Synthesis and antifungal activities of natural and synthetic biflavonoids

Article abstract of DOI:10.1016/j.bmc.2011.04.010

The synthesis of some natural and synthetic biflavonoids was performed in good overall yields starting from readily available materials via high yielding aldol and Ullmann condensations. Some of these compounds, especially bichalcones, display an interesting activity against fungi, higher than that of the corresponding monomers.

Full text of DOI:10.1016/j.bmc.2011.04.010

Bioorganic & Medicinal Chemistry 19 (2011) 3060–3073
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antifungal activities of natural and synthetic biflavonoids
Gabriel Sagrera a, Ana Bertucci b, Alvaro Vazquez b, Gustavo Seoane b,
⇑
a Departamento de Química Orgánica, Facultad de Ciencias, Universidad de la República, C.C.1157 Montevideo, Uruguay
b Departamento de Química Orgánica, Facultad de Química, Universidad de la República, C.C.1157 Montevideo, Uruguay
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of some natural and synthetic biflavonoids was performed in good overall yields starting
from readily available materials via high yielding aldol and Ullmann condensations. Some of these com-
pounds, especially bichalcones, display an interesting activity against fungi, higher than that of the cor-
responding monomers.
Received 25 February 2011
Revised 2 April 2011
Accepted 6 April 2011
Available online 9 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Biflavonoids
Ullmann condensation
Antifungal activity
1. Introduction
Biflavonoids with a 3,800 interflavonyl linkage, 6, are often found
in different species of Garcinia. Ochnaflavone, 7, and hinokiflavone,
The biflavonoids are dimers of C4-carbonyl flavonoids (i.e., chal-
cones, flavanones, flavones, flavanols, flavonols, aurones, and iso-
flavones) which vary at the oxygenation pattern of their
monomers, oxidation level of the C3 moiety, and interflavonyl link-
age.1–4 The interflavonyl linkage may involve the A ring (at posi-
tions 5, 6, 7, or 8), the B ring (at positions 20, 30, 40, 50, or 60) or
the C ring (at positions 2 or 3), through C–C or C–O–C bonds. In this
way, biflavonoids can be classified indicating the rings involved in
the interflavonyl linkage (AA, BB, AB, CC, etc.).5
It has been reported that the class of biflavonoids represents a
library of structurally diverse molecules, which remains to be fully
exploited, since most of them have not yet been found in nature or
else have not been synthesized or else its biological properties
have not been described.6
Most natural biflavonoids contain an interflavonyl linkage be-
tween the B-ring of one and the A-ring of the other flavonoid moi-
ety (AB type) or between two A rings (AA type) and are widely
distributed in Spermatophyta. The first isolated biflavonoid was
ginkgetin, 1, by Furukawa in 1929 from Ginkgo biloba L. (a gymno-
sperm), (Fig. 1).7–11 Also, cupressuflavone ([I-8, II-8]-biapigenin), 3,
and robustaflavone, ([I-6, II-30]-biapigenin), 5, were isolated from
different species of Gymnosperms. In Angiosperms, the following
biflavonoids were isolated from Rhus succedanea (Anacardiaceae)
and Garcinia multiflora (Guttiferae): robustaflavone, amentoflavone
([I-8, II-30]-biapigenin), 2 and agathisflavone ([I-6, II-8]-biapige-
nin), 4. Biflavonoids of the BC type are found in Angiosperms.
([I-6-O-II-40]-biapigenin), 8, are examples of biflavonoids that con-
tains C–O–C bonds.
The interflavonyl linkage between the two B-rings (BB type) is
less common. In Gymnosperms, biflavonoids of this type are very
rare. That is the case of 50,5000-bisdihydroquercetin, 9, which has
been found only in the Douglas-fir (Pseudotsuga menziesii; Pina-
ceae).12 This type of biflavonoids can be found in mosses and ferns.
For example, 30,3000-binaringenin, 10, was isolated from Homalothe-
cium lutescens,13 Selaginella chrysocaulos,14 some species of Pilo-
trichella15,16 and Thuidium kanedae.17
Examples of biflavonoids of the 3,300-CC type are chamaejas-
mine, 11, and its derivatives.18–20
Biflavonoids display several biological activities, namely
antifungal,21–23 antiviral,24–26 antibacterial,27,28 antioxidant,29,30
antitumor,4,24,31–35
antiplasmodial,36
antiallergenic,
anti-
inflammatory,37,38 hepatoprotective,39–41 vasodilating,29,42,43 and
hypotensive26,43–46 activity, sometimes better than that of the cor-
responding monomers.22,37
There is a renewed interest in the biological activities of biflavo-
noids, since, as stated by Rahman et al. in a recent review,6 ‘the
theoretical library of biflavanoids spans a wide range of configura-
tional and conformational space suggesting that possibilities of
interesting biological activity are strong, each of which is capable
of multiple H-bonding and hydrophobic interactions’. In a recent
paper, Kim et al. reported37 that not only naturally-occurring bifl-
avonoids but also synthetic biflavonoids show antibacterial, anti-
fungal and antiviral activity and there have only been a few trials
to synthesize a biflavonoid library. In that sense, Chen et al., pre-
pared a C–C biflavonoid library and showed that the anti-inflam-
matory activity depends on the position of the C–C linkage.47
⇑
Corresponding author. Fax: +598 2924 1906.
E-mail address: gseoane@fq.edu.uy (G. Seoane).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.010

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3061
OH
O
OH
OR
OH
OH
RO
O
HO
OH
O
HO
O
O
OH
OH
O
OH
O
O
O
OH
O
HO
O
O
HO
HO
OH
agathisflavone, 4
R = Me, ginkgetin,1
R = H, amentoflavone,2
cupressuflavone, 3
OH
OH
OH
HO
O
O
OH
O
O
OH
HO
O
O
OH
HO
O
OH
biflavonoids fromGarcinia, 6
OH
OH
O
robustaflavone, 5
HO
OH
O
HO
OH
O
OH
O
O
OH
HO
HO
O
OH
HO
HO
O
OH
OH
O
OH
OH
O
O
O
O
HO
O
O
OH
OH
OH
OH
OH
O
OH
ochnaflavone, 7
hinokiflavone, 8
5',5'''-bisdihidroquercetin,9
O
O
OH
H
H
HO
O
O
OH
O
O
OH
H
H
HO
O
OH
OH
HO
OH
OH
O
3',3''-binaringenin, 10
(d,l)-chamaejasmine,11
HO
O
HO
O
HO
O
O
OH
HO
O
OH
HO
O
OH
OH
lupinalbisone A,12
lupinalbisone B,13
O
O
O
O
OH
OH
OH
OH
OH
OH
Figure 1. Some representative biflavonoids.
Also, it was reported that some synthetic biflavonoids have
anti-inflammatory activity.48,49
certain derivatives of chamaejasmine (neochamaejasmine B and
chamaechromone)23,57 have antifungal action.
Considering the antifungal properties, it was shown that amen-
In some cases the bioactivity of a biflavonoid is greater than
toflavone,21,50–54 isocryptomerin,55 ginkgetin,56 bilobetin,56 and
that of the corresponding monomer.22,37,58 It has been shown that

3062
G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
the dimerization of flavonoids can result in a remarkable increase
of fungitoxicity. That is the case of lupinalbisones A, 12, and B, 13
(two dimers of 20-hydroxygenistein isolated from Lupinus al-
bus),22,59 Figure 1. These compounds are considered post-inhibi-
tins60 since are produced from less fungitoxic precursors in
biologically and/or mechanically injured or stressed plants.
In the biflavone group, it was reported that some dimers of api-
genin (6,600-bigenkwanin; tetradimethoxybigenkwanin; amentof-
lavone and 7,700-dimethoxyagathisflavone)52 are inactive against
Aspergillus flavus but inhibit the production of aflatoxins B1 and
B2 by this fungus.52 With respect to the inhibition of production
of aflatoxins, the four compounds have similar activities, so the
activities are more related to the presence of a dimer than to the
particular location of the interflavonyl linkage.61,62
As part of an ongoing project on the synthesis and biological
evaluation of phenolic compounds,63–67 we became interested in
the preparation of a biflavonoid library containing different inter-
flavonyl linkages, different substitution patterns and different oxi-
dation levels of the C3 moiety, for the evaluation of its antifungal
properties.
Herein we describe the preparation of some biflavonoids with
other types of linkages: BB (20,2000-linkage), 15, AA (6,600-linkage),
16, and CC (3,300-linkage), 17, using a reliable set of conditions from
readily available reagents, and the antifungal activities of some of
them.
2. Methods and results
2.1. Synthesis
2.1.1. Biflavonoids of the BB-type
For the synthesis of BB biflavonoids linked at 20,2000 (Fig. 2, gen-
eral structure 15) it was necessary to prepare the key intermediate
2,20-diformylbiphenyl, 18. The most direct route, consisting of the
Ullmann condensation of 2-bromobenzaldehyde, 19, formed a
complex mixture from which only 21% of 18 was isolated. After
extensive experimentation trying a number of conditions and reac-
tants for the condensation,70 the best procedure involved a clean
condensation of the iodoester 21, obtained from readily available
2-iodobenzoic acid, 20, yielding a 73% of diester 22, which was
then reduced to the diol and oxidized to the desired dialdehyde
18 (Scheme 1). The overall yield of 18 from 20 was 50%.
With the dialdehyde 18 in hand, we prepared the flavanone 27
via aldol condensation with 24, acidic cyclization and debenzyla-
tion (Scheme 2).
As a representative panel of fungi, we included Candida albicans
(ATCC 90028), Candida parapsilosis (ATCC 22019), Cryptococcus neo-
formans (ATCC 14116), Aspergillus niger (ATCC 16404), Aspergillus
oryzae (ATCC 10124), Fusarium solani (ATCC 36031), and Rhizopus
stolonifer (+) (ATCC 6227b), according to the following reasons.
The most common human fungal pathogens are species of the Can-
dida and Aspergillus genera.68,69 Mortality and morbility of infec-
tions caused by them have increased especially in immuno-
compromised and hospitalized patients. C. neoformans is consid-
ered as an opportunistic fungus; infections caused by this species
are rare in those individuals with fully functioning immune sys-
tem. C. neoformans causes primarily lung infections and also fungal
meningitis, especially as a secondary infection in AIDS patients. A.
niger can cause aspergillosis, which produces several pulmonary
effects. F. solani is a plant pathogen and can cause several infec-
tions: superficial (queratitis, onicomycosis), localized (endoftalm-
itis, sinusitis) and disseminated. R. stolonifer (+), the bread mold
fungus, is one of the most common fungi in the world. Many fruits
and vegetables are susceptible to this pathogen, which causes post-
harvest diseases in them.
The unsubstituted 20,2000-biflavone, 33, was obtained in 32% yield
from 28 (Scheme 3). First, the iodoflavone 32 was obtained in three
steps from 28 through esterification with 29, Baker-Venkataraman
rearrangement and acidic cyclization. Then, Ullmann condensation
of 2-iodoflavone 32 formed 33.
2.1.2. Biflavonoids of the AA-type
To obtain bichalcones linked in ring A, we envisioned a route
involving the Ullmann condensation of A-ring iodinated 20-
hydroxychalcones, since it was reported that these compounds
are easily obtained by direct iodination in positions 30 and 50, with-
out formation of secondary products.71 However, in our hands the
treatment of 20-hydroxy-4-methoxychalcone, 34,67 with the I2–
HIO3 system formed different products by iodination of ring A in
position 30 or 50 (ortho and para to free 20-OH), of ring B in position
4 (ortho to OMe), addition of I2 to the double bond and cyclization
to the corresponding flavanone or flavone, depending on the condi-
tions used (Scheme 4). Best conditions for the preparation of
In a previous paper66 we described the preparation of some nat-
ural and unnatural biflavonoids whose B rings are linked at posi-
tion 3 (Fig. 2, structure 14)
O
O
O
O
O
O
O
O
14
15
O
O
O
O
O
O
O
O
16
17
Figure 2. General structures of biflavonoids involved in this paper.

