Synthesis and antimalarial evaluation of novel benzopyrano[4,3-b]benzopyran derivatives

Article abstract of DOI:10.1016/j.bmc.2011.07.009

7-Methoxyflavenes and 5,7,8-trimethoxyflavenes were found to undergo stereoselective acid-catalyzed rearrangement to generate the benzopyrano[4,3-b]benzopyran ring system present in the natural product, dependensin. Dependensin and its analogs were subjected to antimalarial growth inhibition assays against Plasmodium falciparum and found to have IC ₅₀ values ranging between 1.9 and 3.9 μM.

Full text of DOI:10.1016/j.bmc.2011.07.009

Bioorganic & Medicinal Chemistry 19 (2011) 5199–5206
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antimalarial evaluation of novel benzopyrano[4,3-b]benzopyran
derivatives
Ruth Devakaram a, David StC. Black a, Katherine T. Andrews b,c, Gillian M. Fisher b,c, Rohan A. Davis b,
Naresh Kumar a,
⇑
a School of Chemistry, The University of New South Wales, UNSW, NSW, Sydney 2052, Australia
b Eskitis Institute for Cell and Molecular Therapies, Griffith University, Queensland 4111, Australia
c Queensland Institute of Medical Research, Tropical Parasitology Laboratory, 300 Herston Road, Herston, Brisbane 4006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
7-Methoxyflavenes and 5,7,8-trimethoxyflavenes were found to undergo stereoselective acid-catalyzed
rearrangement to generate the benzopyrano[4,3-b]benzopyran ring system present in the natural prod-
uct, dependensin. Dependensin and its analogs were subjected to antimalarial growth inhibition assays
Received 18 April 2011
Revised 30 June 2011
Accepted 6 July 2011
Available online 23 July 2011
against Plasmodium falciparum and found to have IC50 values ranging between 1.9 and 3.9 lM.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dependensin
Dimerization
Flavenes
Acid-catalyzed
Benzopyrans
1. Introduction
low abundance in nature, tedious extraction and purification
procedures, and limited biological data of the biflavonoids class.
Therefore, it is highly desirable to develop efficient synthetic meth-
odologies, which could generate not only the natural products
themselves but also their synthetic analogs for pharmacological
applications.
Flavonoids are a group of heterocyclic polyphenolic compounds
widely distributed in the plant kingdom. They occur naturally in
fruits, vegetables, nuts, seeds and flowers and form a significant
part of our daily diet.1 Flavonoids are known to display a diverse
range of biological and pharmacological activities.1 For example,
baicalein, oroxylin A and wogonin, the three major flavonoids iso-
lated from Scutellaria baicalensis, a traditional Chinese herb are well
noted for their broad spectrum of biological activities, in particular
for their antioxidant properties.2
Biflavonoids are comprised of two identical or non-identical fla-
vonoid units, joined symmetrically or unsymmetrically through
linkers of varying lengths. The large number of possibilities in
the length, position and type of linkages, as well as in the number
and nature of substituents, gives rise to an enormous structural
diversity to the biflavonoids class.3 Biflavonoids have received
increasing recognition due to their wide spectrum of pharmacolog-
ical properties, including anticancer, antiinflammatory, antimicro-
bial, antiviral, and anticlotting activities.3
Dependensin 1 is a dimeric flavonoid isolated from the root
bark of a Tanzanian medicinal plant, Uvaria dependens. The crude
extract of this plant shows potent antimalarial activity.4 It is
well-known that malaria constitutes a major health problem in
tropical and sub-tropical regions of the world, with 200–350 mil-
lion cases annually and mortality reaching 1 million, particularly
among children in sub-Saharan Africa.5 Malaria is caused by proto-
zoan parasites of the phylum Apicomplexa, which are transmitted
by blood feeding Anopheline mosquitoes. There are four main
human malaria parasites, that is, Plasmodium ovale, Plasmodium
malaria, Plasmodium vivax and Plasmodium falciparum, with the
latter two being responsible for the majority of malaria cases
worldwide. P. falciparum, the most virulent of the four main species
infecting humans, has become resistant to nearly all currently em-
ployed antimalarial drugs used for prophylaxis and treatment.6
Even artemisinin combination therapy (ACT), the current first
treatment for drug-resistant P. falciparum malaria is now under
threat.7
Despite their promising biological activities, the development
of biflavonoids as therapeutic agents has been hampered by their
First cases of resistance against P. falciparum were reported
in the 1970s against the popular antimalarials chloroquine and
⇑
Corresponding author. Tel.: +61 2 9385 4698; fax: +61 2 9385 6141.
