E-mail address: berninir@unitus.it (R. Bernini).
and bis-peroxo [CH3ReO(O)2]h
2-rhenium complexes (Scheme 1).23
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.101

7562
R. Bernini et al. / Tetrahedron 64 (2008) 7561–7566
H3CO
O
H3CO
O
O
Re O
O
CH3ReO3 (MTO)
O
Re
O
Re
H2O2
H2O
H2O2
H2O
O
O
O
O
O
O
O
O
H3C
a)
H3C
CH3
+
H
OH
dpRe
mpRe
OH
H
O
O
Scheme 1. H2O2/CH3ReO3 catalytic system.
O
rac-3a
rac-3b
1
For many years, our research group has been investigating the
reactivity of the hydrogen peroxide/methyltrioxorhenium catalytic
system on natural organic compounds in order to get new bioactive
compounds and fine chemicals.24 In particular, we first described
the Bayer–Villiger oxidation of the flavanone C-ring to prepare
b)
a) Oxidant/CH3ReO3, CH3OH, r.t or reflux
b) Ac2O, Py
H3CO
O
25
H3CO
O
new lactones exhibiting apoptotic activity on tumoral cell lines
and the oxidation of the A-ring of catechins to obtain new
p-benzoquinones.26
+
H
OAc
OAc
In this paper, we report the epoxidation–methanolysis of the
double bond of monosubstituted flavones catalyzed by methyl-
trioxorhenium and urea–hydrogen peroxide (UHP) as the oxidant in
the presence of methanol as nucleophilic solvent. After acetylation
of the oxidationproducts, the stable new cis- and trans-3-acetoxy-2-
methoxyflavanone derivatives were tested as antifungal agents
against three fungal strains of common saprotrophic soil and seed
fungi,27 such as Trichoderma koningii, Fusarium solani and Clado-
sporium herbarum, potentially pathogenic for humans.28 Some
considerations on structure–activity relationships are also reported.
H
O
O
rac-3d
rac-3c
Scheme 2. Oxidation–methanolysis of the flavone 1 with the UHP or H2O2/CH3ReO3
catalytic system.
O
O
O
O
O
O
+
H
H
O
O
O
2. Results and discussion
2a
2a
1
Preliminary experiments on oxidation were carried out using
flavone 1 as the substrate model (Scheme 2). Hydrogen peroxide
(50% water solution) or urea–hydrogen peroxide (UHP) was used as
oxidants in the presence of a catalytic amount of methyltrioxo-
rhenium. In these reaction conditions, the corresponding cis- and
trans-3-hydroxy-2-methoxyflavanones 3a and 3b were obtained.
The best conversion of the flavone 1 was obtained using UHP/MTO
in methanol at reflux (conversion and yield: 62%). Due to their
instability during the chromatographic purification, compounds 3a
and 3b were isolated and characterized as acetates after acetylation
of the crude reaction mixture. As expected, the corresponding cis-
and trans-3-acetoxy-2-methoxyflavanones 3c and 3d were isolated
in a 32/68 ratio (3c/3d¼1.0/2.1) and characterized by spectroscopic
analyses.
CH3OH, reflux
H3CO
O
H3CO
O
+
H
OH
OH
H
O
O
rac-3a
rac-3b
Scheme 3. Possible mechanism of oxidation–methanolysis of the flavone 1 with the
UHP/CH3ReO3 catalytic system.
After double bond epoxidation by the catalytic system UHP/
CH3ReO3, the obtained epoxides 2a,b undergo a rapid ring opening
reaction in the solvolytic conditions to get 3a and 3b (Scheme 3), as
reported by Adam for the dioxirane reaction.10 All efforts to isolate
the epoxides 2a,b in a non-nucleophilic solvents failed. In fact, in
dichloromethane and diethyl ether no appreciable conversion of the
flavone 1 occurred. The structural assignation of compounds 3a and
3b was confirmed by NOESY experiments on the corresponding
acetylated products 3c and 3d. An SN1-like mechanism probably
operated in these conditions as suggested by the polarimetric mea-
surements performed on the cis- and trans-3-hydroxy-2-methoxy-
flavanones 3c and 3d, both of them resulted as a racemic mixture.
The 3c/3d¼32/68 ratio was explained by molecular calculations as
an outcome of the different stability of the cis–trans stereoisomers.
After these initial investigations, we extended the epoxidation–
methanolysis to the monosubstituted flavones 4, 6, 8,10 and 12. The
successive acetylation of the crude mixtures allowed the isolation
of the corresponding new cis- and trans-flavanones 5c,d, 7c,d, 9c,d
and 13c,d (Scheme 4, Table 1). The unreactivity of 6-chloroflavone
10 (Table 1, entry 4) with UHP/MTO was attributed to the deacti-
vating effect of the chlorine substituent on the double bond of the
benzopyranic ring.
The oxidation performed on 5,7-dimethoxyflavone 14 in the same
experimental conditions led to the formation of a complex mixture
of products. However, by monitoring the oxidation at room
temperature, despite lower conversion of the substrate (35%), we
obtained a mixture of two compounds, inseparable by column
4, 5c-d: R1=OCH3, R2,=R3=H
6, 7c-d: R1=R3=H, R2=OCH3
8, 9c-d: R1=R3=H, R2=CH3
10, 11c-d: R1=R3=H, R2=Cl
12, 13c-d: R1=R2=H, R3=OCH3
H3CO
O
H3CO
R3
R2
R3
R2
O
R3
R2
O
O
1) UHP/CH3ReO3, CH3OH, reflux
2) Ac2O/Py, r.t
+
H
OAc
OAc
H
R1
O
R1
O
R1
4, 6, 8, 10, 12
rac-5c, 7c, 9c, 11c, 13c
rac-5d, 7d, 9d, 11d, 13d
Scheme 4. Oxidation of the flavones 4, 6, 8, 10 and 12 with the UHP/CH3ReO3 catalytic system in methanol and successive acetylation.