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3063
COOMe
I
COOMe COOMe
CHO
CHO
COOH
CHO
I
Br
b)
c)
d)
e)
a)
20
21
22
18
yield from19 : 21%
19
overall yield from20 (4 steps) : 50%
Scheme 1. Synthesis of dialdehyde 18. Reagents and conditions: (a) Cu (activated), 200–220 °C, 30 min (21%); (b) MeOH–H2SO4, 95:5 v/v (86%); (c) Cu (activated), 200–
220 °C, 30 min (73%); (d) LiAlH4/THF, rt, 1 h (92%); (e) PDC/CH2Cl2, rt, 6 h (85%).
O
HO
OH
O
BnO
OH
O
BnO
OH
O
a)
b)
HO
25
OBn
23
24
c)
O
O
O
HO
O
O
BnO
O
O
d)
OH
O
OBn
27 (overall yield from23 : 16%)
26
Scheme 2. Synthesis of biflavonoids of the BB (20,2000) type. Reagents and conditions: (a) BnCl (1.1 equiv)-K2CO3/DMF, 6 h, reflux, (93%); (b) 18, NaH/THF, rt, 24 h (58%); (c)
AcOH, reflux, 96 h (42%); (d) H2–Pd/C, EtOAc, rt, 12 h (70%).
OH
O
OH
O
a)
b)
+
O
I
Cl
COMe
O
O
I
O
I
28
29
30
31
O
O
O
O
O
c)
d)
I
O
32
33 (32% from28)
Scheme 3. Synthesis of 20,2000-biflavone, 33. Reagents and conditions: (a) Py, 2 h, rt (87%); (b) KOH/Py, 1 h, 60 °C (72%); (c) AcOH/H2SO4, 95:5 v/v, reflux, 1 h (82%); (d) Cu
(activated), 30 min, 200–220 °C (63%).
monoiodinated chalcone 35 involved the use of a 0.5:1:1 molar ra-
tio of I2/HIO3: 34, at 60 °C in aqueous acetic acid during 1 h, giving
35 in 45% isolated yield, together with 40% of 36 and trace amounts
of products resulting from iodination of the double bond.72
Treatment of chalcone 35 with benzyl chloride formed the cor-
responding protected chalcone 40. Unfortunately, the Ullmann
condensation of 40 in order to obtain 41, was unsuccessful
(Scheme 4).
In view of these results, we decided to try the synthesis of
bichalcone 41 by first preparing the AA dimer 42, followed by aldol
condensation. This dimer was conveniently prepared in 42% overall
yield from 4-aminophenol, 43 through a sequence involving diaz-
otization and methylation to 4-iodoanisol, 44, which gave 85% of
the biphenyl 45 through Ullmann condensation, followed by
formation of diacetate 46 and further Fries rearrangement to the
desired dimer 42 (Scheme 5). It is worth mentioning that the more
direct route involving the condensation of 5-iodo-2-benzyloxyace-
tophenone, 47, gave only 5% of the desired dimer when submitted
to the Ullmann condensation.
The aldol condensation of dimer 42 with p-anisaldehyde, 48,
yielded 20,2000-dihydroxy-4,400-dimethoxy-50,5000-bichalcone, 41. This
compound was converted to the corresponding biflavone, 49 which
was deprotected to give 50. The overall yield of 50 from 42 was
55% (23% from 43) (Scheme 6)
2.1.3. Biflavonoids of the CC-type
The synthesis of the 3,300-biflavones 56–58 was performed as
shown in Scheme 7. The treatment of chalcone 34 with the
DMSO–I2 system (using a 1:1 molar ratio of chalcone/I2), formed
the iodochalcone 53, which was then submitted to an Ullmann

3064
G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
OMe
OMe
a)
OMe
OMe
R2
OH
O
OH
O
O
O
I
R3
I
+
+
R1
I
OH
O
34
39 (traces)
38 (traces)
35, R = I, R = R = H (45%)
1
2
3
36, R = R = H, R = I (40%)
1
3
2
37, R = R = R = I (traces)
1
2
3
OMe
OMe
OH
O
c)
OBn
b)
35
O
I
HO
O
40
41
MeO
Scheme 4. Attempted preparation of bichalcone 41 via dimerization of chalcone 34. Reagents and conditions: (a) I2–HIO3/AcOH–H2O–EtOH (10:3:1 v/v), 50–60 °C, 1 h; (b)
BnCl–K2CO3/DMF, reflux, 6 h (92%); (c) Cu, 200–220 °C, 2 h, no reaction.
OMe
OAc
OMe
OH
NH
HO
O
OH
O
a)
b)
d)
e)
c)
f)
I
43
44
45
46
2
42
i)
OAc
OMe
OBn
O
OH
O
g)
h)
I
28
47
Scheme 5. Synthesis of dimer 42. Reagents and conditions: (a1) NaNO2–HCl/H2O, 0–5 °C, (a2) KI/H2O (79%); (b) Me2SO4–K2CO3/acetone, reflux, 24 h (98%); (c) Cu (activated),
200–220 °C, 2 h (85%); (d) BBr3/CH2Cl2, ꢀ60 °C?TA, 6 h (90%); (e) Ac2O–DMAP/Py, 60 °C, 3 h (97%); (f) AlCl3/chlorobenzene, reflux 8 h (72%); (g) BnCl–K2CO3/DMF, reflux, 6 h
(95%); (h) I2–HIO3/AcOH–H2O–EtOH (10:3:1 v/v), reflux, 1 h (79%); (i) Cu, 200–220 °C, 2 h (5%).
OMe
OH
O
OH
O
O
OMe
a)
2
+
OHC
HO
MeO
42
48
41
OH
O
b)
OH
OMe
O
O
O
O
O
O
c)
O
O
49
50 (overall yield from42: 55%)
HO
MeO
Scheme 6. Synthesis of 40,4000-dihydroxy-5,500-biflavone, 50. Reagents and conditions: (a) NaH/THF, rt, 24 h (67%); (b) DMSO–I2 (ratio chalcone/I2 = 100:2), reflux, 1 h (89%); (c)
BBr3/CH2Cl2, ꢀ60 °C?rt, 7 days (92%).

