Ecofriendly synthesis of halogenated flavonoids and evaluation of their antifungal activity

Article abstract of DOI:10.1039/c5nj00258c

Brominated and chlorinated flavonoids belonging to different classes (flavanones, flavones and catechins) were prepared from the corresponding flavonoids by a simple and ecofriendly procedure based on the use of sodium halides, aqueous hydrogen peroxide and acetic acid. Pure samples of substrates and products were tested against Trichoderma koningii, Fusarium oxysporum and Cladosporium cladosporioides, common saprotrophic soil and seed fungi, potential pathogens for humans and their activity was expressed as linear mycelial growth inhibition (%). Among them, 8-chloro-5,7,3′,4′-tetramethoxyepicatechin 29, a novel catechin derivative, exhibited a remarkable effect against all tested fungi also at low concentrations.

Full text of DOI:10.1039/c5nj00258c

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Ecofriendly synthesis of halogenated flavonoids
and evaluation of their antifungal activity
Cite this:DOI: 10.1039/c5nj00258c
Roberta Bernini,*a Marcella Pasqualetti,b Gianfranco Provenzanoa and
Sabrina Tempestab
Brominated and chlorinated flavonoids belonging to different classes (flavanones, flavones and catechins)
were prepared from the corresponding flavonoids by a simple and ecofriendly procedure based on the
use of sodium halides, aqueous hydrogen peroxide and acetic acid. Pure samples of substrates and
products were tested against Trichoderma koningii, Fusarium oxysporum and Cladosporium
cladosporioides, common saprotrophic soil and seed fungi, potential pathogens for humans and their
activity was expressed as linear mycelial growth inhibition (%). Among them, 8-chloro-5,7,30,40-
tetramethoxyepicatechin 29, a novel catechin derivative, exhibited a remarkable effect against all tested
fungi also at low concentrations.
Received (in Montpellier, France)
29th January 2015,
Accepted 2nd February 2015
DOI: 10.1039/c5nj00258c
www.rsc.org/njc
Introduction
Flavonoids are more than 5000 plant secondary metabolites
involved in important biological processes such as nitrogen
fixation, photosensitization, energy transfer, plant growth,
control of respiration, and photosynthesis.1,2 In flowers they
provide colors attractive to plant pollinators; in leaves they
promote physiological survival of the plant, protecting it from
pathogens and UV-B radiation. Flavonoids are found in fruits,
vegetables, propolis, honey, tea, and wine and are of interest
to human health because of their beneficial biological effects
such as anti-inflammatory, antioxidant, antiviral, antibacterial,
anticancer, and antimicrobial activities.3–5
Fig. 1 Basic structure of (a) flavonoids; (b) flavanones; (c) flavones and (d)
flavan-3-ols.
Structurally, these compounds possess a common phenyl-
benzopyrone nucleus (C6–C3–C6) consisting of two aromatic
rings (A and B) linked by an oxygenated central pyranic ring (C) have shown a remarkable antifungal activity against some
and are categorized according to the saturation level of the human pathogens e.g Trichophyton longifusus, Candida albicans,
central ring and the presence of carbonylic or alcoholic func- Aspergillus flavus, Microsporium canis, Fusarium solanii and
tionality. Among them, flavanones, flavones, and flavan-3-ols Candida glaberata even if a structure–activity relationship has
(catechins) are widely diffused in food and beverages (Fig. 1).
The introduction of one or more halogen atoms into the A or
not been described.12–14
Generally, the halogenation of aromatic compounds is
B-ring of flavonoids produced the corresponding halogenated carried out using molecular halogens in chlorinated solvents.
derivatives useful as synthetic intermediates.6–9 In addition, halo- Even if efficient, this procedure has several environmental
gen atoms confer to flavonoids additional biological properties.10,11 drawbacks due to the toxic and hazardous nature of reagents
For example, a series of mono- and di-halogenated flavonoids (chlorine gas and liquid bromine), and solvents. In addition,
pollutant wastes were produced as by-products.