E-mail address: n.kumar@unsw.edu.au (N. Kumar).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.009

5200
R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
sufadoxine-pyrimethamine, barely 10 years after these drugs were
introduced for the treatment of malaria. Chloroquine has also been
the therapy of choice for the treatment of blood stage vivax malar-
ia, but chloroquine resistance in P. vivax has developed since the
nineties of the last century. Besides the increasing drug resistance
in plasmodia strains worldwide, an effective drug employment is
hampered by high costs and major side effects of other antimalari-
als like mefloquine or atovaquone.6
has been commonly employed,10 which has been followed by
cyclization in the presence of strong acids to furnish flavanones
in typically low yields.10 Alternatively, intramolecular oxidative
cyclization of chalcones with I2/DMSO could yield the correspond-
ing flavones in good yields.11 However, in both cases, a further
reduction of the flavones/flavanones to the corresponding flavanols
followed by subsequent dehydration was required to yield the de-
sired flavenes.12
These developments emphasize the need for new strategies in
combating the tropical disease. Hence, our research group is
addressing the need to develop new drugs. Here, we present data
on the development of efficient synthetic methodologies to
dependensin and its analogs, as potential new lead antimalarial
compounds.
Clark-Lewis and Jemison report the direct conversion of
20-hydroxychalcones into the corresponding flav-3-enes by reduc-
tion with NaBH4 in IPA.13 This method of reductive cyclization
could generate the desired flavenes in fewer steps as well as gen-
erate improved overall yields.
Hence, the hydroxy group in the commercially available
20,40-dihydroxyacetophenone 2 was masked by simple methylation
by treatment with anhydrous K2CO3 and CH3I in dry acetone to give
20-hydroxy-40-methoxyacetophenone 3 in 92% yield. Acetophenone
3 was condensed with various para-substituted aryl aldehydes 4a–d
in ethanolic NaOH solution to afford the corresponding chalcones
5a–d in 65–73% yields. Finally, reductive cyclizationof the chalcones
using NaBH4 in IPA as solvent yielded the desired flavenes 6a–d in
50–58% yields (Scheme 1).
MeO
OMe
OMe
MeO
MeO
H
O
MeO
H
H
O
H
The 1H NMR spectrum of 40-bromo-7-methoxyflavene 6b exhib-
ited three doublets of doublets at d 5.61 (J = 3.4, 9.8 Hz, 1H), d 5.83
(J = 1.5, 3.4 Hz, 1H) and d 6.50 (J = 1.5, 9.8 Hz, 1H) corresponding to
H3, H2 and H4 respectively. The 1H NMR spectra of the other fla-
venes were found to exhibit similar coupling patterns in ring A.
Furthermore, the DEPT-135 and the broadband decoupled 13C
NMR spectra of flavenes 6a–d indicated the absence of methylene
(CH2) groups, confirming that the flav-2-ene was not formed. The
structure of 40-bromo-7-methoxyflavene 6b was confirmed by
X-ray crystallography (Fig. 1).14
Dependensin 1
The unique heterocyclic ring system present in dependensin
contains a dense array of functionality and stereochemistry, which
includes two-fused benzopyran rings, four stereocenters and one
trans double bond.4
Nkunya et al. proposed that the natural product, dependensin
originated from the acid-catalyzed reaction of the corresponding
flavene, 5,7,8-trimethoxyflav-3-ene, but were unable to verify it
experimentally, obtaining a ring-opened compound instead.4 How-
ever, the successful synthesis of dependensin via the acid-cata-
lyzed reaction of 5,7,8-trimethoxyflavene has been recently
reported by our research group.8 We have also previously shown
that the benzopyrano[4,3-b]benzopyran ring system present in
dependensin could be synthesized via the acid-catalyzed dimeriza-
tion reaction of 40,7-diacetoxyflav-3-ene.8 Similar acid-catalyzed
reactions of the corresponding 40,5-diacetoxy- and 40,6-diacetoxy-
flavenes produced a complex mixture of products.8
O
O
CHO
a
+
HO
OH
MeO
OH
R
2
3
4a
R = H
4b R = Br
4c
R = Cl
4d R = OMe
O
Encouraged by these results, we targeted the acid-catalyzed
reactions of 7-methoxyflavenes and 5,7,8-trimethoxyflavenes to
generate novel dependensin analogs for biological applications.
Interestingly, we found that the dimerization products obtained
were dependent on the position of the methoxy group in the
flavene nucleus. We have also recently reported the synthesis of
biflavonoids containing a new ring system, a tetrahydrochro-
meno[2,3-b]chromene, generated via the acid-catalyzed reactions
of 5-methoxy- and 6-methoxyflavenes.9
c
b
MeO
OH
5a-d
MeO
O
R
6a-d
R
Scheme 1. Reagents and conditions: CH3I, K2CO3, acetone, reflux, 24 h, 92%; (b)
NaOH, ethanol, 12 h, rt, 65–73%; (c) NaBH4, IPA, 12 h, rt, 50–58%.
We report herein the facile one step methodology to the synthe-
sis of a series of highly functionalized benzopyrano[4,3-b]benzopy-
rans from the acid-catalyzed reactions of 7-methoxyflavenes and
5,7,8-trimethoxyflavenes along with their antimalarial inhibition
assay results.