R. Bernini et al. / Tetrahedron 64 (2008) 7561–7566
7563
Table 1
chromatography on silica gel, assigned as 6-hydroxy-5,7-
dimethoxyflavone 15 (yield: 10%) and 8-hydroxy-5,7-dimethoxy-
flavone 16 (yield: 10%, Scheme 5). A third compound with a lower Rf
was eluted having the structure of 5,8-dihydroxy-7-methoxyflavone
17, probably derived from oxidative demethylation of 16 (yield: 15%).
Therefore, in the presence of two C-5 and C-7 methoxy groups on the
aromatic A ring, the catalytic oxidant system did not attack the C2–C3
double bond, the oxyfunctionalization of the activated A ring being
favoured, as already reported by us for the methylated catechins.26
After methylation of mixture of compounds 15 and 16, the corre-
sponding products 18 and 19 were easily separated by chromato-
graphic column and characterized by spectroscopic analysis. As
expected, the methylation of 16 and 17 gave the same compound 19.
Oxidation of the flavones 4, 6, 8, 10 and 12, and successive acetylation of the
products as depicted in Scheme 4
Entry Substrate Reaction time Conv. (%) Yields (%)b,c
(h)a
Ratio
1
2
3
4
5
4
6
8
10
12
24
24
24
48
24
62
50
66
<5
52
5c: 25, 5d: 75
7c: 29, 7d: 71
9c: 26, 9d: 74
d
5c/5d¼1.0/3.0
7c/7d¼1.0/2.4
9c/9d¼1.0/2.8
d
13c: 22, 13d:78 13c/13d¼1.0/3.5
a
b
c
UHP (6 equiv), CH3ReO3 (0.05 mmol), CH3OH, reflux.
Calculated after chromatographic purification.
Calculated on the converted substrate.
OH
H3CO
HO
O
O
H3CO
O
O
H3CO
O
O
16: R=CH3
17: R=H
+
Oxidation
CH3O
CH3O
14
RO
16, 17
15
Methylation
H3CO
OCH3
H3CO
H3CO
O
O
O
+
CH3O
O
CH3O
19
18
Oxidation: H2O2 (50% water solution, 2 eq.); CH3ReO3 (0.05 mmol), CH3OH, 25 °C, 2h
Methylation: DMF, CH3I (10 eq.), 25 °, 24 h
Scheme 5. Oxidation of the flavones 4, 6, 8 and 10 with the UHP/CH3ReO3 catalytic system in methanol and successive acetylation.
H3CO
O
H3CO
O
O
O
H
OAc
OAc
H
O
O
O
O
rac-3c
1
rac-3d
rac-20
H3CO
O
H3CO
O
O
O
OAc
H
OAc
H3CO
H3CO
H3CO
H3CO
H
O
O
O
O
rac-7d
rac-21
rac-7c
6
H3CO
O
H3CO
O
H3CO
O
H3CO
H3CO
H3CO
O
OAc
H
OAc
H
O
O
O
O
rac-13d
rac-22
rac-13c
12
Figure 2. Compounds tested on the antifungal activity.

7564
R. Bernini et al. / Tetrahedron 64 (2008) 7561–7566
Table 2
,b
Inhibition (%) of linear mycelial growth of T. koningii, F. solani and C. herbaruma
Entry
Compound
T. koningii
F. solani
C. herbarum
8ꢀ10ꢁ4 M
2ꢀ10ꢁ4 M
0.5ꢀ10ꢁ4 M
8ꢀ10ꢁ4 M
2ꢀ10ꢁ4 M
0.5ꢀ10ꢁ4 M
8ꢀ10ꢁ4 M
2ꢀ10ꢁ4 M
0.5ꢀ10ꢁ4 M
1
2
3
4
5
6
7
8
1
79.3
78.3
53.0
40.0
55.8
35.2
74.2
d
85.1
75.0
43.5
10.9
38.7
35.2
85.2
d
26.4
55.6
32.3
4.01
32.0
32.5
98.9
d
63.7
55.0
16.7
9.22
14.2
d
58.2
44.7
7.40
3.21
d
5.80
23.1
3.02
2.20
d
27.1
72.7
48.8
28.8
31.2
11.0
67.0
d
28.0
66.0
32.3
7.30
9.80
13.3
73.5
d
13.3
41.1
26.1
d
3c
3d
5c
5d
6
7c
7d
9c
9d
12
13c
13d
20
21
22
d
d
d
14.5
100
d
13.8
d
4.10
d
3.20
2.50
9.70
d
9
49.2
48.4
62.0
87.8
15.6
68.4
31.0
65.0
48.3
31.8
62.7
98.0
14.7
66.6
30.5
60.0
53.5
23.9
52.2
100
5.30
62.0
33.2
53.0
15.6
5.50
24.3
42.9
2.01
15.8
d
9.60
d
55.3
23.0
59.7
59.1
7.20
8.90
3.50
45.8
44.5
9.70
53.8
100
d
39.1
6.40
48.6
100
d
10
11
12
13
14
15
16
23.9
46.0
3.80
11.7
d
12.4
50.0
4.81
13.2
d
11.4
4.60
43.2
d
d
24.0
14.5
2.80
42.4
a
Data were evaluated by analysis of the variance; the probability of single differences was calculated at 5% level; all data were statistically significant at this level.