In order to overcome these problems, in the last few years
several papers have reported green procedures based on the
a Dipartimento di Scienze e Tecnologie per l’Agricoltura, le Foreste, la Natura e
`
generation of the electrophilic reagent X+ (e.g. Br+, Cl+) by an
ecofriendly oxidation of halide ions. The most used benign
oxidants are dimethyldioxirane (DMD), potassium peroxymono-
sulfate (Oxones) and hydrogen peroxide (H2O2). Although DMD
l’Energia (DAFNE), Universita degli Studi della Tuscia, Via S. Camillo De Lellis,
01100 Viterbo, Italy. E-mail: berninir@unitus.it; Fax: +39 0761 357242;
Tel: +39 0761 357452
b
`
Dipartimento di Scienze Ecologiche e Biologiche (DEB), Universita degli Studi della
`
Tuscia, Largo dell’Universita, 01100 Viterbo, Italy
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is an efficient oxidizing agent and its preparation is an easy for humans, e.g. Trichoderma koningii, Fusarium oxysporum, and
and safe operation, Oxones is more readily accessible being Cladosporium cladosporioides.26–28
commercially available and H2O2 appears as the best candidate
producing water as the only by-product.15,16 In combination
Results and discussion
Synthesis of halogenated flavonoids
with hydrochloric acid or sodium halides, DMD and Oxones
were very efficient for the halogenation of phenols, methoxy-
benzenes17–19 and flavonoids;20,21 similarly, the selective halo-
genation of phenols and methoxybenzene derivatives was carried
out providing the products in good yields using H2O2 in the
presence of catalysts or in acetic acid.22,23 To the best of our
knowledge, no papers on the halogenation of flavonoids under
similar conditions have been published.
We firstly investigated the efficiency of the selected halogenating
system on flavanone 1, 5-methoxyflavanone 4, 7-methoxyflavanone
8, 5,7,40-trimethoxyflavanone (methylated naringenin) 12 and
5,7,30,40-tetramethoxyflavanone (methylated hesperetin) 15
(Scheme 1). All experimental data are reported in Table 1. In
acetic acid, in the presence of NaX (X = Br, Cl) and H2O2 (30%
water solution), flavanone 1 was converted into the corre-
sponding 6-halogenated derivatives 2 and 3 in 70% and 62%
yields (Table 1, entries 1 and 3). According to our previous
results,20 flavanone 1 was regioselectively brominated and chlori-
nated at position C-6 activated by the para ethereal oxygen of the
heterocylic C-ring. In our hands, the bromination and chlorina-
tion of flavanone 1 failed in ethanol (Table 1, entries 2 and 4).
As part of our research on the chemical modifications of
natural phenols to obtain bioactive compounds by ecofriendly
procedures,24,25 we prepared several halogenated flavonoids
using a simple and environmentally benign way based on the
use of sodium halides, aqueous solution of hydrogen peroxide
and acetic acid as a solvent. Final compounds were tested against
common saprotrophic soil and seed fungi, potential pathogens
Scheme 1 Halogenation of flavanones 1, 4, 8, 12 and 15.
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These results confirmed the crucial role of acetic acid in promot-
These data were appealing because the H2O2–NaX–CH3COOH
ing the formation of the electrophilic species X+ (Br+ and Cl+) system resulted in being efficient with different classes of flavo-
responsible for the attack of the aromatic A-ring of flavanone and noids producing the corresponding halogenated derivatives in
the success of halogenation.22 In the presence of H2O2–NaX– good to excellent yields. In addition, to the best of our knowledge,
CH3COOH, the reaction proceeded with good regioselectivity also catechin derivatives 22–25 and 27–30 are novel compounds.