2. Results and discussion
2.1. Synthesis of 7-methoxyflavenes
Flavenes are direct precursors to the dimeric flavonoids and
therefore, an efficient route to their synthesis was considered
highly desirable for this study. The base catalyzed condensation
of hydroxyacetophenones with aldehydes to generate chalcones
Figure 1. ORTEP diagram of compound 6b.

R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
5201
2.1.1. Acid-catalyzed reactions of 7-methoxyflavenes
We have previously proposed a rationale for the observed acid-
catalyzed dimerization reaction.8 It is assumed that the flavene 8
protonates and ring opens resulting in the formation of a stable
benzylic carbocation 9. A second molecule of flavene 8 could pos-
sibly attack this benzylic carbocation 9 in such a way as to generate
a second stable benzylic carbonium ion 10, which upon cyclization,
by loss of a proton yields the dimeric system 12 (Scheme 3).
Alternatively, the low stability of flav-3-enes 8 containing the
2H-pyran ring may facilitate a Claisen-type rearrangement to oc-
cur, leading to the formation of reactive orthoquinone methide
11. A Diels–Alder reaction of the intermediate orthoquinone met-
hide 11 with the second molecule of flavene 8, followed by ring
closure could also possibly result in the formation of the dimer
12 (Scheme 3).
The 7-methoxyflavenes 6a–d were subjected to the acid-cata-
lyzed dimerization reaction, using HCl, TFA or glacial AcOH as cat-
alysts in MeOH, giving bisflavonoids 7a–d as analogs of the natural
product dependensin (Scheme 2). The dimerization products ob-
tained were the same irrespective of the catalyst utilized for these
reactions.
A similar acid-catalyzed dimerization reaction of
40,7-diacetoxyflavene has been reported previously.8
These dimers 7a–d contain four tertiary aliphatic protons, two
of which are attached to oxygenated carbon atoms. Thus, the iden-
tification of protons H6, H7, H6a and H12a were of prime impor-
tance in assigning the structure of the product. In the dimeric
flavonoid 7b, a doublet of doublet of doublets at d 2.43 (J = 2.1,
2.3, 10.9 Hz, 1H) correlated to H6a, a doublet of doublets at d
3.13 (J = 2.1, 6.4 Hz, 1H) correlated to H7, and two doublets at d
5.03 (J = 10.9 Hz, 1H) and d 5.05 (J = 2.3 Hz, 1H) correlated to H6
and H12a, respectively. The protons present on the trans double
2.2. Synthesis of 5,7,8-trimethoxyflavenes
bond, represented as Hb and H , were identified as a doublet at d
The synthesis of 5,7,8-trimethoxyflav-3-enes 14a–d was under-
taken with the aim of producing direct analogs of the natural prod-
uct, dependensin. The 5,7,8-trimethoxyflavenes 14a–d were
synthesized from the corresponding chalcones 13a–d in 56–63%
yields by the method of reductive cyclization in a similar fashion
as before (Scheme 4).
a
5.99 (J = 15.8 Hz, 1H) and a doublet of doublets at d 6.22 (J = 6.4,
15.8 Hz, 1H), respectively. The dimeric flavonoids were obtained
in moderate yields of 65–71% (Table 1).
The chalcones 13a–d were synthesized by base catalyzed
Claisen–Schmidt condensation of 20-hydroxy-30,40,60-trimethoxy-
acetophenone with para-substituted benzaldehydes, 4b–e (4e
R = Me).
OMe
H
MeO
H+, methanol
O
12a
H
H
O
6
7
H
MeO
O
α
65-71%
2'
β
2.2.1. Acid-catalyzed reactions of 5,7,8-trimethoxyflavenes
The 5,7,8-trimethoxyflavenes were then subjected to acid-cata-
lyzed reactions in MeOH as before, which furnished direct analogs
of the natural product 15a–d as desired (Scheme 5). The optimum
yields were obtained with the use of TFA as catalyst and it was ob-
served that use of glacial acetic acid as catalyst resulted in longer
reaction times.
R
6a-d
2''
R
R = H,Br, Cl, OMe
7a-d
R
Scheme 2.
The 1H NMR spectra of these dimers were found to have cou-
pling patterns similar to those resulting from the dimerization of
7-methoxyflavenes. The dimeric flavonoids were obtained in good
yields of 68–73% (Table 2).
Table 1
Dimerization products of 7-methoxyflavenes
Flavenea
R
Benzopyranobenzopyran
Yieldsb (%)
The X-ray crystal structure15 of compound 15c confirmed the
stereochemistry of the product, which was found to be identical
to that of the parent dependensin as reported in the literature
(Fig. 2).3
6a
6b
6c
6d
H
Br
Cl
OMe
7a
7b
7c
7d
65
71
69
66
a
Catalysed by HCl.
b
Yields of isolated pure product.