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3065
R1
R2
R1
R1
O
O
O
R2
R2
c)
R1
R2
OH
O
OH
O
O
O
a)
+
b)
O
OHC
I
R2
28
R1
R1= H, R = OMe
34 (89%)
51 (89%)
52 (88%)
53 (83%)
54 (79%)
55 (72%)
56(72%)
57 (71%)
58 (64%)
2
R1 = R2 = H
R1 = R2 = OMe
Scheme 7. Synthesis of 3,300-biflavones 56–58. Reagents and conditions: (a) NaH/THF, rt, 16 h; (b) DMSO–I2 (1 equiv), reflux, 12 h; (c) Cu (activated), 205–220 °C, 30 min.
O
OBn
OBn
BnO
2
OH
O
BnO
OH
O
CHO
+
HO
OBn
OHC
OH
a)
BnO
BnO
24
59
60
b)
O
O
O
OBn
HO
O
O
BnO
c)
O
O
O
OH
OBn
HO
BnO
62
61
Scheme 8. Synthesis of 5-deoxyanalog of 30,3000-binaringenin, 62. Reagents and conditions: (a) NaH/THF, rt, 24 h, 72%; (b) AcOH, reflux, 96 h, 50%; (c) H2–Pd/C 10%/EtOAc, rt,
6 h, 76%.
condensation to yield the biflavone 56. In a similar way were ob-
tained the biflavones 57 and 58 from chalcones 51 and 52. The glo-
bal yield of 56–58 from 28 was 40–53%.
57, 62, 63, and 65–68 and their corresponding monomers were
determined, as shown in Table 2. The MIC value of 60 against C.
neoformans was not determined due to decomposition of the prod-
uct during the assay.
Finally, using the methodology previously reported by us,66 the
5-deoxyanalog of 30,3000-binaringenin was prepared (Scheme 8). To
this end, protected resacetophenone, 24, was condensed with dial-
dehyde 59 to form bichalcone 60, which was then cyclized and
debenzylated to biflavanone 62. Dialdehyde 59 was prepared by
us in a previous work,66 by a four step synthesis from p-anisalde-
hyde (54%). Considering this fact, the global yield of 62 from read-
ily available p-anisaldehyde was 15%.
3. Discussion
The antifungal activities (MIC values) are typical for a variety of
flavonoids described in the literature.74 Despite the reduced size of
the set of compounds tested, some general trends seem to emerge
from the inspection of Tables 1 and 2.
(a) Considering the microorganisms used:
2.2. Biological evaluation
The strains used showed differential susceptibility toward
monomers and dimers, but no general trend (i.e., a higher suscep-
tibility for either monomers o dimers) could be derived for any of
the microorganism used. The most susceptible strain used was A.
niger, and the majority of the biflavonoids that showed activity in
the qualitative screening were active against this microorganism.
Conversely, it is interesting to note that none of the compounds
tested showed activity against A. oryzae.
In addition to compounds 25–27, 33, 49, 50, 57, and 60–62 pre-
pared in this work, the previously synthesized compounds 10
(binaringenin) and 63–69,66 were evaluated (Fig. 3). The figure also
shows the corresponding monomers 70–86, which are all known
compounds and were prepared according to procedures previously
reported by us.65–67,73
On the synthesized biflavonoids, as well as their monomers, a
preliminary screening was carried out by a modified Agar Diffusion
Bioautography method (Table 1).
Half of the biflavonoids whose MIC was determined (five out of
ten) were active against only one of the five strain of the panel, and
this strain was A. niger in four cases.
None of the compounds were active against A. oryzae nor F.
solani so these strains were not considered in further assays.
Then, the subset of active compounds was used in a quantita-
tive screening by Microdilution to compare the antifungal activity
of the biflavonoids to the corresponding monomers. Thus, the Min-
imum Inhibitory Concentration (MIC) of the compounds 25, 33, 50,
(b) Regarding the interflavonyl linkage:
Biflavonoids of the BB-type represented the biggest set of com-
pounds assayed, comprising four compounds linked through the 2-
position (compounds 25–27 and 33) and eleven through the 3-po-
sition (compounds 10 and 60–69). Two of the four representatives
of the 2,20-linkage and seven of the other group were active against

3066
G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
O
R2
R3
bichalcones :
OH
chalcones :
R1
R3
OH
O
R1
HO
R1
R3
R2
R2
O
R
R
R
1 = R2 = R3 = OMe,63
1 = R2 = R3 = OBn,64
1 = R2 = R3 = OH,65
R
1 = R2 = R3 = OMe,70
R1 = R2 = R3 = OBn,71
R1 = R2 = R3 = OH,72
R1 = R3 = OBn, R = H,60
R1 = R3 = OBn, R2 = H,73
2
O
BnO
OH
O
R1 = OBn, R2 = R3 = H,74
25
HO
OBn
R3
biflavanones :
R1
flavanones :
O
O
R2
R3
R1
O
O
O
O
R1
R2
R3
R
R
R
R
1 = R2 = R3 = OH,10 (binaringenin)
1 = R3 = OBn, R2 = OH,66
1 = R3 = OBn, R2 = H,61
1 = R3 = OH, R2 = H,62
R
1 = R2 = R3 = OH,75
R2
R1 = R3 = OBn, R2 = OH,76
R1 = R3 = OBn, R2 = H,77
1 = R3 = OH, R2 = H,78
R
O
R
O
O
R
1 = OBn, R2 = R3 = H,79
R = OBn,26
R = OH,27
R1 = OH, R2 = R3 = H,80
O
R
O
O
R2
biflavones :
flavones :
O
R3
R3
R1
R1
O
O
R1
R3
R2
O
R1 = R2 = R3 = OMe,67
R1 = R2 = R3 = OH,68
R2
R1 = R2 = R3 = OMe,81
R1 = R2 = R3 = OH,82
O
O
O
O
O
O
O
O
R1 = R2 = R3 = H,83
33
57
R
O
O
O
R1 = R2 = H, R3 = OMe,84
R = OMe,49
R = OH,50
R1 = R2 = H, R3 = OH,85
O
R
O
O
OH
biflavonols :
flavonols :
HO
OH
OH HO
O
HO
O
O
OH
OH
69
OH HO
86
OH
O
OH
Figure 3. Biflavonoids tested for their antifungal activity and the corresponding monomers.
at least one microorganism of the panel, Table 1. The activity of the
biflavonoids with the 2,20-linkage was poor and lower than that of
the corresponding monomers, Table 2. On the other hand, some
members of the group linked through the 3-position showed an
interesting activity, higher than that of the corresponding
monomers (compounds 62, 63, 65, and 67), Table 2.
qualitative screening, even though the MIC values were poor. The
corresponding monomer displayed a better or similar activity,
Table 2
Regarding the two AA-type biflavonoid tested, only one showed
a slight activity against Cp, 50, comparable to the corresponding
monomer.
The only CC-type biflavonoid tested, 57, was one of the two
compounds showing activity against three strains in the
(c) Considering the C3 moiety, five 20,2000-dihydroxybichalcones,
six biflavanones, six biflavones, and one biflavonol were tested:

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3067
Rs
Table 1
Fungal activity of biflavonoids against seven isolates of pathogen fungi, determined by Agar Diffusion Bioautography method
Entry
Site of union
Ring A
Ring B
Compound
Ca
Cp
Cn
An
Ao
Fs
20,2000-Dihydroxybichalcones
1
2
3
4
5
2,200
3,300
3,300
3,300
3,300
40-OBn
H
25
60
63
64
65
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
40-OBn
4-OBn
4-OMe
4-OBn
4-OH
ꢀ
40,60-DiOMe
40,60-DiOBn
40,60-DiOH
+
ꢀ
ꢀ
Biflavanones
6
7
8
9
10
11
30,3000
20,2000
20,2000
30,3000
30,3000
30,3000
5,7-DiOH
7-OBn
7-OH
7-OBn
7-OH
4-OH
H
H
4-OBn
4-OH
4-OBn
10
26
27
61
62
66
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5-OH, 7-OBn
ꢀ
Biflavones
12
13
14
15
16
17
20,2000
6,600
H
H
H
H
H
33
49
50
57
67
68
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
40-OMe
40-OH
H
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
+
6,600
3,300
30,3000
30,3000
+
5,7-DiOMe
5,7-DiOH
40-OMe
40-OH
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
Biflavonols
18
30,3000
5,7-DiOH
4-OH
69
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Controls
Amphotericine B
Acetone
+
ꢀ
+
ꢀ
+
ꢀ
+
ꢀ
+
ꢀ
+
ꢀ
+
ꢀ
Ca: Candida albicans (ATCC 90028), Cp: Candida parapsilosis (ATCC 22019), Cn: Cryptococcus neoformans (ATCC 14116), An: Aspergillus niger (ATCC 16404), Ao: Aspergillus
oryzae (ATCC 10124), Fs: Fusarium solani (ATCC 36031), Rs: Rhizopus stolonifer (+) (ATCC 6227b).
Table 2
Minimum Inhibitory Concentration (MIC,
lmol/mL) of selected biflavonoids and their corresponding monomers
Entry
1
Site of union
2,200
Ring A
Ring B
H
Compound
Ca
Cp
Cn
An
Rs
40-OBn
40,60-DiOMe
40,60-DiOH
7-OH
25
74
63
70
65
72
62
78
66
76
33
83
50
85
57
83
67
81
68
82
>0.8
>0.8
>0.8
0.1
0.013
>0.8
2
3,300
4-OMe
4-OH
4-OH
4-OBn
H
3
3,300
0.8
>0.8
0.013
0.4
0.2
0.8
4
30,3000
30,3000
20,2000
6,600
0.2
>0.8
5
5-OH, 7-OBn
H
>0.8
>0.8
6
>0.8
0.8
0.8
0.1
7
H
40-OH
H
0.4
0.2
0.4
0.2
8
3,300
H
>0.8
0.1
>0.8
0.1
9
30,3000
30,3000
5,7-DiOMe
5,7-DiOH
40-OMe
40-OH
0.4
>0.8
0.1
10
0.006
Ca: Candida albicans (ATCC 90028), Cp: Candida parapsilosis (ATCC 22019), Cn: Cryptococcus neoformans (ATCC 14116), An: Aspergillus niger (ATCC 16404), Rs: Rhizopus
stolonifer (+) (ATCC 6227b).
All assayed 20,2000-dihydroxybichalcones were active at least
against one fungus, except for 64, an hexabenzyloxy-substituted
bichalcone, Table 1 This observation is consistent with the work
of Zacchino and co-workers about the antifungal activity of differ-
planarity could be related to the strength of the hydrogen bond
of the 20-OH proton, which is involved in intramolecular hydrogen
bond to the carbonyl of the C3 moiety. For this system, it is re-
ported that the magnitude of the proton chemical shift is directly
proportional to the strength of the hydrogen bond,78,79 so a com-
parison of the 20-OH shifts in different 20-hydroxychalcones could
give an indication of the planarity of the system through the
strength of the hydrogen bond. In our case (using CDCl3 as solvent)
we observed the 20-OH protons as sharp singlets for compounds 63
and 65, at d >14 ppm, whereas the corresponding proton in 64 ap-
pears as a broad singlet at d <14 ppm, indicating a smaller degree
of coplanarity, resulting in the loss of antifungal activity. In addi-
ent chalcones.75 They found that the presence of the
a,b-conju-
gated system and a high degree of coplanarity between the A- or
B-rings and the C3 moiety are required for the antifungal activity.
The presence of electron donating groups in 40 increases the pla-
narity by favoring the electronic delocalization,76 whereas substi-
tution in 60 diminishes the planarity by a steric effect.77 Thus the
presence of a bulky benzyloxy group in 60 could be responsible
for the loss of antifungal activity in 64. In the same direction, the