Non-reported data indicated as ‘‘d’’ correspond to no significant growth inhibition.
b
The obtained products are the methylated derivatives of natural
4. Conclusions
bioactive compounds. In fact, 5,6,7-trimethoxyflavone 17 is the
trimethyl ether of baicalein, the main component of a traditional
Chinese herbal medicine Scutellaria baicalensis having multiple
biological activities including antiallergic, anticarcinogenic and
anti-HIV properties.29 Recent studies report that this compound
has an important role in the cure of Parkinson’s disease inhibiting
New cis- and trans-3-hydroxy-2-methoxyflavanones have been
obtained by the oxidation of the flavone double bond with urea–
hydrogen peroxide (UHP)/methyltrioxorhenium (CH3ReO3, MTO)
catalytic system in methanol as nucleophilic solvent. After acetyla-
tion of the reaction products and chromatographic purification, the
corresponding cis- and trans-3-acetoxy-2-methoxyflavanones were
isolated. These compounds have been tested as antifungal agents
against three fungal strains of common saprotrophic soil and seed
fungi, such as T. koningii, F. solani and C. herbarum, potentially
pathogenic for humans. A structure–activity relationship showed
that the introduced chemical modification influences the antifungal
activity. In fact, the reaction products were generally more active
than the corresponding starting materials (commercial flavones)
and the structural analogues (commercial flavanones). Moreover,
the relative stereochemistry of the 2-methoxy and 3-acetoxy groups
was an important factor. In fact, the cis isomers showed higher ac-
tivity than the corresponding trans isomers. In particular, the cis
derivatives 3c, 7c and 13c possessed the highest activity against all
three fungal strains tested, even at low concentration (0.5ꢀ10ꢁ4 M).
the fibril formation of
fibrils;30 5,7,8-trimethoxyflavone 18 is the 5,7-dimethyl ether of
wogonin,31
component of pharmaceutical preparations for
a-synuclein or disaggregating its existing
a
treatment of gastrointestinal diseases, regeneration of nerve and
neural stem cells and antiinflammatory agents.32
3. Antifungal activities
The new cis- and trans-3-acetoxy-2-methoxyflavanones 3c–3d,
5c–5d, 7c–7d, 9c–9d and 13c–13d were screened in vitro for their
antifungal activity against three fungal strains of common sapro-
27
trophic soil and seed fungi,
such as T. koningii, F. solani and C.
herbarum, potentially pathogenic for humans.28 The antifungal
activity of the most active compounds 3c, 7c and 13c was compared
with those of the corresponding starting materials (flavones 1, 6
and 12) and of the structurally analogous flavanones 20–22 (Fig. 2).
All new compounds were tested at three different concentrations
(0.5, 2.0 and 8.0ꢀ10ꢁ4 M). As reported in Table 2, a significant an-
tifungal activity against the three fungal strains has been observed
for all tested compounds. The only exception is flavanone 7d.
According to Weidenborner et al. and Silva et al.,4 no linear
relationship between the concentration and the antifungal activity
has been observed for some tested compounds. As shown, all new
cis derivatives are more active than the corresponding trans de-
rivatives (compare entry 2 with entry 1; entry 6 with entry 5; entry
10 with entry 9; entry 14 with entry 13; entry 16 with entry 15).
Between them, the cis derivatives 3c, 7c and 13c are the most active
compounds against the three fungal strains tested. In particular,
also at low concentration (0.5ꢀ10ꢁ4 M) compounds 7c and 13c
totally inhibited the growth of T. koningii and C. herbarum (see
entries 6 and 10). Their activity was compared with those of the
corresponding starting materials such as flavones 1, 6 and 12 and of
the structurally analogous flavanones 20–22 (Fig. 2). As reported in
Table 2, the antifungal activities of flavones 1, 6 and 12 against T.
koningii, F. solani and C. herbarum were similar to flavanones 20–22
(compare entry 1 with entry 14; entry 6 with entry 15; entry 12
with entry 22). These activities appeared to increase in the new cis-
synthesized flavanones 3c, 7c and 13c.
5. Experimental
5.1. General
All reagents were of the highest grade available and were used
as such (Sigma–Aldrich–Fluka). Chromatographic purifications
were performed on columns packed with Merck silica gel 60, 230–
400 mesh. Thin layer chromatography was carried out using Merck
platen Kieselgel 60 F254. 1H and 13C NMR spectra were recorded on
a Bruker 200 MHz. Mass spectra were recorded with a GC Shimadzu
GC-17A equipped with an electron beam of 70 eV, an SPB column
(25 mꢀ0.30 mm and 0.25 mm film thickness) and a mass-selective
detector QP 6000. The injector temperature was 280 ꢂC. An iso-
thermal temperature profile of 60 ꢂC for 5 min, followed by a 10 ꢂC/
min temperature gradient to 250 ꢂC for 10 min was used. Chro-
matographic grade helium was used as the carrier gas.
5.2. Oxidation of flavones: general procedure
A 10 ml reaction flask was charged with methyltrioxorhenium
(0.05 mmol) and urea–hydrogen peroxide (2.0 mmol) in methanol
(5 ml). The stirred solution became yellow due to the formation of
peroxo complexes and after 5 min the flavone derivative (0.5 mmol)

R. Bernini et al. / Tetrahedron 64 (2008) 7561–7566
7565
was added. The mixture was stirred at reflux for 8–24 h. The oxi-
dationwas monitored by TLC and GC–MS analysis. Depending on the
substrate, successive equivalents of urea–hydrogen peroxide were
added. At the end of the reaction, the solvent was removed under
reduced pressure; the residue was extracted with dichloromethane
(3ꢀ10 ml), dried over Na2SO4 and concentrated to affordayellowoil.