MeO
O
MeO
a
OMe
MeO
OH
MeO
O
MeO
MeO
R
R
14a-d
13a-d
O
R = Br, Cl, OMe, Me
Ar
Ar
O
OMe
8
Ar
Ar
Scheme 4. Reagents and conditions: (a) NaBh4, IPA, 12 h, rt, 56–63%.
MeO
O
Ar
MeO
O
H
MeO
O
8
9
H
10
MeO
OMe
OMe
MeO
MeO
H
MeO
MeO
MeO
H+, methanol
O
H
H
H
R
Ar
O
Ar
MeO
O
Ar
MeO
O
H
Ar
68-73%
O
R
11
O
14a-d
OMe
MeO
O
R = Br, Cl, OMe, Me
8
OMe
15a-d
12
R
Scheme 3.
Scheme 5.

5202
R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
Table 2
found to be the most active with an IC50 value of 1.9 lM. However,
Dimerization products of 5,7,8-trimethoxyflavenes
all analogs showed similar antimalarial activity thus identifying
that C40 and C400 substituents have minimal effect on the ability
of these molecules to inhibit malaria parasite growth. Likewise,
all compounds showed similar cytotoxicity against NFF cells which
are commonly used as a normal cell control (Table 3). All com-
pounds, except 15b, have around 10 fold selectivity for malaria
parasites versus the NFF cells. The synthesis of further dimeric ana-
logs that incorporate substituent variation at other positions
around the tetracyclic dependensin skeleton are required in order
to determine whether this structure class has the potential to pro-
vide antimalarial lead molecules.
Flavenea
R
Benzopyranobenzopyran
Yieldsb (%)
14a
14b
14c
14d
Br
Cl
OMe
Me
15a
15b
15c
15d
68
70
71
73
a
Catalysed by TFA.
Yields of isolated pure product.
b
3. Conclusion
A facile simple one step methodology to the synthesis of a series
of novel benzopyrano[4,3-b]benzopyrans has been developed via
the acid-catalyzed dimerization reactions of 7-methoxyflavenes
and 5,7,8-trimethoxyflavenes. Moderate antimalarial activity cou-
pled with synthetic tractability of several synthetic bisflavonoids
suggests that this structure class may warrant further investigation
as potential antimalarial agents.
4. Experimental section
4.1. Materials and methods
Figure 2. ORTEP diagram of compound 15c.
All reagents and solvents were obtained from commercial
sources and purified if necessary. Melting points were measured
using a Mel-Temp melting point apparatus and are uncorrected.
Microanalyses were performed on a Carlo Erba Elemental Analyser
EA 1108 at the Campbell Microanalytical Laboratory, University of
Otago, New Zealand. 1H and 13C NMR spectra were obtained on
Bruker DPX300 (300 MHz). Mass spectra were recorded on either
a Bruker FT-ICR MS (EI) or a Micromass ZQ2000 (ESI). Infrared
spectra were recorded with a Thermo Nicolet 370 FTIR Spectrom-
eter using KBr discs. Column chromatography was carried out
using Merck 230–400 mesh ASTM silica gel, and preparative thin
layer chromatography was performed using Merck silica gel 7730
Importantly, the acid-catalyzed reactions of 7-methoxyflavenes
and 5,7,8-trimethoxyflavenes can give rise to different stereoiso-
mers, owing to the presence of four stereocenters in their struc-
tures. Interestingly, we observed the formation of a single isomer
in all cases, evident from the coupling constants of the aliphatic
protons. Hence, these acid-catalyzed reactions were highly stereo-
selective; one of the reasons may be attributed to the exceptionally
high stability of the carbocation intermediates generated during
the course of their rearrangement.
The identity and purity (>98%) of all compounds were
confirmed by 1H and 13C NMR spectroscopy (1D and 2D NMR spec-
troscopy), IR spectroscopy, mass spectrometry, thin layer chroma-
tography and elemental analyzes.
60GF254
.
4.1.1. 20-Hydroxy-40-methoxyacetophenone (3)
To
solution of 20,40-dihydroxyacetophenone
2.3. Biological activity of dependensin analogs
a
2
(5.0 g,
32.86 mmol) in acetone (75 mL), was added K2CO3 (9.1 g,
65.72 mmol). The reaction mixture was cooled to 0 °C and MeI
(2.1 mL, 32.86 mmol) was added to it slowly. The reaction mixture
was then refluxed for 24 h. The solvent was evaporated, and the res-
idue was acidified using 2 M HCl to pH 3. The mixture was extracted
with EtOAc (3 ꢀ 100 mL), washed with brine (100 mL), dried over
anhydrous Na2SO4 and evaporated under vacuum. The residue was
recrystallized from EtOAc/light petroleum (2:8) to afford the title
compound as off-white crystals (5.0 g, 92%). Mp 53–55 °C, lit.16
52–54 °C.