with activated substrates; the combination of the ortho and para
Evaluation of the antifungal activity
effects of both the ethereal oxygen of the heterocylic C-ring and
methoxyl groups present in the A-ring was responsible for the Pure samples of flavonoids 4, 8, 12, 15, 18, 21, 26 and the
formation of the exclusive (or main) halogenated regioisomer. corresponding halogenated derivatives 5, 6, 9, 10, 11, 13, 14, 16,
The bromination of 5-methoxyflavanone 4 produced the corre- 17, 19, 20, 22, 23, 24, 25, 27, 28 and 29 were screened in vitro at
sponding 8-bromoderivative 5 in quantitative yield (Table 1, three different concentrations 0.5, 2.0 and 8.0 ꢀ 10ꢁ4 M 13,14,25
entry 5); chlorination gave 8-chloroderivative 6 as the main for their growth inhibitory activity against Trichoderma koningii,
product (72%) and 6-chloroderivative 7 as the secondary product Fusarium oxysporum and Cladosporium cladosporioides, common
(Table 1, entry 6). Similarly, 7-methoxyflavanone 8 afforded the saprotrophic soil and seed fungi, potential pathogens for
corresponding 6-bromoderivative 9 in quantitative yield (Table 1, humans.26–28 All experimental results, expressed as linear
entry 7); 6-chloroderivative 10 and 8-chloderivatives 11, respec- mycelial growth inhibition (%), are reported in Table 2 and
tively, in 70 and 18% yields (Table 1, entry 8). Finally, methylated grouped in the MDS plot depicted in Fig. 2. The MDS approach
naringenin 12 and methylated hesperetin 15 gave the corre- showed the main distribution trend of the flavonoids along the
sponding 8-bromo and 8-chloroderivatives 13 and 14, respec- first axis related to their antifungal activity that increases from
tively, in 95 and 85% yields; 16 and 17 in 88 and 76% yields right to left. 7-Methoxyflavanone 8 and 8-chloro-5,7,30,40-
(Table 1, entries 9–12). Compared to DMD/HCl and Oxones/NaX tetramethoxyepicatechin 29, located on the right side of the
systems, halogenated flavanones were obtained in better yields plot (group A), were the most active compounds against all
with the only exception of 6-bromo and 6-chloroflavanones 2 and species tested and the percentage of radial growth inhibition
3. The absence of 6,8-dihalogenated derivatives evidencing a was high also at a lower tested concentration (0.5 ꢀ 10ꢁ4 M)
better control of the regioselectivity of reaction is worth noting. against Trichoderma koningii (respectively, 49.4% and 51.5%,
On the basis of these experimental results, we extended the Table 2). 8-Bromo-5-methoxyflavanone 5, 6-bromo-7-methoxy-
use of the H2O2–NaX–CH3COOH system to biologically relevant flavanone 9, 8-chloro methylated naringenin 14, methylated
flavonoids such as 5,7-dimethoxyflavone (chrysin) 18 and hesperetin 15, 8-bromo methylated hesperetin 16, 8-chloro
catechin derivatives 21 and 26. As depicted in Scheme 2, methylated hesperetin 17, 8-bromo methylated chrysin 19,
compound 18 was converted into the corresponding 8-bromo 8-bromo-5,7,30,40-tetramethoxycatechin 22, 6,8-dibromo-5,7,30,40-
and 8-chloroderivatives 19 and 20 in 92 and 74% yields (Table 1, tetramethoxycatechin 23, 8-chloro-5,7,30,40-tetramethoxycatechin
entries 13 and 14) whereas 5,7,30,40-tetramethylated catechin 21 24, 6,8-dichloro-5,7,30,40-tetramethoxycatechin 25 and 6,8-
and 5,7,30,40-tetramethylepicatechin 26 resulted in being more dibromo-5,7,30,40-tetramethoxyepicatechin 28 (group D) showed
reactive producing, respectively, the corresponding 8-halogenated low antifungal activity against all tested fungi (mean growth
compound 22, 24, 27, 29 as main products and 6,8-derivatives 23, reduction o25%, Table 2). Flavonoids distributed in the central
25, 28, 30 as secondary products (Table 1, entries 15–18).