The reaction mixture was dissolved in dry pyridine (1 ml) and then
a large excess of acetic anhydride (20 mmol) was added dropwise.
The mixture was stirred at room temperature overnight. At the end
of the reaction, the solution was neutralized with a solution of 1 M
HCl and then extracted with ethyl acetate (3ꢀ10 ml). After washing
with a saturated solution of NaCl, the crude was dried over Na2SO4.
The solvent was removed under reduced pressure to afford a yellow
oil. The residue was purified by preparative TLC or by flash chro-
matography on silica gel using dichloromethane/methanol or ethyl
acetate/hexane mixtures as eluents. Products were identified by 1H
NMR, 13C NMR, GC–MS and elemental analyses.
134.8 (C), 150.6 (C), 154.8 (C), 167.9 (C), 187.2 (C]O). m/z (EI) 342
(Mþ). Anal. Calcd for C19H18O6 (342.35): C, 66.66; H, 5.30; O, 28.04.
Found: C, 66.62; H, 5.28; O, 28.10.
5.2.6. trans-3-Acetoxy-2,6-dimethoxyflavanone (7d)
Colourless oil. 1H NMR,
d
(ppm): 1.74 (s, 3H, CH3), 3.06 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 5.46 (m, 1H, H-3), 7.07–7.18 (m, 2H, PhH),
7.35–7.42 (m, 4H, PhH), 7.60–7.64 (m, 2H, PhH). 13C NMR,
(ppm):
d
20.1 (CH3), 50.2 (OCH3), 55.8 (OCH3), 71.9 (CH),104.7 (C),108.0 (CH),
119.4 (CH), 119.7 (C), 125.5 (CH), 127.1 (CH), 128.3 (CH), 129.4 (CH),
134.9 (C), 151.2 (C), 154.8 (C), 167.9 (C), 187.2 (C]O). m/z (EI) 342
(Mþ). Anal. Calcd for C19H18O6 (342.35): C, 66.66; H, 5.30; O, 28.04.
Found: C, 66.60; H, 5.24; O, 28.16.
5.2.7. cis-3-Acetoxy-2-methoxy-6-methylflavanone (9c)
Yellow oil. 1H NMR,
d
(ppm): 2.09 (s, 3H, CH3), 2.32 (s, 3H, CH3),
3.10 (s, 3H, OCH3), 5.74 (m, 1H, H-3), 7.04 (d, 1H, J¼8.3 Hz, PhH),
7.37–7.44 (m, 4H, PhH), 7.64–7.69 (m, 3H, PhH). 13C NMR,
(ppm):
Spectroscopic and analytical data of these compounds are given
below.
d
20.1 (CH3), 20.3 (CH3), 51.1 (OCH3), 77.6 (CH), 106.0 (C), 114.0 (C),
117.7 (CH), 120.3 (C), 126.5 (CH), 127.1 (CH), 128.3 (CH), 129.4 (CH),
132.0 (C), 137.5 (CH), 154.3 (C), 169.3 (C), 187.4 (C]O). m/z (EI) 326
(Mþ). Anal. Calcd for C19H18O5 (326.35): C, 69.93; H, 5.56; O, 24.51.
Found: C, 69.99; H, 5.50; O, 24.51.
5.2.1. cis-3-Acetoxy-2-methoxyflavanone (3c)
Yellow oil. 1H NMR,
d
(ppm): 2.10 (s, 3H, OCOCH3), 3.11 (s, 3H,
OCH3), 5.77 (s, 1H, H-3), 7.13–7.18 (m, 2H, PhH), 7.42–7.45 (m, 3H,
PhH), 7.59–7.70 (m, 3H, PhH), 7.89 (m, 1H, PhH). 13C NMR,
(ppm):
d
20.4 (CH3), 51.2 (OCH3), 77.4 (CH), 106.2 (C), 118.0 (CH), 120.7 (C),
122.6 (CH), 126.8 (CH), 128.3 (CH), 128.7 (CH), 129.5 (CH), 134.7 (C),
136.4 (CH), 156.4 (C), 169.2 (C), 187.2 (C]O). m/z (EI) 270 (Mþꢁ42).
Anal. Calcd for C18H16O5 (312.32): C, 69.22; H, 5.17; O, 25.62. Found:
C, 69.24; H, 5.16; O, 25.60.
5.2.8. trans-3-Acetoxy-2-methoxy-6-methylflavanone (9d)
Colourless oil. 1H NMR,
d (ppm): 1.73 (s, 3H, CH3), 2.34 (s, 3H,
CH3), 3.06 (s, 3H, OCH3), 5.46 (s, 1H, H-3), 7.07 (d, 1H, J¼8.4 Hz,
PhH), 7.38–7.43 (m, 4H, PhH), 7.60–7.65 (m, 2H, PhH), 7.73 (s, 1H,
PhH). 13C NMR,
d (ppm): 20.1 (CH3), 20.4 (CH3), 50.2 (OCH3), 72.1
(CH), 104.7 (C), 117.9 (CH), 119.4 (C), 127.0 (CH), 128.3 (CH), 129.4
(CH), 132.0 (C), 134.9 (C), 137.6 (CH), 154.8 (C), 167.9 (C), 187.3
(C]O). m/z (EI) 326 (Mþ). Anal. Calcd for C19H18O5 (326.35): C,
69.93; H, 5.56; O, 24.51. Found: C, 69.90; H, 5.50; O, 24.60.