This paper is the first report of antimalarial activity for the nat-
ural product, dependensin 1 and it analogs 15a–d. These novel
benzopyrano[4,3-b]benzopyran derivatives were screened for anti-
malarial activity against a chloroquine sensitive P. falciparum line
(3D7) using standard in vitro growth inhibition assays. The antima-
larial drug, chloroquine was used as the positive growth inhibition
control. Table 3 gives the IC50 values of the tested compounds.
Among the compounds screened for antimalarial activity, com-
pound 15d, which bears methyl substituent at C40 and C400, was
4.1.2. General procedure for the synthesis of chalcones (5a–d)
To a solution of the appropriate acetophenone 3 (1.0 equiv) in
EtOH (50 mL) was added the corresponding aldehydes 4a–d
(1.0 equiv). This was followed by slow addition of crushed NaOH
pellets (2.5 equiv). The reaction mixture was stirred for 12 h at
rt, poured into ice (250 g) and acidified using conc. HCl to pH 3.
The solid so obtained was filtered and air-dried. Recrystallization
from EtOH afforded the desired chalcones as yellow/orange
crystals.
Table 3
Antimalarial activities for compound 1, 15a–d
Compound
P. falciparum 3D7
NFFa cytotoxicity % inhibition
( SD) at 50 lM
IC50 ( SD) (
lM)
1
15a
15b
15c
15d
Chloroquine
3.9 ( 1.5)
2.9 ( 1.7)
3.3 ( 1.2)
3.2 ( 0.6)
1.9 ( 0.50
46.9 ( 0.3)
46.3 ( 5.4)
98.4 ( 0.1)
43.2 ( 3.8)
47.2 ( 0.6)
nd
0.02 ( 0.01)
In cases when the chalcones did not precipitate out on acidifi-
cation, the aqueous layer was extracted with DCM (3 ꢀ 100 mL).
a
NFF, neonatal foreskin fibroblast cells; nd, not determined.

R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
5203
The organic layer was washed with brine (100 mL), dried over
anhydrous Na2SO4 and evaporated under reduced pressure. The
residue so obtained was then recrystallized from EtOH.
107.1 (C6), 114.4 (C4a), 121.1 (C4), 124.0 (C3), 127.3 (C5), 128.4
(C20, C60), 128.7 (C30, C50), 134.1 (C40), 139.3 (C10), 154.0 (C8a),
160.9 (C7); MS (TOF-ESI) m/z calcd for C16H13ClO2 (M+1)+ 273.07.
Found 273.08; Anal. Calcd for C16H13ClO2ꢂ1/10H2O: C, 70.00; H,
4.85. Found: C, 70.09; H, 4.47.
4.1.2.1. 20-Hydroxy-40-methoxychalcone (5a). Yellow crystals,
yield: 72%; mp 107–108 °C, lit.17 104–105 °C.
4.1.4.3. 40,7-Dimethoxyflav-3-ene (6d). White solid, yield: 54%;
mp 79–81 °C, lit.22 81–82 °C.
4.1.2.2. 4-Bromo-20-hydroxy-40-methoxychalcone (5b). Yellow
crystals, yield: 65%; mp 138–140 °C, lit.18 138–140 °C.
4.1.4.4. 40-Bromo-5,7,8-trimethoxyflav-3-ene (14a). Yellow
4.1.2.3. 4-Chloro-20-hydroxy-40-methoxychalcone (5c). Yellow
crystals, yield: 73%; mp 100–102 °C, lit.19 109–110 °C.
sticky residue, yield: 56%; UV (MeOH): kmax 204 (e 61,481
cmꢁ1 Mꢁ1), 223 (49,023), 296 (15,338) nm; IR (KBr): mmax 3438,
3010, 2933, 2840, 1609, 1561, 1504, 1464, 1437, 1347, 1239,
4.1.2.4. 20-Hydroxy-4,40-dimethoxychalcone (5d). Yellow crys-
tals, yield: 71%; mp 114–116 °C, lit.20 116–118 °C.
1206, 1138, 1116, 1070, 1010, 811 cmꢁ1 1H NMR (300 MHz,
;
CDCl3): d 3.66, 3.82 and 3.86 (3s, 9H, 3 ꢀ CH3O), 5.66 (dd, J = 3.8,
10.2 Hz, 1H, H3), 5.84 (dd, J = 1.5, 3.8 Hz, 1H, H2), 6.06 (s, 1H,
H6), 6.82 (dd, J = 1.5, 10.2 Hz, 1H, H4), 7.34 (d, J = 8.3 Hz, 2H, H20,
H60), 7.46 (d, J = 8.3 Hz, 2H, H30, H50); 13C NMR (75.6 MHz, CDCl3):
d 55.8, 56.0 and 61.2 (3 ꢀ CH3O), 75.7 (C2), 89.3 (C6), 105.1 (C4a),
119.1 (C4), 122.2 (C40), 126.8 (C3), 128.8 (C20, C60), 130.4 (C30, C50),
139.3 (C8), 146.2 (C8a), 146.5 (C10), 151.7 (C5), 153.2 (C7); MS
(TOF-ESI) m/z calcd for C18H17BrO4 (M+1)+ 377.04 (Br79). Found
376.96 (Br79); Anal. Calcd for C18H17BrO4: C, 57.31; H, 4.54. Found:
C, 57.53; H, 4.83.