part of the plot showed a relatively high activity and could be
Table 1 Halogenation of flavonoids depicted in Schemes 1 and 2
Entry
Substratea
Experimental conditions
Producta (yield%)
1
2
3
4
5
6
7
8
1
1
1
1
4
4
8
8
12
12
15
15
18
18
21
21
26
26
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 24 h
H2O2 (3.0 eq.), EtOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 24 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (1.0 eq.), 40 1C, 48 h
H2O2 (6.0 eq.), EtOH (2.5 mL), NaCl (1.0 eq.), 40 1C, 48 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 4 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaCl (3.0 eq.), 25 1C, 24 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 4 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (6.0 eq.), 40 1C, 48 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 4 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (6.0 eq.), 25 1C, 8 h
H2O2 (1.0 eq.), AcOH (2.5 mL), NaBr (3.0 eq.), 25 1C, 4 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (6.0 eq.), 25 1C, 24 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 24 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (6.0 eq.), 40 1C, 24 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 4 h
H2O2 (6.0 eq.), AcOH (2.5 mL), NaCl (6.0 eq.), 40 1C, 4 h
H2O2 (3.0 eq.), AcOH (2.5 mL), NaBr (1.0 eq.), 25 1C, 4 h
H2O2 (6.0 eq.), AcOH (2.5 mL), AcOH, NaCl (10.0 eq.), 25 1C, 4 h
2: 70
2: —
3: 62
3: —
5: 98
6: 72; 7: 20
9: 95
10: 70; 11: 18
13: 95
14: 85
16: 88
17: 76
19: 92
20: 74
22: 70; 23: 22
24: 60 25: 18
27: 78; 28: 20
29: 62; 30: 10
9
10
11
12
13
14
15
16
17
18
a
Calculated after chromatographic purification.
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Scheme 2 Halogenation of flavonoids 18, 21 and 26.
Table 2 Antifungal activity of tested flavonoids expressed as inhibition (%) of linear mycelial growth; significant differences at P o 0.05; no significant
values are unreported
Trichoderma koningii
Fusarium oxysporum
Cladosporium cladosporioides
Flavonoid 8.0 ꢀ 10ꢁ4 M 2.0 ꢀ 10ꢁ4 M 0.5 ꢀ 10ꢁ4 M 8.0 ꢀ 10ꢁ4 M 2.0 ꢀ 10ꢁ4 M 0.5 ꢀ 10ꢁ4 M 8.0 ꢀ 10ꢁ4 M 2.0 ꢀ 10ꢁ4 M 0.5 ꢀ 10ꢁ4
M
4
5
6
8
45.3
15.1
48.4
82.2
21.9
48.9
43.3
74.6
70.4
22.5
8.9
19.5
15.9
52.9
23.6
80.2
38.2
23.7
20.0
32.8
29.3
70.2
68.7
25.4
73.2
43.0
16.3
25.0
70.7
13.1
34.6
30.2
61.5
20.8
15.6
10.7
—
41.1
21.3
23.2
49.4
5.0
31.3
28.3
18.4
10.3
—
6.9
4.9
5.9
34.5
—
32.4
22.8
21.0
6.7
6.5
6.8
29.6
19.2
22.3
55.8
11.1
28.6
24.4
60.0
27.5
20.4
15.2
12.3
13.9
27.0
17.1
41.8
16.4
22.8
40.6
—
28.4
15.1
20.4
49.7
6.9
21.4
16.3
12.5
27.1
6.7
24.4
12.3
14.1
14.9
15.5
9.8
10.8
8.3
18.2
—
14.9
9.6
6.8
47.1
21.2
26.5
70.3
7.8
38.3
23.9
17.4
24.1
7.8
36.8
15.0
23.1
49.6
14.8
32.0
25.1
18.6
9.0
—
7.3
8.6
—
21.5
—
18.9
17.1
4.5
8.6
5.6
25.6
13.2
17.4
26.4
10.5
20.7
5.8
10.2
—
—
—
4.7
—
16.5
—
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
35.4
18.9
52.8
18.7
20.7
12.5
21.7