5.2.2. trans-3-Acetoxy-2-methoxyflavanone (3d)
Colourless oil. 1H NMR,
d (ppm): 1.74 (s, 3H, OCOCH3), 3.08 (s,
3H, OCH3), 5.48 (s, 1H, H-3), 7.09–7.20 (m, 2H, PhH), 7.39–7.43 (m,
3H, PhH), 7.60–7.66 (m, 3H, PhH), 7.90 (m, 1H, PhH). 13C NMR,
d
(ppm): 20.1 (CH3), 50.3 (OCH3), 72.1 (CH), 104.9 (C), 118.1 (CH),
5.2.9. cis-3-Acetoxy-2,7-dimethoxyflavanone (13c)
119.8 (C), 122.4 (CH), 127.0 (CH), 127.4 (CH), 128.4 (CH), 129.4 (CH),
134.8 (C), 136.6 (CH), 156.8 (C), 167.9 (C), 187.0 (C]O). m/z (EI) 270
(Mþꢁ42). Anal. Calcd for C18H16O5 (312.32): C, 69.22; H, 5.17; O,
25.62. Found: C, 69.18; H, 5.15; O, 25.67.
Yellow oil. 1H NMR (CDCl3/CD3OD),
d (ppm): 2.09 (s, 3H, CH3),
3.13 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.71 (s,1H, H-3), 6.60–6.71 (m,
2H, PhH), 7.38–7.44 (m, 3H, PhH), 7.63–7.68 (m, 2H, PhH), 7.83 (d,
1H, J¼8.6 Hz, PhH). 13C NMR (CDCl3/CD3OD),
d (ppm): 20.1 (CH3),
51.2 (OCH3), 55.8 (OCH3), 72.1 (CH), 101.8 (CH), 106.2 (C), 110.1 (CH),
114.3 (C), 127.1 (CH), 128.3 (CH), 129.2 (CH), 129.4 (CH), 134.9 (C),
158.4 (C), 166.4 (C), 169.3 (C), 185.7 (C]O). m/z (EI) 342 (Mþ). Anal.
Calcd for C19H18O6 (342.35): C, 66.66; H, 5.30; O, 28.04. Found: C,
66.62; H, 5.34; O, 28.04.
5.2.3. cis-3-Acetoxy-2,5-dimethoxyflavanone (5c)
Colourless oil. 1H NMR,
d
(ppm): 2.10 (s, 3H, CH3), 3.11 (s, 3H,
OCH3), 3.90 (s, 3H, OCH3), 5.62 (s, 1H, H-3), 6.68 (dd, 2H, PhH), 7.35–
7.50 (m, 4H, PhH), 7.62–7.68 (m, 2H, PhH). 13C NMR,
(ppm): 20.1
d
(CH3), 51.2 (OCH3), 56.2 (OCH3), 77.5 (CH), 105.3 (CH), 105.2 (C),
109.9 (CH), 115.9 (C), 127.0 (CH), 128.4 (CH), 129.5 (CH), 134.8 (C),
136.6 (CH), 158.4 (C), 161.2 (C), 169.4 (C), 184.7 (C]O). m/z (EI) 300
(Mþꢁ42). Anal. Calcd for C19H18O6 (342.35): C, 66.66; H, 5.30; O,
28.04. Found: C, 66.56; H, 5.30; O, 28.14.
5.2.10. trans-3-Acetoxy-2,7-dimethoxyflavanone (13d)
d (ppm): 1.72 (s, 3H, CH3), 3.09 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 5.45 (m,1H, H-3), 6.62–6.70 (m, 2H, PhH),
Colourless oil. 1H NMR,
7.36–7.46 (m, 3H, PhH), 7.57–7.63 (m, 2H, PhH), 7.86 (d,1H, J¼8.6 Hz,
PhH). 13C NMR,
d (ppm): 20.1 (CH3), 50.4 (OCH3), 55.7 (OCH3), 72.1
5.2.4. trans-3-Acetoxy-2,5-dimethoxyflavanone (5d)
(CH), 101.7 (CH), 105.2 (C), 110.3 (CH), 113.5 (C), 127.1 (CH), 128.3
(CH), 129.2 (CH), 129.4 (CH), 134.9 (C), 158.9 (C), 166.6 (C), 167.9 (C),
185.7 (C]O). m/z (EI) 342 (Mþ). Anal. Calcd for C19H18O6 (342.35): C,
66.66; H, 5.30; O, 28.04. Found: C, 66.60; H, 5.26; O, 28.14.
Colourless oil. 1H NMR,
d
(ppm): 1.74 (s, 3H, CH3), 3.08 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 5.41 (s, 1H, H-3), 6.77 (dd, 2H, PhH), 7.35–