4.1.3. General procedure for the synthesis of flavenes (6a–d,
14a–d)
To a solution of the appropriate chalcone (1.0 equiv) in IPA
(30 mL) at 50 °C was slowly added NaBH4 (3.0 equiv) in small por-
tions over 15 min. The reaction mixture was cooled to rt and left to
stir overnight. The solvent was evaporated partially, ice (50 g) was
added and the resulting solution was acidified using 10% AcOH to
pH 5. The solution was extracted with DCM (2 ꢀ 150 mL), and the
organic layer was washed with brine (100 mL), dried over anhy-
drous Na2SO4 and evaporated under reduced pressure. Purification
of the residue by column chromatography over silica gel using
DCM/light petroleum (20:80) eluted the desired flavene and fur-
ther elution using DCM/light petroleum (40:60) gave the unre-
acted chalcone. The flavenes so obtained were either low melting
white solids or yellow sticky oily residues. As the flavenes were
found to be relatively unstable, they were immediately used in
the next subsequent step of dimerization. Otherwise, they were
dissolved in MeOH and stored at room temperature to avoid
decomposition.
4.1.4.5. 40-Chloro-5,7,8-trimethoxyflav-3-ene (14b). Yellow
sticky residue, yield: 58%; UV (MeOH): kmax 204 (
e 103,221
cmꢁ1 Mꢁ1), 221 (74,779), 297 (18,053) nm; IR (KBr): mmax 3431,
3015, 2934, 2838, 1610, 1505, 1465, 1435, 1351, 1239, 1214,
1136, 1116, 1066, 1031, 809 cmꢁ1 1H NMR (300 MHz, CDCl3): d
;
3.80, 3.83 and 3.86 (3s, 9H, 3 ꢀ CH3O), 5.66 (dd, J = 3.0, 9.8 Hz,
1H, H3), 5.85 (dd, J = 1.5, 3.0 Hz, 1H, H2), 6.05 (s, 1H, H6), 6.85
(dd, J = 1.5, 9.8 Hz, 1H, H4), 7.30 (d, J = 8.6 Hz, 2H, H20, H60), 7.40
(d, J = 8.6 Hz, 2H, H30, H50); 13C NMR (75.6 MHz, CDCl3): d 55.8,
56.0 and 61.1 (3 ꢀ CH3O), 75.7 (C2), 89.3 (C6), 105.1 (C4a), 119.0
(C4), 128.4 (C20, C60), 128.5 (C3), 128.6 (C30, C50), 134.0 (C40),
138.8 (C8), 138.9 (C10), 146.5 (C8a), 151.2 (C5), 153.7 (C7); MS
(TOF-ESI) m/z calcd for C18H17ClO4Na (M+Na)+ 355.07. Found
355.01; Anal. Calcd for C18H17ClO4ꢂH2O: C, 61.63; H, 5.46. Found:
C, 61.80; H, 5.42.
4.1.4. 7-Methoxyflav-3-ene (6a)
White solid, yield: 58%; mp 103–105 °C, lit.21 102–103 °C.
4.1.4.1. 40-Bromo-7-methoxyflav-3-ene (6b). Yellow sticky resi-
due, yield: 53%; UV (MeOH): kmax 202 (e
46,842 cmꢁ1 Mꢁ1), 230
(50,861), 306 (11,829) nm; IR (KBr): mmax 3448, 3022, 2975,
4.1.4.6. 5,7,8,40-Tetramethoxyflav-3-ene (14c). Yellow sticky
2934, 2843, 1614, 1503, 1444, 1308, 1275, 1198, 1154, 1110,
residue; yield: 63%; UV (MeOH): kmax 204 (e
25,961 cmꢁ1 Mꢁ1),
1026, 968, 812, 708 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 3.75 (s,
232 (20,630), 274 (8299) nm; IR (KBr): mmax 3434, 3008, 2935,
3H, CH3O), 5.61 (dd, J = 3.4, 9.8 Hz, 1H, H3), 5.83 (dd, J = 1.5,
3.4 Hz, 1H, H2), 6.37 (d, J = 2.3 Hz, 1H, H8), 6.43 (dd, J = 2.3,
8.3 Hz, 1H, H6), 6.50 (dd, J = 1.5, 9.8 Hz, 1H, H4), 6.92 (d,
J = 8.3 Hz, 1H, H5), 7.32 (d, J = 8.7 Hz, 2H, H20, H60), 7.49 (d,
J = 8.7 Hz, 2H, H30, H50); 13C NMR (75.6 MHz, CDCl3): d 55.2
(CH3O), 76.4 (C2), 101.8 (C8), 107.1 (C6), 114.4 (C4a), 121.3 (C4),
122.3 (C40), 124.0 (C3), 127.3 (C5), 128.7 (C20, C60), 131.7 (C30,
C50), 139.8 (C10), 154.0 (C8a), 160.9 (C7); (TOF-ESI) m/z calcd
for C16H13BrO2Na (M+Na)+ 339.00 (Br79). Found 339.04 (Br79);
Anal. Calcd for C16H13BrO2: C, 60.59; H, 4.13. Found: C, 60.87; H,
4.35.