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G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
tion to weaken the hydrogen bond, the existence of other effects on
the antifungal activity caused by the presence of six benzyloxy
groups can not be ruled out.
hexanes as initial eluent followed by different eluent mixtures
(CH2Cl2/hexanes; ethyl acetate/hexanes or ethyl acetate/CH2Cl2/
hexanes).
All biflavanones were inactive, except for 66 and 62 which were
active against only one microorganism, Table 1. The poor anti-
fungal activity of these compounds can be explained by the ab-
sence of a double bond in the C3 moiety. This observation is
consistent with the fact that compounds obtained by conjugate
addition of diethyl malonate or malononitrile to a chalcone were
inactive.77 On the other hand, it was reported a slight activity of
7-hydroxyflavanone, 80, against different fungi.80 However biflav-
anone 27, dimer of 80, displayed no activity when submitted to the
panel, Table 1.
All assayed biflavones were active at least against one fungus,
except for 49, Table 1.
Biflavonol 69 resulted inactive against the whole panel. This
observation is consistent with the fact that monomeric flavonols
such as 3-hydroxyflavone and quercetin are inactive or slightly ac-
tive against different fungi.81
Considering the MIC values, Table 2, bichalcones (entries 1–3)
gave the smallest values of MIC and were generally more active
than their corresponding monomers (compounds 63, 65, excep-
tion: 25). On the other hand, biflavones (entries 6–10) show an
opposite trend, being the monomers generally more active than
the dimers (compounds 33, 50, 57, 68, exception: 67). For the bif-
lavanones, 66 and 62 (entries 4 and 5), no trend could be derived.
(d) The most active biflavonoids tested were two bichalcones of
the BB-type, 63 and 65, showing different activity profiles:
whereas 65 was active against three microorganisms of the panel
(C. albicans, C. neoformans, and R. stolonifer (+)), 63 was active only
against A. niger. Both dihydroxybichalcones were more active than
the corresponding monomers and displayed the same substitution
pattern (4,40,60), but the substituents are OH for 65 and MeO for 63.
1H and 13C NMR spectra were recorded at 30 °C on a Bruker
DPX-400 spectrometer (at 400 and 100 MHz, respectively). 1H
NMR were recorded using TMS as the internal reference. 13C
NMR were recorded using the residual solvent as the internal ref-
erence (the central peak of the CDCl3 triplet was assigned to
77.00 ppm). Assignments of H and C were done on the basis of
2D NMR (1H–1H COSY, 1H,13C HMQC and 1H,13C HMBC) experi-
ments. Mass spectra were recorded on a Shimadzu GC–MS QP
1100 EX spectrometer at 70 eV. HRMS were recorded on an Auto-
SpecQ spectrometer at 70 eV (EI, positive mode). Melting points
were determined using a Gallenkamp capillary melting point appa-
ratus and are uncorrected. Elemental analyses were obtained from
vacuum-dried samples (over phosphorus pentoxide at 3–4 mmHg,
24 h at room temperature) and performed on a Fisons EA1108
CHNS–O analyzer.
Assignments of H and C are given according to the following
numbering scheme (in flavanones, C@O means C4):
5'
2'
5
2
4
3
6'
4'
3'
6
8
5
3'
O
OH
8a
4a
2'
2
4'
5'
7
6
1'
1
3
4
6'
O
O
5.1.1. Synthesis of chalcones and 20-hydroxychalcones. General
procedure 1 (adapted from a procedure reported by Stout)82
This procedure is suitable to compounds that not contain free
phenolic groups or when the phenolic groups are protected as
methyl or benzylethers.
4. Conclusions
We have prepared a small library of natural and unnatural bifl-
avonoids in good yields using a reliable set of conditions, based on
aldol and Ullmann condensations of suitable precursors. Both reac-
tions were optimized according to the type of desired biflavanoid.
In particular, the order of events is dependent on the structure of
the target compound (AA-, BB-, or CC-type), that is, the Ullmann
condensation can be performed either on the whole monomer or
an early intermediate previous to the aldol reaction.
The results of the antifungal activity on 18 biflavonoids (and
their monomers) indicate that various compounds such as bichal-
cones 63 and 65 display interesting in vitro activity against some
human pathogenic fungi. The pronounced differential activity
found between monomers and dimers in the more active bichal-
cones merits further studies, which are being conducted in our
laboratory.
To a solution of the corresponding 20-hydroxyacetophenone
(10 mmol) in dry THF (25 mL), NaH (25 mmol, 1 g of 60% disper-
sion in mineral oil) was added in portions, under a nitrogen
atmosphere and with vigorous stirring. When the evolution of
H2 ceased,
a solution of the corresponding benzaldehyde
(10 mmol) in dry THF (25 mL) was added dropwise over
15 min and the reaction mixture was stirred at room tempera-
ture for 16 h, except otherwise stated. After this, the mixture
was poured cautiously over ice-water (100 mL) to destroy the
excess of NaH and stirred until the evolution of H2 subsided.
The mixture was acidified with 25% HCl and extracted with ethyl
acetate (3 ꢁ 50 mL). The organic layer was washed with water
(3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous MgSO4.
The solution was concentrated under vacuum at 40 °C, until it
reached 1/3 of the original volume. After cooling at 0 °C for
12 h the crystalline product was separated by filtration. The 20-
hydroxychalcones obtained in this way are pure enough for most
purposes. Analytical samples were obtained after purification by
column chromatography.
5. Experimental
5.1. Chemistry
Using this procedure, good yields of chalcones were obtained.
An additional amount of product can be obtained from the
mother liquors. To this end, the solvent was removed under vac-
uum and the compound was purified by column
chromatography.
All reagents were purchased from commercial sources and used
without purification, unless otherwise indicated. All solvents were
dried and distilled prior to use. THF was dried by refluxing with
benzophenone over sodium wire until a blue color persisted, then
distilled and collected. CH2Cl2 was dried by refluxing over P2O5 for
3 h, then distilled and collected. All reactions were monitored by
TLC. TLC was carried out on AlugramÒ Sil G/UV254 on polyester
plates using different solvent systems. Column chromatography
(CC) was carried out on silica gel (Merck, 60–230 mesh) using
5.1.2. Synthesis of polyhydroxychalcones. General procedure 2
(adapted from a procedure reported by Sogawa)83
This procedure is suitable to compounds with phenolic groups
protected as tetrahydropyranylethers.