17.4
23.7
10.2
26.5
18.2
7.1
26.0
4.2
4.5
10.2
22.3
5.9
6.7
16.9
8.1
—
59.5
21.1
48.1
48.4
5.3
17.4
17.5
23.8
12.5
66.9
29.0
72.2
28.2
12.6
47.7
14.1
16.5
10.8
13.3
—
13.8
11.3
—
5.1
—
5.7
—
6.8
—
6.5
—
19.7
9.3
—
—
—
36.3
32.5
6.2
48.9
40.2
—
21.5
27.3
—
5.9
17.6
—
22.4
22.8
51.5
43.7
39.3
55.2
47.4
19.3
separated in two main groups: group B including 8-chloro-5- active against all fungal species. Finally, methylated naringenin
methoxyflavanone 6, 8-chloro-7-methoxyflavanone 11 and 5,7,30,40- 12 resulted in being active only against Trichoderma koningii and
tetramethoxycatechin 21, active mainly against Trichoderma koningii; Fusarium oxysporum; 8-bromo methylated naringenin 13 and
group C including 5-methoxyflavanone 4, 6-chloro-7-methoxy- 5,7,30,40-tetramethoxyepicatechin 26 were active, respectively,
flavanone 10, methylated chrysin 18, 8-chloro-5,7-methylated against Trichoderma koningii and all fungi but only at higher
chrysin 20 and 8-bromo-5,7,30,40-tetramethoxyepicatechin 27, concentrations.
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Fig. 2 MDS analysis on fungal activity of tested compounds.
Conclusions
GC-17A equipped with an electron beam of 70 eV, a SPB column
(25 m ꢀ 0.30 mm and 0.25 mm film thickness) and a mass-
selective detector QP 6000. The injector temperature was
280 1C. An isothermal temperature profile of 60 1C for 5 min,
followed by a 10 1C minꢁ1 temperature gradient to 250 1C for
10 min, was used. Chromatographic grade helium was used as
the carrier gas. High Resolution Mass Spectrometry (HRMS)
analyses were performed using a Micromass Q-TOF micro Mass
Spectrometer (Waters).
A simple and environmentally benign bromination and chlori-
nation procedure based on the use of aqueous solutions of
hydrogen peroxide, sodium halides (NaBr and NaCl) and acetic
acid was applied for the first time to flavonoids to obtain the
corresponding halogenated derivatives, useful synthetic inter-
mediates and potentially bioactive compounds. The procedure
was efficient with different representative classes of flavonoids;
the halogenation proceeded with good regioselectivity induced
by a combination of the effects of both the ethereal oxygen of the
heterocylic C-ring and methoxyl groups present in the aromatic
A-ring. To the best of our knowledge, catechin derivatives 22–25
and 27–30 appear as novel compounds. Substrates and the
corresponding halogenated derivatives were tested against
Trichoderma koningii, Fusarium oxysporum, and Cladosporium
cladosporioides, common saprotrophic soil and seed fungi,
potential pathogens for humans. Although experimental data
did not evidence a structure–activity relationship, almost all
flavonoids showed an inhibitory activity; in addition the effect
of 8-chloro-5,7,30,40-tetramethoxyepicatechin 29 which resulted
in being active against all tested fungi also at low concentrations
is remarkable.