2839, 1607, 1511, 1464, 1439, 1347, 1246, 1211, 1135, 1114,
1064, 1032, 811 cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.61, 3.78,
;
3.82 and 3.84 (4s, 12H, 4 ꢀ CH3O), 5.68 (dd, J = 3.8, 10.2 Hz, 1H,
H3), 5.84 (dd, J = 1.5, 3.8 Hz, 1H, H2), 6.06 (s, 1H, H6), 6.85 (dd,
J = 1.5, 10.2 Hz, 1H, H4), 6.87 (d, J = 8.3 Hz, 2H, H30, H50), 7.38 (d,
J = 8.3 Hz, 2H, H20, H60); 13C NMR (75.6 MHz, CDCl3): d 55.2, 55.8,
56.0 and 61.1 (4 ꢀ CH3O), 76.2 (C2), 89.1 (C6), 105.3 (C4a), 113.6
(C30, C50), 119.7 (C4), 127.4 (C3), 128.7 (C20, C60), 131.7 (C10),
139.3 (C8), 146.7 (C8a), 151.1 (C5), 153.5 (C7), 159.5 (C40); MS
(TOF-ESI) m/z calcd for
C
19H20O5Na (M+Na)+ 351.12. Found
351.04; Anal. Calcd for C19H20O5ꢂ1/2H2O: C, 67.64; H, 6.27. Found:
C, 67.59; H, 6.17.
4.1.4.2. 40-Chloro-7-methoxyflav-3-ene (6c). White solid, yield:
50%; mp 76–78 °C; UV (MeOH): kmax 203
(e
26,199
4.1.4.7. 40-Methyl-5,7,8-trimethoxyflav-3-ene (14d). Yellow
cmꢁ1 Mꢁ1), 230 (27,838), 306 (6703) nm; IR (KBr): mmax 3442,
sticky residue, yield: 63%; UV (MeOH): kmax 205 (e 15,503
3022, 2978, 2933, 2842, 1614, 1503, 1444, 1313, 1275, 1252,
cmꢁ1 Mꢁ1), 257 (5150) nm; IR (KBr): mmax 3363, 3003, 2926,
1198, 1154, 1110, 1026, 969, 812, 705 cmꢁ1
;
1H NMR (300 MHz,
2840, 1606, 1510, 1460, 1425, 1352, 1240, 1214, 1139, 1113,
CDCl3): d 3.75 (s, 3H, CH3O), 5.63 (dd, J = 3.8, 9.8 Hz, 1H, H3),
5.85 (dd, J = 1.9, 3.8 Hz, 1H, H2), 6.37 (d, J = 2.3 Hz, 1H, H8), 6.43
(dd, J = 2.3, 8.3 Hz, 1H, H6), 6.51 (dd, J = 1.9, 9.8 Hz, 1H, H4), 6.92
(d, J = 8.3 Hz, 1H, H5), 7.31–7.39 (m, 4H, H20, H30, H50, H60); 13C
NMR (75.6 MHz, CDCl3): d 55.2 (CH3O), 76.3 (C2), 101.8 (C8),
1068, 1032, 795 cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.64 (s, 3H,
;
CH3), 3.81, 3.83 and 3.87 (3s, 9H, 3 ꢀ CH3O), 5.69 (dd, J = 3.8,
9.8 Hz, 1H, H3), 5.87 (dd, J = 1.5, 3.8 Hz, 1H, H2), 6.05 (s, 1H, H6),
6.83 (dd, J = 1.5, 9.8 Hz, 1H, H4), 7.14 (d, J = 8.3 Hz, 2H, H30, H50),
7.35 (d, J = 8.3 Hz, 2H, H20, H60); 13C NMR (75.6 MHz, CDCl3): d

5204
R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
21.3 (CH3), 55.8, 56.0 and 61.0 (3 ꢀ CH3O), 76.5 (C2), 89.3 (C6),
105.4 (C4a), 120.5 (C4), 126.8 (C3), 127.1 (C20, C60), 129.1 (C30,
C50), 137.4 (C40), 137.9 (C10), 139.8 (C8), 146.8 (C8a), 151.1 (C5),
153.5 (C7); MS (TOF-ESI) m/z calcd for C19H20O4Na (M+Na)+
335.13. Found 335.06; Anal. Calcd for C19H20O4ꢂ3/4MeOH: C,
70.52; H, 6.89. Found: C, 70.82; H, 7.12.