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3069
5.1.2.1. Synthesis of tetrahydropyranylethers.
To a stirred
5.1.5. Synthesis of flavones. General procedure 5 (adapted from
a procedure reported by Song et al.)84
suspension of the corresponding phenol (0.1 mol) in dry CH2Cl2
(100 mL), dry p-TsOH (0.85 g, 0.005 mol) and dry pyridine (re-
cently distilled from KOH) (0.40 mL, 0.39 g, 0.005 mol) were added
To
a
solution of the corresponding 20-hydroxychalcone
(1 mmol) in DMSO (10 mL), iodine (2.5 mg, 0.01 mmol) was added.
The reaction mixture is placed in a pre-heated sand bath (190 °C)
and refluxed for 1 h. After this time, water (50 mL) was added
and the mixture was extracted with EtOAc (3 ꢁ 50 mL). The organ-
ic layer was washed with a 10% solution of NaHSO3 (50 mL), water
(3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous MgSO4. The
solvent was removed under vacuum and the compound was puri-
fied by column chromatography.
under a nitrogen atmosphere. After this, 3,4-dihydro-a-pyran
(DHP, 10.5 g, 0.125 mol, 11.4 mL per OH group) was added drop-
wise. The reaction mixture was stirred at room temperature for
30 min, and after this time, a clear solution was obtained. The sol-
vent was removed under vacuum and the residue was dissolved in
EtOAc (500 mL). The organic layer was washed with water
(3 ꢁ 100 mL), brine (100 mL), separated and dried over anhydrous
MgSO4. The solvent was removed under vacuum and the com-
pound was purified by column chromatography.\
5.1.6. Synthesis of flavonols. General procedure 6
To a solution of the corresponding flavone (1 mmol) in MeOH
(15 mL), a solution of KOH (170 mg, 3 mmol) in MeOH (15 mL)
was added with stirring at room temperature. After this, iodoben-
zene diacetate (IBD) (350 mg, 1.1 mmol) was added in four por-
tions. The reaction mixture was stirred at room temperature for
72 h. After this, water (50 mL) was added and the mixture was ex-
tracted with EtOAc (3 ꢁ 50 mL). The organic layer was washed
with water (3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous
MgSO4. The solvent was removed under vacuum and the com-
pound was purified by column chromatography.
5.1.2.2. Synthesis of 20-hydroxy-tetrahydropyranyloxychal-
cones.
To a solution of the corresponding 20-hydroxyaceto-
phenone (10 mmol) and benzaldehyde (10 mmol) in absolute
MeOH (25 mL), barium hydroxide octahydrate (3.2 g, 0.01 mol)
was added under a nitrogen atmosphere and with vigorous stir-
ring. The reaction mixture was stirred at 60 °C for 12 h. After this,
the mixture was neutralized with 10% HCl and extracted with ethyl
acetate (3 ꢁ 50 mL). The organic layer was washed with water
(3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous MgSO4. The
solvent was removed under vacuum and the compound was puri-
fied by column chromatography.
5.1.7. Deprotection of methoxy groups in methylphenylethers.
General procedure 7
The corresponding methoxyflavone or methoxyflavonol
(1 mmol) was dissolved in dry CH2Cl2 (15 mL) under a nitrogen
atmosphere. The solution was cooled to ꢀ60 °C and BBr3 (3 mmol
per OMe group; 3 mL of a 1 M solution in CH2Cl2) was added drop-
wise from an equalizer. The reaction mixture was stirred at room
temperature for the indicated time. After this, the mixture was
poured cautiously over ice-water (50 mL) and extracted with
EtOAc (3 ꢁ 50 mL). The organic layer was washed with water
(3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous MgSO4. The
solvent was removed under vacuum and the compound was puri-
fied by column chromatography.
5.1.2.3. Deprotection of 20-hydroxy-tetrahydropyranyloxychal-
cones.
To a solution of the corresponding 20-hydroxy-tetrahy-
dropyranyloxychalcone (1 mmol) in absolute MeOH (20 mL), p-
toluenesulfonic acid (10 mg, 0.058 mmol) was added. The reaction
mixture was stirred for 2 h at room temperature and then the sol-
vent was removed under vacuum. After this, water (50 mL) was
added and the mixture was extracted with EtOAc (3 ꢁ 50 mL).
The organic layer was washed with water (3 ꢁ 50 mL), brine
(50 mL), and dried over anhydrous MgSO4. The solvent was re-
moved under vacuum and the compound was purified by column
chromatography.
5.1.8. Hydrogenation of benzyloxychalcones and
benzyloxyflavanones. General procedure 8
5.1.3. Synthesis of flavanones using AcOH as solvent. General
procedure 3
To a solution of the corresponding benzyloxychalcone or ben-
zyloxyflavanone (0.5 mmol) in ethyl acetate (50 mL), 50 mg of
10% Pd–C was added and the mixture was stirred at room temper-
ature under a H2 atmosphere. After 1 h, the mixture was filtered
and washed with ethyl acetate (3 ꢁ 50 mL). The solvent was re-
moved under vacuum. The compound was purified by column
chromatography.
A solution of the corresponding 20-hydroxychalcone (1 mmol)
in glacial AcOH (15 mL) was refluxed for 72 h under nitrogen atmo-
sphere. Then, the solution was poured in 100 mL of cold water and
extracted with ethyl acetate (3 ꢁ 50 mL). The organic layer was
washed with water (3 ꢁ 50 mL), brine (50 mL), separated and dried
over anhydrous MgSO4. Cyclohexane (10 mL) was added and the
solvent was removed under vacuum. The compound was purified
by column chromatography.
5.1.9. Synthesis of alkylethers. General procedure 9
To a solution of the corresponding phenol (0.01 mol) in anhyd
DMF (15 mL), the suitable alkyl halide (0.011 mol per OH group)
and K2CO3 (0.011 mol per OH group) were added. The reaction
mixture is vigorously stirred and refluxed for 6 h under a nitrogen
atmosphere. After this, ice-water (100 mL) was added and the mix-
ture was stirred until all carbonate was dissolved. Concd HCl was
added until pH 1 and stirred until the evolution of CO2 subsided.
The mixture was extracted with EtOAc (3 ꢁ 100 mL). The organic
layer was washed with water (3 ꢁ 100 mL), brine (100 mL), and
dried over anhydrous MgSO4. The solvent was removed under vac-
uum and the compound was purified by column chromatography.
5.1.4. Synthesis of flavanones by microwave heating. General
procedure 465
This procedure is suitable for compounds that not contain ben-
zyloxy groups.
To a solution of desired chalcone (0.1 mmol) in dry CH2Cl2
(5 mL), TFA (0.2 mL, 0.307 g, 2.69 mmol) and 1 g of silica gel (1 g)
were added. The mixture was evaporated under vacuum and the
resulting powder was poured into a loosely stoppered test tube.
The tube was placed vertically in the center of the microwave oven
besides a vial with ice (20 g), and irradiated for three successive
periods of 3 min with cooling intervals of 5 min. After irradiation
was finished, the solid mixture was extracted with EtOAc
(3 ꢁ 15 mL) and filtered. The organic layer was washed with water,
brine, separated and dried over anhydrous MgSO4. The mixture
was purified by column chromatography.
5.1.10. Synthesis of aryl iodides. General procedure 10
To a solution of the corresponding arene (0.1 mol) in a mixture
of AcOH (250 mL), H2O (75 mL) y EtOH (25 mL), I2 (0.06 mol), and
HIO3 (0.1 mol) were added. The reaction mixture was refluxed for
3 h. After this, the mixture was poured cautiously over ice-water