7.56 (m, 4H, PhH), 7.58–7.68 (m, 2H, PhH). 13C NMR,
(ppm): 20.1
d
(CH3), 50.4 (OCH3), 56.1 (OCH3), 73.4 (CH), 104.4 (C), 105.0 (CH),
110.0 (CH), 113.3 (C), 127.0 (CH), 128.3 (CH), 129.3 (CH), 134.9 (C),
136.4 (CH), 158.5 (C), 161.0 (C), 167.9 (C), 185.6 (C]O). m/z (EI) 300
(Mþꢁ42). Anal. Calcd for C19H18O6 (342.35): C, 66.66; H, 5.30; O,
28.04. Found: C, 66.60; H, 5.32; O, 28.08.
5.2.11. Mixture (1/1) of 6-hydroxy-5,7-dimethoxyflavone (15) and
8-hydroxy-5,7-dimethoxyflavone (16)
Yellow oil. 1H NMR (CDCl3/CD3OD),
d (ppm): 3.85 (s, 6H, 2OCH3),
3.93 (s, 6H, 2OCH3), 6.39 (s, 1H, H-6, compound 16), 6.58 (s, 1H, H-3,
compound 16), 6.62 (s, 1H, H-8, compound 15), 6.78 (s, 1H, H-3,
5.2.5. cis-3-Acetoxy-2,6-dimethoxyflavanone (7c)
compound 15), 7.38–7.46 (m, 6H, PhH), 7.79–7.90 (m, 4H, PhH). 13
C
Colourless oil. 1H NMR,
d
(ppm): 2.09 (s, 3H, CH3), 3.09 (s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 5.75 (m, 1H, C-3), 7.04–7.19 (m, 2H, PhH),
7.32–7.45 (m, 4H, PhH), 7.64–7.69 (m, 2H, PhH). 13C NMR,
(ppm):
NMR (CDCl3/CD3OD), d (ppm): 56.2 (OCH3), 56.4 (OCH3), 62.1 (OCH3),
92.5 (CH), 96.1 (CH), 107.7 (CH), 108.6 (C), 112.1 (C), 125.9 (CH), 126.1
(CH), 128.0 (C), 128.9 (CH), 131.2 (CH), 137.4 (C), 144.0 (C), 146.5 (C),
150.7 (C), 151.8 (C), 152.9 (C), 153.2 (C), 161.1 (C), 161.7 (C), 177.7
(C]O), 178.5 (C]O).
d
20.1 (CH3), 51.1 (OCH3), 55.8 (OCH3), 77.5 (CH), 106.1 (C), 107.9 (CH),
119.4 (CH), 120.7 (C), 124.8 (C), 127.1 (CH), 128.3 (CH), 129.4 (CH),

7566
R. Bernini et al. / Tetrahedron 64 (2008) 7561–7566
5.2.12. 5,8-Dihydroxy-7-methoxyflavone (17)
References and notes
Yellow oil. 1H NMR (CDCl3/CD3OD),
d
(ppm): 3.90 (s, 3H, OCH3),
6.39 (s, 1H, H-6), 6.58 (s, 1H, H-3), 7.36–7.47 (m, 3H, PhH), 7.88–7.93
(m, 2H, PhH). 13C NMR,
(ppm): 56.0 (CH3), 93.3 (CH), 101.9 (C),
1. (a) Middleton, E., Jr.; Kandaswami, C. The Flavonoids: Advances in Research Since
1986; Harborne, J. B., Ed.; Chapman and Hall: London, 1993; pp 619–652; (b)
Hackett, A. M. In Plant Flavonoids in Biology and Medicine: Biochemical, Phar-
macological and Structure–Activity Relationship; Cody, V., Middleton, E., Har-
borne, J. B., Eds.; Liss, A.: New York, NY, 1986; pp 177–194.
2. (a) Brouillard, R.; Dangles, O. The Flavonoids: Advances in Research Since 1986;
Harborne, J. H., Ed.; Chapman and Hall: London, 1993; pp 565–588; (b) Har-
borne, J. B.; Grayer, R. J. The Flavonoids: Advances in Research Since 1986; Har-
borne, J. H., Ed.; Chapman and Hall: London, 1993; pp 589–618; (c) Harborne, J.
B.; Williams, C. A. Phytochemistry 2000, 55, 481–504 and references therein.
3. Weidenboerner, M.; Jha, H. C. Pestic. Sci. 1993, 38, 347–351.
4. (a) Wedenborner, M.; Jha, H. C. Mycol. Res. 1997, 101, 733–736; (b) Silva, A. M. S.;
Weidenborner, M.; Cavaleiro, J. A. S. Mycol. Res. 1998, 102, 638–640.
5. (a) Donnelly, J. A.; Keegan, J. R.; Quigley, K. Tetrahedron 1980, 36, 1671–1680; (b)
Bernini, R.; Mincione, E.; Coratti, A.; Fabrizi, G. Tetrahedron 2004, 60, 967–971.
6. (a) Kurasawa, K.; Ashihara, Y. Bull. Chem. Soc. Jpn. 1978, 51, 1175–1177; (b)
Ashihara, Y.; Nagata, Y.; Kurosawa, K. Bull. Chem. Soc. Jpn. 1977, 50, 3298–3301.
7. Evans, D. L.; Minster, D. K.; Jordis, U.; Hecht, S. M.; Mazzur, A. L.; Meyers, A. I.
J. Org. Chem. 1979, 44, 497–501.
8. Nadkami, D. R.; Wheeler, T. S. J. Chem. Soc. 1938, 1320–1322.
9. Singh, O. V.; Kapoor, R. P. Tetrahedron Lett. 1990, 31, 1459–1462.
10. Adam, W.; Golsch, D.; Hadjiarapoglou, L. J. Org. Chem. 1991, 56, 7292–7297.
11. Yang, D.; Wong, M.-K.; Yip, Y.-C. J. Org. Chem. 1995, 60, 3887–3889.
12. (a) Beattie, R.; Jones, P. J. Inorg. Chem. 1979, 18, 2318–2319; (b) Herrmann, W. A.;
Kuhn, F. E.; Fischer, R. W.; Thiel, W. R.; Romao, C. C. Inorg. Chem. 1992, 31, 4431–
4432; (c) Herrmann, W. A.; Kuhn, F. E. Acc. Chem. Res. 1997, 30, 169–180; (d)
Romao, C. C.; Kuhn, F. E.; Herrmann, W. A. Chem. Rev. 1997, 97, 3197–3246.