Methylation reactions
5,7,40-Trimethoxyflavanone (methylated naringenin) 12; 5,7,30,40-
tetramethoxyflavanone (methylated hesperetin) 15; 5,7,30,40-
tetramethylcatechin 21 and 5,7,30,40-tetramethylepicatechin 26
were prepared as already reported by us.29,30
5,7,40-Trimethoxyflavanone (methylated naringenin) (12)
Yield: 90%; colorless oil; spectroscopic data are according to
those reported in the literature.29
5,7,30,40-Tetramethoxyflavanone (methylated hesperetin) (15)
Yield: 88%; colorless oil; spectroscopic data are according to
those reported in the literature.29
Experimental section
Materials and methods
5,7,30,40-Tetramethoxycatechin (21)
All reagents and flavonoids were of the highest grade available
and used as such (Sigma-Aldrich, Milan, Italy). Chromato-
graphic purifications were performed on columns packed with
a Merck silica gel 60, 230–400 mesh. Thin layer chromatography
Yield: 92%; yellow oil; spectroscopic data are according to those
reported in the literature.30
5,7,30,40-Tetramethoxyepicatechin (26)
was carried out using Merck platen Kieselgel 60 F254. 1H and 13
C
NMR spectra were recorded on a Bruker 200 and 400 MHz. Mass Yield: 90%; yellow oil; spectroscopic data are according to those
spectra were recorded using a gas chromatograph Shimadzu reported in the literature.30
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Halogenation reactions
8-Bromo-5,7,30,40-tetramethoxyflavanone (16)
A 30% aqueous solution of hydrogen peroxide (3.0–6.0 eq.) was Yield: 88%; yellow oil; spectroscopic data are according to those
added to the solution of flavonoid (0.5 mmol) and sodium reported in the literature.31
halide (1.0–3.0 eq.) in acetic acid (2.5 mL); then, the mixture
8-Chloro-5,7,30,40-tetramethoxyflavanone (17)
was stirred at room temperature or 40 1C for 4–48 h depending
on the substrate. The reaction was monitored by thin layer
chromatography (TLC) and gas-mass chromatography (GC-MS).
At the end, the crude was treated with sodium thiosulfate and
extracted with ethyl acetate (3 ꢀ 10 mL). The reunited organic
fractions were dried over anhydrous sodium sulfate; after
filtration, the solvent was evaporated under reduced pressure.
Final products were isolated and purified by chromatographic
column using CH2Cl2/CH3OH = 9.5/05 as an eluent and charac-
Yield: 76%; colorless oil; spectroscopic data are according to
those reported in the literature.31
8-Bromo-5,7-dimethoxyflavone (19)
Yield: 92%; colorless oil; spectroscopic data are according to
those reported in the literature.32
8-Chloro-5,7-dimethoxyflavone (20)
1
terized by H NMR, 13C NMR, and HR-MS analysis.
Yield: 74%; colorless oil; spectroscopic data are according to
those reported in the literature.32
6-Bromoflavanone (2)
8-Bromo-5,7,30,40-tetramethoxycatechin (22)
Yield: 70%; yellow oil; spectroscopic data are according to those
reported in the literature.20
1
Yield: 70%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.41–2.54
(dd, J1 = 8.1 Hz, J2 = 18.3 Hz, CH), 2.82–2.94 (dd, J1 = 5.3 Hz, J2 =
18.4 Hz, 1H, CH), 3.70 (s, 3H, OCH3), 3.86 (s, 9H, 3OCH3), 4.01–
4.11 (m, 1H, CH), 4.75 (d, J = 7.7 Hz, 1H, CH), 6.13 (s, 1H, Ph-H),
6.83–6.96 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 161.8, 148.5,
148.1, 134.5, 134.0, 120.5, 113.9, 110.9, 110.6, 93.6, 80.2, 79.5,
73.1, 57.0, 56.1, 55.8, 46.9, 35.2. C19H21BrO6 requires C, 53.66;
H, 4.98; O, 22.57. Found: C, 53.02; H, 5.01; O, 22.67.