3070
G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
(500 mL) and the solid was separated by means of a Buchner fun-
nel. The solid is dissolved in EtOAc (150 mL) and a saturated solu-
tion of Na2SO3 (50 mL) was added. The organic layer was separated
and washed with water (3 ꢁ 50 mL), brine (50 mL), and dried over
anhydrous MgSO4. The solvent was removed under vacuum and
the compound was purified by column chromatography.
(3 ꢁ 30 mL), brine (30 mL), and dried over anhydrous MgSO4. The
solvent was removed under vacuum and the compound was puri-
fied by column chromatography.
5.1.14. 20,2000-Dihydroxy-40,4000-dibenzyloxy-2,200-bichalcone, 25
Prepared according general method 1, from 24 (484 mg,
2 mmol) and 18 (210 mg, 1 mmol), stirred at rt for 24 h. Purified
by CC (hexanes?EtOAc/CH2Cl2/hexanes 1:5:5). Yield: 382 mg
(58%).
5.1.11. Synthesis of biaryls by Ullmann reaction. General
procedure 11
Yellow solid, mp: 129.0–130.5 °C.
To a solution of the corresponding aryl iodide (0.01 mol) in
EtOAc (50 mL), activated Cu85 (2.5 g, 0.04 mol) was added and
the solvent was removed under vacuum. A reflux condenser was
connected and the mixture was heated for 30 min at 205–220 °C, un-
der N2 atm, with stirring. The mixture was allowed to cool under N2
atm. EtOAc (100 mL) was added and the mixture was vigorously stir-
red at rt under N2 atm until a fine suspension was obtained. The sus-
pension was filtered through a sintered glass pad and washed with
EtOAc (250 mL, in portions) until absence of product (checked by
TLC in the mother liquors). The solvent was removed under vacuum
and the compound was purified by column chromatography.
1H NMR (CDCl3, 400 MHz): d = 13.28 (1H, s, 20-OH), 8.21 (1H, d,
0
Jb = 15.4, Hb), 7.81 (1H, d, J6´ 5´ = 8.0, H6 ), 7.72 (1H, dd, J65 = 7.8,
a
J64 = 1.4, H6), 7.64 (1H, dd, J34 = 8.0, J35 = 1.0, H3), 7.49 (1H, d,
J
a
b = 15.4, H ), 7.44–7.32 (6 H, m, phenyl + H5), 7.25 (1H, ddd,
a
0
0
J43 = 8.0, J45 = 7.3, J46 = 1.6, H4), 6.57–6.54 (2H, m, H3 + H5 ), 5.11
(2H, s, CH2).
13
0
C NMR (CDCl3, 100 MHz): d = 191.5 (C@O), 166.7 (C4 ), 165.5
0
(C2 ), 150.0 (C2), 142.7 (C1), 135.8 (Cb), 135.0 (C1phenyl), 133.6 (C4),
0
131.4 (C6), 128.7 (C6 ), 128.3, 128.0, 127.7, 127.5, 126.0
0
0
(C3 + C5 + (C2–C6)phenyl), 123.3 (C ), 114.2 (C1 ), 108.3 (C5 ), 102.2
a
0
(C3 ), 70.3 (CH2).
MS (EI, 70 eV): m/z (%) = 658 (26.2, M+), 568 (15.4,
M+ꢀC6H5CH2þ), 478 (2.4, M+ꢀ2C6H5CH2þ), 91 (100.0, C6H5CH2þ).
Anal. Calcd for C44H34O6: C, 80.23; H, 5.20. Found: C, 80.41; H,
5.11.
5.1.12. Synthesis of flavones by the Baker–Venkataraman
method. General procedure 12
5.1.12.1. Synthesis of esters of 20-hydroxyacetophenones.
A
solution of the corresponding 20-hydroxyacetophenone (10 mmol)
and acid chloride (10 mmol) in dry pyridine (15 mL), was stirred at
rt for 2 h. After this time, the mixture was neutralized with 10% HCl
and extracted with EtOAc (3 ꢁ 50 mL). The organic layer was
washed with water (3 ꢁ 50 mL), brine (50 mL), and dried over
anhydrous MgSO4. The solvent was removed under vacuum and
the compound was purified by column chromatography.
5.1.15. 7,700-Dibenzyloxy-20,2000-diflavanone, 26
Prepared according to general method 3, from 25 (329 mg,
0.5 mmol) and refluxing for 96 h. Purified by CC (hex-
anes?EtOAc/CH2Cl2/hexanes 1:5:5). Yield: 138 mg (42%).
White solid, mp: 118–120 °C.
1H NMR (CDCl3, 400 MHz): d = 7.85 (1H, d, J56 = 8.7, H5), 7.79–
0
0
7.60 (2H, m, H3 + H6 ), 7.45–7.31 (6 H, m, phenyl + H5´ ), 7.28 (1H,
ddd, J4´ 5´ = 7.2, J4´ 3´ = 7.9, J4´6´ = 1.6, H4´ ), 6.60 (1H, dd, J65 = 8.7,
J68 = 2.3, H6), 6.45 (1H, d, J86 = 2.3, H8), 5.44 (1H, dd, J2,3ax = 13.2,
J2,3eq = 2.9 H2), 5.13 (2H, s, CH2), 3.04 (1H, dd, J3ax,3eq = 16.8,
J3ax,2 = 13.2, H3ax), 2.83 (1H, dd, J3eq,3ax = 16.8, J3eq,2 = 2.9 H3eq).
13C NMR (CDCl3, 100 MHz): d = 190.2 (C@O), 164.6 (C7), 163.9
5.1.12.2. Synthesis of 1,3-diaryl-1,3-diketones by Baker-Venka-
taraman rearrangement.
To a solution of the corresponding
ester of 20-hydroxyacetophenone (1 mmol) in dry pyridine (15 mL),
KOH (850 mg, 15 mmol) was added. The reaction mixture was
heated at 60 °C for 30 min.
0
0
0
(C8a), 148.2 (C2 ), 140.3 (C1 ), 136.1 (C1phenyl), 131.0 (C3 ), 129.3,
After this time, the mixture was neutralized with 10% HCl and
extracted with ethyl acetate (3 ꢁ 50 mL). The organic layer was
washed with water (3 ꢁ 50 mL), brine (50 mL), and dried over
anhydrous MgSO4. The solvent was removed under vacuum and
the compound was purified by column chromatography.
0
0
128.6, 128.3, 128.1, 128.0, 127.5, 127.1, (C4 + C5 + C6´ + C5 + ((C2–
C6)phenyl), 116.2 (C4a), 107.4 (C6), 100.3 (C8), 78.3 (C2), 70.0 (CH2),
44.1 (C3).
MS (EI, 70 eV): m/z (%) = 658 (21.2, M+), 568 (10.1,
M+ꢀC6H5CH2þ), 91 (100.0, C6H5CH2þ).
Anal. Calcd for C44H34O6: C, 80.23; H, 5.20 Found: C, 80.05; H,
5.35.
5.1.12.3. Acid cyclization of 1,3-diaryl-1,3-diketones.
A
solution of the corresponding 1,3-diaryl-1,3-diketone (1 mmol) in
AcOH–H2SO4 (10 mL; 95:5 v/v), was refluxed for 1 h. After this
time, water (100 mL) was added the mixture was extracted with
ethyl acetate (3 ꢁ 50 mL). The organic layer was washed with
water (3 ꢁ 50 mL), brine (50 mL), and dried over anhydrous
MgSO4. The solvent was removed under vacuum and cyclohexane
(10 mL) was added. The solvent was removed under vacuum and
the compound was purified by column chromatography.
5.1.16. 7,700-Dihydroxy-20,2000-diflavanone, 27
Prepared according to general method 8, from 26 (132 mg,
0.2 mmol) with 12 h of stirring at rt Purified by CC (hex-
anes?EtOAc/hexanes 1:1). Yield: 67 mg (70%).
White solid, mp: 135–136.5 °C.
1H NMR (CDCl3, 400 MHz): d = 7.83 (1H, d, J56 = 8.7, H5), 7.50–
0
0
7.30 (4H, m, H3 –H6 ), 6.55 (1H, dd, J65 = 8.7, J68 = 2.3, H6), 6.40
(1H, d, J86 = 2.3, H8), 5.43 (1H, dd, J2,3ax = 13.2 J2,3eq = 3.0, H2), 3.03
(1H, dd, J3ax,3eq = 16.8, J3ax,2 = 13.2, H3ax), 2.83 (1H, dd,
J3eq,3ax = 16.8, J3eq,2 = 3.0, H3eq).
5.1.13. Acetylation of phenols. General procedure 13
To a solution of the corresponding phenol (1 mmol) in dry pyr-
idine (5 mL) Ac2O (freshly distilled, 130 mg, 1.25 mmol per OH
group) and N,N-dimethylaminopyridine (DMAP, 10 mg), were
added. The mixture was heated at 60 °C for 3 h with stirring. After
this time, H2O (50 mL) was added and the mixture was stirred for
15 min to destroy the excess of Ac2O. The mixture was extracted
with EtOAc (3 ꢁ 30 mL). The organic layer was washed with water
13C NMR (CDCl3, 100 MHz): d = 190.3 (C@O), 166.7 (C7), 162.9
0
0
0
(C8a), 148.5 (C2 ), 142.5 (C1 ), 131.4 (C3 ), 128.2, 128.0, 127.6,
0
0
127.2 (C4 , C5 , C6´ , C5), 117.1 (C4a), 107.4 (C6), 101.0 (C8), 77.9 (C2),
44.3 (C3).
MS (EI, 70 eV): m/z (%) = 659 (25.7, M++1), 658 (100.0, M+), 657
(58.4, M+ꢀ1), 137 (33.2), 136 (10.0), 121 (10.1), 129 (63.8).
Anal. Calcd for C30H22O6: C, 75.30; H, 4.63. Found: C, 75.58; H,
4.38.
This step is performed to obtain an intimate mixture of the reactants and is
advantageous over the use of a mortar.