13. Heaney, H. Top. Curr. Chem. 1993, 164, 1–19.
d
105.1 (CH), 126.3 (CH), 129.1 (CH), 130.0 (C), 131.9 (CH), 151.4 (C),
156.7 (C), 158.2 (C), 162.9 (C), 181.7 (C]O). m/z (EI) 284 (Mþ). Anal.
Calcd for C16H12O5 (284.27): C, 67.60; H, 4.26; O, 28.14. Found: C,
67.50; H, 4.28; O, 28.22.
5.2.13. 5,6,7-Trimethoxyflavone (Baicalein trimethyl ether 18)
Yellow oil. 1H NMR (CDCl3),
d (ppm): 3.89 (3H, s, OCH3), 3.96 (3H,
s, OCH3), 3.97 (3H, s, OCH3), 6.64 (s, 1H, H-8), 6.79 (s, 1H, H-3), 7.45–
7.52 (m, 3H, PhH), 7.82–7.87 (m, 2H, PhH). 13C NMR (CDCl3),
d
(ppm): 56.2 (OCH3 in C-7), 61.2 (OCH3 in C-6), 61.4 (OCH3 in C-5),
96.2 (CH), 108.3 (CH), 112.9 (C), 125.9 (CH), 128.9 (CH), 131.2 (CH),
131.5 (C), 140.3 (C), 152.5 (C), 154.5 (C), 157.7 (C), 161.0 (C), 177.1
(C]O). m/z (EI) 312 (Mþ). Anal. Calcd for C18H16O5 (312.32): C,
69.22; H, 5.16; O, 25.62. Found: C, 69.18; H, 5.14; O, 25.68.
5.2.14. 5,7,8-Trimethoxyflavone (Wogonin dimethyl ether 19)
Yellow oil. 1H NMR (CDCl3),
d (ppm): 3.93 (3H, s, OCH3), 3.96 (3H,
s, OCH3), 3.98 (3H, s, OCH3), 6.42 (s, 1H, H-6), 6.67 (s, 1H, H-3), 7.45–
7.52 (m, 3H, PhH), 7.88–7.93 (m, 2H, PhH). 13C NMR (CDCl3),
14. (a) Herrmann, W. A.; Ding, H.; Krtazer, R. M.; Kuhn, F. E.; hiader, j. J.; Fischer, R.
W. J. Organomet. Chem. 1997, 549, 319–322; (b) Rudolph, J.; Reddy, L.; Chiang, J.
P.; Sharpless, K. B. J. Am. Chem. Soc. 1997, 119, 6189–6190.
15. Tan, H.; Espenson, J. H. Inorg. Chem. 1998, 37, 467–472; Gregorio, J. R.; Gerbase,
A. E.; Martinelli, M.; Jacobi, M. A. M.; De Luca Freitas, L. V.; Holleben, M. L. A.;
Marcico, P. D. Macromol. Rapid Commun. 2000, 21, 401–405.
d
(ppm): 56.2 (OCH3 in C-5), 56.5 (OCH3 in C-7), 61.5 (OCH3 in C-8),
92.6 (CH), 108.3 (CH), 109.1 (C), 125.9 (CH), 128.9 (CH), 130.8 (C),
131.2 (CH), 152.0 (C), 156.3 (C), 156.6 (C), 160.6 (C), 177.8 (C]O); m/z
(EI) 312 (Mþ). Anal. Calcd for C18H16O5 (312.32): C, 69.22; H, 5.16; O,
25.62. Found: C, 69.25; H, 5.12; O, 25.63.
16. Zhu, Z.; Espenson, J. H. J. Org. Chem. 1995, 60, 7728–7732.
17. (a) Adam, W.; HerrmannLin, J.; Saha-Moller, C. R.; Fischer, R. W.; Correia, J. D. G.
Angew. Chem., Int. Ed. Engl. 1994, 33, 2475–2477; (b) Adam, W.; Herrmann, W.
A.; Lin, J.; Saha-Moller, C. R. J. Org. Chem. 1994, 59, 8281–8283; (c) Herrmann,
W. A.; Haider, J. J.; Fischer, R. W. J. Mol. Catal. 1999, 138, 115–121.
18. Adam, W.; Mitchell, C. M.; Saha-Moller, C. R. Tetrahedron 1994, 50, 13121–13124.
19. Zhu, Z.; Espenson, J. H. Synthesis 1998, 417–420.
5.3. Assay of antifungal activity of flavanones derivatives
Before testing, compoundsweredissolvedin100%acetonesothat
the final concentration of solvent in the test medium did not exceed
1% of the total solution composition. All compounds were used in
three different concentrations: 0.5, 2.0 and 8.0ꢀ10ꢁ4 M. All solutions
were prepared immediately before testing. T. koningii, F. solani and C.
herbarum was used for the antifungal screening test. Prior to testing,
each isolate was subcultured on MEA (DIFCO) to ensure optimal
growth characteristics andpurity. Theisolates had been grown for 4–
14 days, on MEA at 25 ꢂC. Conidia suspensions were prepared in
sterilewatersupplementedwith0.01%ofTween80. Eachsuspension
was diluted to obtain the final inoculum, which ranged from 0.5ꢀ104
to 1.0ꢀ104 CFU/ml. The inoculum size was determined microscopi-
20. Eager, M. D.; Espenson, J. H. Inorg. Chem. 1999, 38, 2533–2535.
21. (a) Murray, R. W.; Iyanar, K.; Chen, J. T.; Wearing, J. T. Tetrahedron Lett. 1995, 36,
6415–6418; (b) Adam, W.; Mitchell, C. M.; Saha-Moller, C. R.; Weichold, O. J. Am.