6-Chloroflavanone (3)
Yield: 62%; colorless oil; spectroscopic data are according to
those reported in the literature.20
8-Bromo-5-methoxyflavanone (5)
Yield: 98%; Yellow oil; spectroscopic data are according to
those reported in the literature.20
6,8-Dibromo-5,7,30,40-tetramethoxycatechin (23)
1
8-Chloro-5-methoxyflavanone (6)
Yield: 22%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.80–2.92
(dd, J1 = 8.5 Hz, J2 = 16.6 Hz, CH), 3.07–3.19 (dd, J1 = 5.2 Hz, J2 =
16.6 Hz, 1H, CH), 3.72 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.86 (s,
6H, 2OCH3), 4.10–4.18 (m, 1H, CH), 4.75 (d, J = 7.7 Hz, 1H, CH),
6.83–6.86 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 162.7, 157.6,
149.7, 149.5, 134.1, 120.1, 112.6, 111.3, 107.6, 100.3, 99.4, 83.1,
67.1, 60.5, 59.9, 56.1, 56.0, 55.9, 27.5. C19H20Br2O6 requires C,
45.26; H, 4.00; O, 19.04. Found: C, 45.89; H, 4.09; O, 18.87.
Yield: 72%; yellow oil; spectroscopic data are according to those
reported in the literature.20
6-Chloro-5-methoxyflavanone (7)
Yield: 20%; colorless oil; spectroscopic data are according to
those reported in the literature.20
6-Bromo-7-methoxyflavanone (9)
8-Chloro-5,7,30,40-tetramethoxycatechin (24)
Yield: 95%; colorless oil; spectroscopic data are according to
those reported in the literature.20
1
Yield: 60%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.42–2.56
(dd, J1 = 8.1 Hz, J2 = 18.3 Hz, CH), 2.80–2.94 (dd, J1 = 5.3 Hz, J2 =
18.4 Hz, CH), 3.72 (s, 3H, OCH3), 3.86 (s, 9H, 3OCH3), 4.00–4.12
(m, 1H, CH), 4.72 (d, J = 7.7 Hz, 1H, CH), 5.83 (s, 1H, CH), 6.85–
6.98 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 161.8, 148.5, 148.1,
134.5, 134.0, 120.5, 113.9, 110.9, 110.6, 93.6, 80.2, 79.5, 73.1,
57.0, 56.1, 55.8, 46.9, 35.2. C19H21ClO6 requires C, 59.92;
H, 5.56; O, 25.21. Found: C, 60.02; H, 5.26; O, 24.99.
6-Chloro-7-methoxyflavanone (10)
Yield: 70%; yellow oil; spectroscopic data are according to those
reported in the literature.20
8-Chloro-7-methoxyflavanone (11)
Yield: 18%; colorless oil; spectroscopic data are according to
those reported in the literature.20
6,8-Dichloro-5,7,30,40-tetramethoxycatechin (25)
1
Yield: 18%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.78–2.90
8-Bromo-5,7,40-trimethoxyflavanone (13)
(dd, J1 = 8.5 Hz, J2 = 16.6 Hz, 1H, CH), 3.06–3.20 (dd, J1 = 5.2 Hz,
J2 = 16.6 Hz, 1H, CH), 3.74 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.86
(s, 6H, 2OCH3), 4.10–4.20 (m, 1H, CH), 4.76 (d, J = 7.7 Hz, 1H,
CH), 6.82–6.88 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 150.7, 148.8,
148.2, 134.9, 134.0, 120.6, 113.9, 110.8, 105.5, 97.8, 79.8, 79.6,
Yield: 95%; colorless oil; spectroscopic data are according to
those reported in the literature.20
8-Chloro-5,7,40-trimethoxyflavanone (14)
Yield: 85%; colorless oil; spectroscopic data are according to 73.2, 59.5, 60.0, 55.9, 55.8, 55.7, 37.0. C19H20Cl2O6 requires C,
those reported in the literature.20
54.95; H, 4.85; O, 23.12. Found: C, 55.25; H, 4.98; O, 23.42.