G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
3071
5.1.17. Iodination of 20-hydroxy-4-methoxychalcone
5.1.22. 20-Hydroxy-
Yellowish solid, mp: 95–97 °C.
1H NMR (CDCl3, 400 MHz): d = 11.86 (1H, s, 20-OH), 7.76 (1H, dd,
a,b-diiodo-4-methoxy-dihydrochalcone, 39
According to general method 10, 34 (254 mg, 1 mmol) was trea-
ted with iodine (254 mg, 1 mmol) and HIO3 (90 mg, 0.5 mmol) and
heated at 50–60 °C for 1 h. The mixture was separated by CC (hex-
anes?EtOAc/hexanes 2:8). The following products were obtained:
35 (45%), 36 (40%), 37 (1%), 38 (2%), and 39 (1%).
0
0
0
0
0
0
0
0
0
J6´ 5´ = 8.1, J6 4 = 1.1, H6 ), 7.50 (1H, ddd, J4 3 ꢂ J4 5 = 7.8, J4 6 = 1.3,
0
0
0
0
H4 ), 7.42–7.38 (2H, m, H2 + H6), 7.01 (1H, d, J3 4 = 8.3, H3 ), 6.95–
0
6.88 (3H, m, H3 + H5 + H5 ), 5.53 (1H, d, J b = 8.5, H ), 5.34 (1H, d,
a
a
Jb = 8.5, Hb), 3.82 (3H, s, OMe).
a
13
5.1.18. 20-Hydroxy-50-iodo-4-methoxychalcone, 35
Yellow solid, mp: 178–180 °C (lit.: 175 °C).86
C NMR (CDCl3, 100 MHz): d = 204.3 (C@O), 158.2 (C2 ), 157.0
0
0
0
(C4), 132.8 (C6 ), 131.3 (C4 ), 130.4 (C1), 122.1 (C2 + C6), 119.4
1H NMR (CDCl3, 400 MHz): d = 12.88 (1H, s, 20-OH), 8.16 (1H, d,
(C5 ), 116.6 (C1 ), 115.4 (C3 ), 111.2 (C3 + C5), 56.0 (OMe), 36.2
0
0
0
0
0
0
0
0
J6 5 = 2.1, H6 ), 7.92 (1H, d, Jb = 15.4, Hb), 7.71 (1H, dd, J4 3 = 8.8,
(C ), 31.4 (Cb).
a
a
MS (EI, 70 eV): m/z (%) = 509 (5.4, M++1), 508 (33.6, M+), 507
(14.8, M+ꢀ1), 127 (8.8, I), 121 (100.0, C6H4(OH)CO+).
Anal. Calcd for C16H14I2O3: C, 37.82; H, 2.78. Found: C, 37.60; H,
2.66.
0
0
0
J4 6 = 2.1, H4 ), 7.67–7.62 (2H, m, H2 + H6), 7.42 (1H, d, J b = 15.3,
a
0
0
0
H ), 7.01–6.94 (2H, m, H3 + H5), 6.81 (1H, d, J3 4 = 8.8, H3 ), 3.88
a
(3H, s, OMe).
13
0
C NMR (CDCl3, 100 MHz): d = 192.5 (C@O), 163.1 (C2 ), 162.4
0
0
(C4), 146.4 (C4 ), 144.3 (Cb), 137.8 (C6 ), 130.8 (C2 + C6), 127.1 (C1),
0
0
0
122.3 (C1 ), 121.0 (C ), 116.9 (C3 ), 114.6 (C3 + C5), 79.6 (C5 ), 55.5
(OMe).
5.1.23. 20,2000-Dihydroxy-4,400-dimethoxy-50,5000-dichalcone, 41
Prepared according to general method 1, from 42 (135 mg,
0.5 mmol) and 48 (136 mg, 1 mmol), with stirring at rt for 24 h.
Yield: 169 mg (67%). An analytical sample was obtained by purifi-
cation by CC (hexanes?EtOAc/CH2Cl2/hexanes 1:5:5).
Yellow solid, mp: 131–132.5 °C.
a
MS (EI, 70 eV): m/z (%) = 381 (10.2, M++1), 380 (100.0, M+), 254
(M+ꢀI, 23.2), 127 (9.4, I).
Anal. Calcd for C16H14IO3: C, 50.55; H, 3.45. Found: C, 50.72; H,
3.28.
1H NMR (CDCl3, 400 MHz): d = 12.85 (1H, s, 20-OH), 8.25 (1H, d,
5.1.19. 20-Hydroxy-30-iodo-4-methoxychalcone, 36
Yellow solid, mp: 153–154 °C (lit.: 152.0–152.5 °C).87
1H NMR (CDCl3, 400 MHz): d = 13.92 (1H, s, 20-OH), 7.98–7.92
J6 4 = 2.2, H6 ), 7.80 (1H, dd, J4 3 = 8.7, J4 6 = 2.2, H4 ), 7.74 (1H, d,
Jb = 15.5, Hb), 7.68–7.59 (2H, m, H2 + H6), 7.52 (1H, d, J b = 15.5,
0
0
0
0
0
0
0
0
a
a
0
0
0
H ), 7.13 (1H, d, J3 4 = 8.7, H3 ), 6.94–6.81 (2H, m, H3 + H5), 3.89
a
0
0
(3H, m, Hb + H6 + H4 ), 7.66–7.58 (2H, m, H2 + H6), 7.52 (1H, d,
(s, 3H, OMe).
13
0
J
b = 15.4, H ), 7.00–6.93 (2H, m, H3 + H5), 6.74 (1H, t,
C NMR (CDCl3, 100 MHz): d = 193.5 (C@O), 161.9, 161.8 (C2 ,
a
a
0
0
0
0
0
0
0
0
J5 4 = J5 6 = 7.8, H5 ), 3.88 (3H, s, OMe).
C4), 145.8, 145.5, 145.2 (Cb, C4 , C5 ), 131.0 (C6 ), 130.9 (C2 + C6),
13
0
0
0
C NMR (CDCl3, 100 MHz): d = 191.4 (C@O), 160.0 (C4 + C2 ),
127.1 (C1), 119.1 (C3 ), 119.3 (C1 ), 117.1 (C ), 114.8 (C3 + C5), 55.9
a
0
0
0
146.4 (C4 ), 145.4 (Cb), 130.7 (C6 ), 129.7 (C2 + C6), 120.4 (C1 + C5 ),
(OMe).
0
0
127.0 (C ), 116.8 (C1 ), 114.6 (C3 + C5), 86.9 (C3 ), 55.5 (OMe).
MS (EI, 70 eV): m/z (%) = 507 (15.0, M++1), 506 (83.1, M+), 134
(100.0), 161 (13.2), 147 (17.8), 119 (10.1), 121 (52.3).
Anal. Calcd for C32H26O6: C, 75.88; H, 5.17. Found: C, 75.59; H,
5.31.
a
MS (EI, 70 eV): m/z (%) = 381 (14.2, M++1), 380 (100.0, M+), 254
(M+ꢀI, 17.9), 127 (6.7, I).
5.1.20. 20-Hydroxy-3,30,50-triiodo-4-methoxychalcone, 37
Yellow solid, mp: 97.5–100 °C.
5.1.24. 40,4000-Dimethoxy-6,600-diflavone, 49
1H NMR (CDCl3, 400 MHz): d = 13.79 (1H, s, 20-OH), 8.23 (1H, d,
Prepared according to general method 5, from 41 (152 mg,
0.3 mmol). Purified by CC (hexanes?EtOAc/hexanes 2:8). Yield:
134 mg (89%).
0
0
0
0
J4 6 = 2.0, H4 ), 8.16–8.14 (2H, m, H2 + H6 ), 7.86 (1H, d, Jb = 15.3,
Hb), 7.62 (1H, dd, J65 = 8.5, J62 = 2.1, H6), 7.39 (1H, d, J b = 15.3,
H ), 6.87 (1H, d, J56 = 8.5, H5), 3.96 (3H, s, OMe).
a
a
Yellowish solid, mp: 310 °C (dec) (lit.: 315–320 °C).87,89
a
13
1H NMR (CDCl3, 400 MHz): d = 8.35 (1H, d, J57 = 2.1, H5), 7.92–
0
C NMR (CDCl3, 100 MHz): d = 191.8 (C@O), 162.0 (C2 ), 160.7
0
0
0
0
0
(C4), 152.4 (C4 ), 145.7 (Cb), 139.5 (C2), 138.0 (C6 ), 131.5 (C6 ),
7.86 (2H, m, H2 + H6 ), 7.75 (1H, dd, J78 = 8.7, J75 = 2.1, H7), 7.28
0
0
0
0
0
128.9 (C1), 121.7 (C1 ), 117.2 (C ), 110.9 (C5), 88.0 (C3 o C5 ), 86.9
(1H, d, J87 = 8.6, H8), 7.08–7.01 (2H, m, H3 + H5 ), 6.71 (1H, s, H3),
a
0
0
(C3), 80.2 (C3 o C5 ), 56.6 (OMe).
3.88 (3H, s, OMe).
C NMR (CDCl3, 100 MHz): d = 178.2 (C@O), 162.7 (C4 ), 162.0
(C2), 159.4 (C8a), 149.0 (C6), 145.2 (C7), 128.3 (C2 + C6 ), 125.3
MS (EI, 70 eV): m/z (%) = 633 (13.2, M++1), 632 (100.0, M+), 254
(M+ꢀ3I, 16.2), 127 (7.4, I).
13
0
0
0
0
0
0
Anal. Calcd for C16H11I3O3: C, 30.41; H, 1.75. Found: C, 30.50; H,
2.01.
(C5), 124.5 (C1 ), 123.5 (C4a), 117.0 (C8), 114.0 (C3 + C5 ), 106.8
(C3), 55.9 (OMe).
MS (EI, 70 eV): m/z (%) = 503 (13.8, M++1), 502 (100.0, M+), 501
(34.5, M+ꢀ1), 251 (5.6, M+/2).
5.1.21. 3-Iodo-4-methoxyflavanone, 38
Yellowish solid, mp: 162–163 °C (lit.: 160 °C).88
Anal. Calcd for C32H22O6: C, 76.48; H, 4.41. Found: C, 76.25; H,
4.69.
1H NMR (CDCl3, 400 MHz): d = 7.93 (1H, dd, J56 = 7.6, J57 = 1.6,
0
0
0
H5), 7.92 (1H, d, J2 6 = 2.2, H2 ), 7.51 (1H, ddd, J78 = 8.3, J76 = 7.2,
5.1.25. 40,4000-Dihydroxy-6,600-diflavone, 50
0
0
0
0
0
J75 = 1.7, H7), 7.41 (1H, dd, J6 5 = 8.5, J6 2 = 2.1, H6 ), 7.09–7.01 (2H,
0
0
0
m, H6 + H8), 6.86 (1H, d, J5 6 = 8.4, H5 ), 5.39 (1H, dd, J2ax,3ax = 13.3,
J2ax,3eq = 2.8, H2), 3.91 (3H, s, OMe), 3.06 (1H, dd, J3ax,3eq = 16.8,
Prepared according to general method 7, from 49 (50 mg,
0.1 mmol), with stirring at rt for 7 days. Purified by CC (hex-
anes?EtOAc/hexanes 1:1). Yield: 43 mg (92%).
J3ax,2ax = 13.3, H3ax), 2.86 (1H, dd, J3eq,3ax = 16.8, J3eq,2ax = 2.9, H3eq).
C NMR (CDCl3, 100 MHz): d = 191.7 (C@O), 161.4 (C4 ), 158.5
(C8a), 139.5 (C2 ), 137.6 (C7), 132.9 (C1 ), 127.6 (C6 ), 127.1 (C5),
13
0
Whitish solid, mp: 265–267 °C.
1H NMR (CDCl3, 400 MHz): d = 9.88 (1H, s, OH), 8.34 (1H, d,
0
0
0
0
0
0
121.7 (C4a), 120.9 (C6), 118.1 (C8), 110.8 (C5), 86.3 (C3 ), 78.4 (C2),
56.5 (OMe), 44.5 (C3).
J57 = 2.2, H5), 7.90–7.85 (2H, m, H2 + H6 ), 7.76 (1H, dd, J78 = 8.6,
J75 = 2.2, H7), 7.27 (1H, d, J87 = 8.6, H8), 6.98–7.03 (2H, m,
MS (EI, 70 eV): m/z (%) = 381 (11.6, M++1), 380 (100.0, M+), 254
H3 + H5 ), 6.70 (1H, s, H3).
0
0
(M+ꢀI, 13.2), 127 (3.7, I).
C NMR (CDCl3, 100 MHz): d = 177.9 (C@O), 162.5, 162.1 (C4 ,
C2), 160.7 (C8a), 149.0 (C6), 144.9 (C7), 127.8 (C2 + C6 ), 126.1 (C5),
125.0 (C1 ), 121.5 (C4a), 117.5 (C8), 113.1 (C3 + C5 ), 106.2 (C3).
13
0
0
0
Anal. Calcd for C16H13IO3: C, 50.55; H, 3.45. Found: C, 50.72; H,
3.68.
0
0
0