Chem. Soc. 1999, 121, 2097–2113; (c) Stankovic, S.; Espenson, J. J. J. Org. Chem.
2000, 65, 5528–5530.
22. Herrmann, W. A.; Fischer, R. W.; Correia, J. D. G. J. Mol. Catal. 1994, 36, 6415–
6418.
23. Herrmann, W. A.; Fischer, R. W.; Scherer, W.; Rouch, M. U. Angew. Chem., Int. Ed.
Engl. 1993, 32, 1157–1160.
24. (a) Saladino, R.; Neri, V.; Mincione, E.; Marini, S.; Coletta, M.; Fiorucci, C.; Fil-
ippone, P. J. Chem. Soc., Perkin Trans. 1 2000, 4, 581–586; Saladino, R.; Neri, V.;
Mincione, E.; Filippone, P. Tetrahedron 2002, 58, 8493–8500; (b) Saladino, R.;
Neri, V.; Pelliccia, A. R.; Mincione, E. Tetrahedron 2003, 59, 7403–7408; (c)
Saladino, R.; Mincione, E.; Attanasi, O. A.; Filippone, P. Pure Appl. Chem. 2003,
75, 265–272; (d) Bernini, R.; Coratti, A.; Provenzano, G.; Fabrizi, G.; Tofani, D.
Tetrahedron 2005, 61, 1821–1825; (e) Bernini, R.; Mincione, E.; Barontini, M.;
Fabrizi, G.; Pasqualetti, M.; Tempesta, S. Tetrahedron 2006, 62, 7733–7737.
25. (a) Bernini, R.; Mincione, E.; Cortese, M.; Aliotta, G.; Oliva, A.; Saladino, R.
Tetrahedron Lett. 2001, 42, 5401–5404; (b) Bernini, R.; Mincione, E.; Cortese, M.;
Saladino, R.; Gualandi, G.; Belfiore, M. C. Tetrahedron Lett. 2003, 44, 4823–4825;
(c) Bernini, R.; Coratti, A.; Fabrizi, G.; Goggiamani, A. Tetrahedron Lett. 2003, 44,
8991–8994.
cally using Bu¨rker’s chamber and verified by plating 100 mL of serial
dilutions of each inoculum onto an MEA plate and incubation until
growth became visible. Each Petri dishes (90 mm) containing
12 ml of the medium including the products in required concentra-
tions (added to the agar at temperature below 50 ꢂC) was inoculated
26. Bernini, R.; Mincione, E.; Provenzano, G.; Fabrizi, G. Tetrahedron Lett. 2005, 46,
2993–2996.
with 2 ml of the inoculum suspensions; 5–6 replications for each
concentration and fungus were made. The Petri dishes were in-
cubated at 25 ꢂC in the dark to a clearly visible growth on drug-free
control. Evaluation of linear growth was conducted by measuring
mycelial diameters of each inoculated plate at broadest, medium and
smallest diameter, and compared with the corresponding control.
The inhibition (%) of linear mycelial mean growth of T. koningi, C.
herbarum and F. solani was calculated after incubation for 3, 5 and 6
days, respectively. The data were evaluated by analysis of variance
and probability of single differences was calculated at the 5% level.
27. Watanabe, T. Pictorial Atlas of Soil and Seed Fungi, 2nd ed.; CRC: Tsukuba city,
Japan, 2002; pp 1–484.
28. Hoog, G. S.; deGuarro, J.; Gene´, J.; Figueras, M. J. Atlas of Clinical Fungi, 2nd ed.;
Centraalbureau voor Schimmelcultures: Utrecht, The Netherlands, 2000;
pp 1–1126.
29. (a) Tomas-Barberan, F. A.; Msonthi, J. D.; Hostettmann, K. Phytochemistry 1988,
27, 753–755; (b) Inuma, M.; Matsuura, S.; Kusuda, K. Chem. Pharm. Bull. 1980,
28, 708–716.
30. (a) Li, B. Q.; Gong, W. H.; Dunlop, N.; Kung, H.; Yan, Y.; Kang, J.; Wang, J. M.
Immunopharmacology 2000, 49, 295–306; (b) Wu, J. A.; Attele, A. S.; Zhang, L.;
Yuan, C. S. Am. J. Chin. Med. 2001, 29, 69–81; (c) Ikezoe, T.; Chen, S. S.; Heber, D.;
Tagushi, H.; Koeffler, H. P. Prostate 2001, 49, 285–292; (d) Gao, Z.; Huang, K.; Xu,
H. Pharm. Res. 2001, 43, 173–178; (e) Shieh, D. E.; Liu, L. T.; Lin, C. C. Anticancer
Res. 2000, 20 2861–2685.
31. Zhu, M.; Rajamani, S.; Kaylor, J.; Han, S.; Zhou, F.; Fink, A. L. J. Biol. Chem. 2004,
279, 26846–26857.
Acknowledgements
32. (a) Ryo Wom, C.; Soo Jin, P. KR patent 2005039189, 2005; (b) Byung Kil, C.; Dae
Seong, P. KR Patent 2004013997, 2004; (c) Jinhee, J.; Kwan-Seog, S.; Hyun Pyo,
K.; Haeil, P. Arch. Pharm. Res. 2005, 28, 877–884.
Italian Project ‘Valorolio’ of the Ministero delle Politiche Agri-
cole e Forestali (MIPAF) is acknowledged for the financial support.