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Paper
8-Bromo-5,7,30,40-tetramethoxyepicatechin (27)
dish (90 mm) containing 12 mL of the medium including the
products in required concentrations (added to the agar at tem-
perature below 50 1C) was inoculated with 2 mL of the inoculum
suspensions; 5–6 replications for each concentration and fungus
were made. The Petri dishes were incubated at 25 1C in the dark to
a clearly visible growth. Evaluation of linear growth was conducted
by measuring mycelial diameters of each inoculated plate at
broadest, medium and smallest diameters, and compared with
the corresponding control. The inhibition (%) of linear mycelial
mean growth of T. koningii, C. cladosporioides and F. oxysporum
was calculated after incubation for 3, 5 and 6 days respectively.
The data were evaluated by analysis of variance, probability of
single differences was calculated at the 5% level. The data were
also ordered by Multi-Dimensional Scaling (MDS) analysis using
SYSTAT 10.0 Statistics35,36 to group the antifungal activity of all
tested compounds; an input matrix of the Euclidean distance
(calculated on linear mycelial growth) and the Kruskal loss
function was utilized.37
1
Yield: 78%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.85–2.88
(m, 2H, CH2), 3.71 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.85
(s, 9H, 3OCH3), 3.88 (s, 9H, 3OCH3), 4.21–4.24 (m, 1H, CH), 4.91
(d, J = 7.7 Hz, 1H, CH), 6.13 (s, 1H, Ph-H), 6.85–7.05 (m, 3H,
Ph-H); 13C NMR (CDCl3), d: 161.9, 148.5, 148.2, 134.7, 134.2,
120.8, 113.9, 110.9, 110.8, 93.6, 80.4, 79.6, 73.1, 57.2, 56.0, 55.9,
46.8, 35.4. C19H21BrO6 requires C, 53.66; H, 4.98; O, 22.57.
Found: C, 54.02; H, 5.04; O, 22.87.
6,8-Dibromo-5,7,30,40-tetramethoxyepicatechin (28)
1
Yield: 20%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.85–2.88
(m, 2H, CH2), 3.70 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.86
(s, 6H, 2OCH3), 4.10–4.18 (m, 1H, CH), 4.75 (d, J = 7.7 Hz, 1H,
CH), 6.83–6.86 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 162.8, 157.6,
149.7, 149.5, 134.1, 120.1, 112.6, 111.3, 107.6, 100.4, 99.4, 83.2,
67.2, 60.4, 59.9, 56.2, 56.1, 55.9, 27.4. C19H20Br2O6 requires C,
45.26; H, 4.00; O, 19.04. Found: C, 45.79; H, 4.12; O, 18.87.
8-Chloro-5,7,30,40-tetramethoxyepicatechin (29)
Acknowledgements
1
Yield: 62%; yellow oil; H NMR (400 MHz, CDCl3), d: 2.85–2.88
The authors would like to thank the ‘‘Complex Equipment
Center’’ of Tuscia University for the use of the NMR spectrometer.
(m, 2H, CH2), 3.72 (s, 3H, OCH3), 3.86 (s, 9H, 3OCH3), 4.02–4.12
(m, 1H, CH), 4.74 (d, J = 7.7 Hz, 1H, CH), 5.88 (s, 1H, Ph-H),
6.86–7.00 (m, 3H, Ph-H); 13C NMR (CDCl3), d: 157.8, 148.5,
148.1, 137.9, 134.2, 120.6, 113.9, 110.8, 102.5, 92.8, 80.4, 79.8,
73.0, 57.0, 56.2, 55.9, 55.8, 54.2, 34.8. C19H21ClO6 requires C,
59.92; H, 5.56; O, 25.21. Found: C, 60.02; H, 5.26; O, 24.99.
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