3072
G. Sagrera et al. / Bioorg. Med. Chem. 19 (2011) 3060–3073
MS (EI, 70 eV): m/z (%) = 476 (11.4, M++2), 475 (23.0, M++1), 474
Anal. Calcd for C58H46O8: C, 79.98; H, 5.32. Found: C, 79.65; H,
5.21.
(100.1, M+), 474 (50.3, M+ꢀ1), 120 (17.4).
Anal. Calcd for C30H18O6: C, 75.94; H, 3.82. Found: C, 76.15; H,
3.49.
5.1.29. 7,700,40,4000-Tetrahydroxy-30,3000-biflavanone, 62
Prepared according to general method 8, from 61 (87 mg,
0.1 mmol), with stirring at rt for 6 h. Purified by CC (hex-
anes?EtOAc). Yield: 39 mg (76%).
5.1.26. 30,3000,40,4000-Tetramethoxy-3,300-biflavone, 58
Prepared according to general method 11, from 55 (41 mg,
0.1 mmol). Purified by CC (hexanes?EtOAc/hexanes 2:8). Yield:
18 mg (64%)
1H NMR (CDCl3, 400 MHz): d = 7.85 (1H, d, J56 = 8.8, H5), 7.03
0
0
0
(1H, m, H5 ), 6.95 (2H, m, H2 + H6 ), 6.64 (1H, dd, J65 = 8.7,
J68 = 2.3, H6), 6.51 (1H, d, J86 = 2.3, H8), 5.32 (1H, dd, J2,3ax = 13.0,
J2,3eq = 2.8, H2), 2.96 (1H, dd, J3ax,3eq = 16.8, J3ax,2 = 13.0, H3ax), 2.75
(1H, dd, J3eq,3ax = 16.8, J3eq,2 = 2.9, H3eq).
1H NMR (CDCl3, 400 MHz): d = 8.30 (1H, dd, J56 = 8.0, J57 = 1.5,
H5), 7.70 (1H, ddd, J78 = 8.6, J76 = 7.1, J75 = 1.5, H7), 7.63–7.58 (4H,
0
0
0
0
0
m, H6 + H8 + H2 + H6 ), 7.02 (1H, d, J5 6 = 8.2, H5 ), 3.97 (3H, s,
OMe), 3.95 (3H, s, OMe).
13C NMR (acetone-d6, 100 MHz): d = 190.4 (C@O), 161.7, 161.3
13C NMR (CDCl3, 100 MHz): 175.8 (C@O), 173.6 (C2), 155.4 (C8a),
(C8a, C7), 155.6 (C4 ), 135.0 (C1´ ), 131.1 (C2 ), 128.7 (C5), 124.8
0
0
0
0
0
0
0
150.3 (C3 ), 146.4 (C4 ), 134.1 (C7), 126.7, 126.4, 125.0, 123.8, 123.0
(C3 + C6 ), 118.0 (C5 ), 116.0 (C4a), 113.9 (C6), 99.1 (C8), 76.6 (C2),
44.7 (C3).
0
0
0
0
(C6, C1 , C5, C4a, C3), 119.4, 119.2 (C6 , C8), 110.7, 109.2 (C2 , C5 ), 56.9
(OMe), 56.7 (OMe).
MS (EI, 70 eV): m/z (%) = 511 (17.4, M++1), 510 (100.0, M+), 509
(56.3, M+ꢀ1), 492 (47.9, M+ꢀ18), 255.8 (7.1, M+/2).
Anal. Calcd for C30H22O8: C, 70.58; H, 4.34. Found: C, 70.25; H,
4.23
MS (EI, 70 eV): m/z (%) = 564 (3.8, M++2), 563 (27.2, M++1), 562
(100.0, M+), 561 (57.4, M+ꢀ1), 544 (11.4, M+ꢀH2O), 281 (10.0, M+/
2), 121 (47.2, 1,3A+), 92 (17.4, C6H5O+).
Anal. Calcd for C34H26O8: C, 72.59; H, 4.66. Found: C, 72.25; H,
4.92.
5.2. Biology
5.1.27. 20,2000-Dihydroxy-4,40,400,4000-tetrabenzyloxy-3,300-
dichalcone, 60
For the antifungal evaluation fresh cultures of C. albicans (Ca)
ATCC 90028, C. parapsilosis (Cp) ATCC 22019, C. neoformans (Cn)
ATCC 14116; A. niger (An) ATCC 16404, R. stolonifer (Rs) ATCC
6227b were used. For each extract, the MIC value was determined
by using broth microdilution techniques according to the guide-
lines of the NCCLS for yeasts (M27-A2) and for filamentous fungi
(M 38 A).90,91 MIC values were determined in Sabouraud dextrose
broth (SDB) (Sigma, St. Louis, MO, USA) buffered to pH 7.0 with
MOPS. Microtiter trays were incubated at 28–30 °C for all strains
in a moist, dark chamber. MICs were visually recorded at 48 h for
yeasts, 72 h for C. neoformans.
Prepared according to general method 1, from 24 (484 mg,
2 mmol) and 59 (422 mg, 1 mmol), with stirring at rt for 24 h.
Yield: 626 mg (72%). A sample for elemental analysis was purified
by CC (hexanes?EtOAc/CH2Cl2/hexanes 1:5:5).
Yellow solid, mp: 143–144.5 °C.
1H NMR (CDCl3, 400 MHz): d = 13.45 (1H, s, 20-OH), 7.78 (1H, d,
0
J6´ 5´ = 8.0, H6 ), 7.77 (1H, d, Jb = 15.5, Hb), 7.48–7.29 (11 H, m, phe-
a
nyl ꢁ 2 + H )à, 7.22 (1H, d, J26 = 1.8, H2), 7.19 (1H, dd, J65 = 8.3,
a
J62 = 1.7, H6), 6.95 (1H, d, J56 = 8.3, H5), 6.57–6.55 (2H, m, H3´ + H5´ ),
5.21 (2H, s, CH2), 5.11 (2H, s, CH2).
For the assay, 40
(50 mg/mL) was diluted with 960
2 mg/mL. Two hundred microliters of this new solution was
poured into the first well and then, 100 L were transferred to
the next well containing 100 L of SDB. The same procedure was
performed for all wells of the same file obtaining twofold dilutions
of the extract. A volume of 100 L of inoculum suspension was
l
L of a stock solution of each extract in DMSO
13C NMR (CDCl3, 100 MHz): d = 191.8 (C@O), 166.6, 165.2 (C2
lL of SDB giving a solution of
0
0
C4 ), 161.0 (C4), 151.6 (C1), 144.3 (Cb), 137.0, 136.7, 135.9, 131.1,
128.7,
128.6,
128.3,
128.0,
127.5,
127.4,
127.2
l
0
(C6 + C2 + C3 + C5 + (C1–C6)phenyl), 123.6 (C6), 118.4 (C ), 114.4
l
a
0
0
0
(C1 ), 108.1, 102.1, (C3 , C5 ), 71.7 (CH2), 71.0 (CH2).
MS (EI, 70 eV): m/z (%) = 690 (15.2, M+ꢀ2Bn), 600 (8.7,
M+ꢀ3Bn), 510 (30.1, M+ꢀ4Bn), 91 (100.0, Bn+).
Anal. Calcd for C58H46O8: C, 79.98; H, 5.32. Found: C, 80.09; H,
5.12.
l
added to each well (with the exception of the sterility control
where sterile water was added to the well instead) giving extract0s
concentrations from 1000 to 0.98 lg/mL and a final DMSO concen-
tration 61%. Amphotericin B was used as positive control for yeasts
and Aspergillus spp. End-points were defined as the lowest concen-
tration of drug resulting in total inhibition (MIC) of visual growth
compared to the growth in the control wells containing no anti-
fungal. Tests were carried out by duplicate.
5.1.28. 7,700,40,4000-Tetrabenzyloxy-30,3000-biflavanone, 61
Prepared according to general method 3, from 60 (435 mg,
0.5 mmol), refluxing for 96 h. Purified by CC (hexanes?EtOAc/
CH2Cl2/hexanes 1:5:5). Yield: 217 mg (50%).
1H NMR (CDCl3, 400 MHz): d = 7.86 (1H, d, J56 = 8.8, H5), 7.48–
0
7.24 (10 H, m, phenyl ꢁ 2), 7.07 (1H, m, H5 ), 6.96 (2H, m,
Acknowledgments
0
0
H2 + H6 ), 6.68 (1H, dd, J65 = 8.8, J68 = 2.3, H6), 6.53 (1H, d,
J86 = 2.3, H8), 5.33 (1H, dd, J2,3ax = 13.1, J2,3eq = 2.8, H2), 5.17 (2H, s,
CH2), 5.08 (2H, s, CH2), 2.96 (1H, dd, J3ax,3eq = 16.8, J3ax,2 = 13.1,
The authors wish to thank Facultad de Química, UdelaR, Uru-
guay. The assistance of Mr. Horacio Pezaroglo for recording NMR
spectra is kindly acknowledged.
H
3ax), 2.74 (1H, dd, J3eq,3ax = 16.9, J3eq,2 = 2.9, H3eq).
13C NMR (CDCl3, 100 MHz): d = 190.6 (C@O), 165.2 (C8a), 163.4
0
(C7), 149.5 (C4 ), 137.1, 137.0, 135.9, ((C1phenyl) ꢁ 2 + C1´ ), 131.9
Supplementary data
0
(C3 ), 128.8, 128.7, 128.5, 128.3, 128.0, 127.9, 127.8, 127.5, 127.4
0
0
(C2 + C6 + C5 + (C2–6 phenyl
)
ꢁ 2), 115.0 (C4a), 113.5 (C5), 110.8 (C6),
Supplementary data (experimental procedures and spectral
data for known compounds) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2011.04.010.
101.9 (C8), 79.8 (C2), 71.5 (CH2), 71.3 (CH2), 44.1 (C3).
MS (EI, 70 eV): m/z (%) = 690 (13.0, M+ꢀ2Bn), 600 (7.8,
M+ꢀ3Bn), 510 (25.3, M+ꢀ4Bn), 91 (100.0, Bn+).
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à
H
a
appears as a doublet at 7.33 ppm (J b = 15.3 Hz), partly overlapped with the
a
signals corresponding to phenyls.

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