4.1.5.3. 6a,12a-Dihydro-3,10-dimethoxy-6-(40-chlorophenyl)-7-
[(1E)-2-(400-chlorophenylethenyl)]-6H,7H-[1]benzopyrano[4,3-b]
[1]benzopyran (7c). White solid, yield: 69%; mp 131–133 °C; UV
(MeOH): kmax 206 (
mmax 3454, 3021, 2958, 2910, 2835, 1610, 1587, 1504, 1443, 1267,
1198, 1160, 1130, 1111, 1034, 1013, 959, 836, 803 cmꢁ1 1H NMR
e
44,459 cmꢁ1 Mꢁ1), 261 (10,880) nm; IR (KBr):
;
(300 MHz, CDCl3): d 2.43 (ddd, J = 2.1, 2.3, 10.6 Hz, 1H, H6a), 3.12
(dd, J = 2.1, 6.4 Hz, 1H, H7), 3.76 and 3.78 (2s, 6H, 2 ꢀ CH3O),
5.03 (d, J = 10.6 Hz, 1H, H6), 5.06 (d, J = 2.3 Hz, 1H, H12a), 6.00 (d,
4.1.5. General procedure for acid-catalyzed dimerization
reactions (7a–d, 15a–d)
J = 15.8 Hz, 1H, Hb), 6.20 (dd, J = 6.4, 15.8 Hz, 1H, H ), 6.49–6.54
a
To a solution of the appropriate flavene in MeOH (20 mL) was
added 10 drops of acid (HCl, TFA or AcOH) and the solution was
heated at 60–70 °C for 12 h. The solvent was partially removed un-
der reduced pressure and EtOAc (25 mL) was added. The organic
layer was washed with saturated NaHCO3 solution (20 mL), dried
over anhydrous Na2SO4 and evaporated under reduced pressure.
Purification of the crude product by column chromatography over
silica gel using DCM/light petroleum (50:50) gave the desired di-
mer. The dimers were recrystallized twice from absolute EtOH to
yield analytically pure products.
(m, 3H, H4, H9, H11), 6.60 (dd, J = 2.3, 8.3 Hz, 1H, H2), 6.83 (d,
J = 8.3 Hz, 1H, H8), 7.19 (d, J = 8.3 Hz, 2H, H20, H60), 7.26 (d,
J = 8.3 Hz, 2H, H30, H50), 7.30 (d, J = 8.3 Hz, 1H, H1), 7.32 (d,
J = 8.6 Hz, 2H, H300, H500), 7.40 (d, J = 8.6 Hz, 2H, H200, H600); 13C
NMR (75.6 MHz, CDCl3): d 37.9 (C7), 41.4 (C6a), 55.2 and 55.3
(2 ꢀ CH3O), 66.9 (C12a), 76.1 (C6), 101.2 (C11), 101.3 (C4), 108.2
(C9), 108.5 (C2), 111.5 (C7a), 113.4 (C12b), 127.4 (C30, C50), 128.5
(C20, C60), 128.6 (C300, C500), 128.9 (Cb), 130.9 (C200, C600), 131.1
(C40), 133.0 (C400), 133.7 (C1), 133.9 (C ), 134.5 (C8), 135.1 (C100),
a
137.1 (C10), 153.4 (C4a), 155.5 (C11a), 160.0 (C3), 161.6 (C10);
HRMS (ESI) m/z calcd for C32H26Cl2O4Na (M+Na)+ 567.1108. Found
567.1090; Anal. Calcd for C32H26Cl2O4ꢂ1/4MeOH: C, 69.99; H, 4.92.
Found: C, 70.26; H, 5.22.
4.1.5.1.
6a,12a-Dihydro-3,10-dimethoxy-6-phenyl-7-[(1E)-2-
(phenylethenyl)]-6H,7H-[1]benzopyrano[4,3-b][1]benzopyran
(7a). White solid; yield: 65%; mp 110–112 °C; UV (MeOH): kmax
207 (
3026, 2954, 2910, 2834, 1610, 1587, 1504, 1443, 1268, 1198,
1160, 1130, 1112, 1033, 1010, 958, 834 cmꢁ1 1H NMR (300 MHz,
e
39,892 cmꢁ1 Mꢁ1), 285 (3618) nm; IR (KBr): mmax 3421,
4.1.5.4. 6a,12a-Dihydro-3,10-dimethoxy-6-(40-methoxyphenyl)-
7-[(1E)-2-(400-methoxyphenylethenyl)]-6H,7H-[1]benzopyrano[4,
3-b][1]benzopyran (7d). White solid, yield: 66%; mp 113–
;
e
137,728 cmꢁ1 Mꢁ1), 272 (29,148)
CDCl3): d 2.50 (ddd, J = 2.1, 2.3, 10.6 Hz, 1H, H6a), 3.17 (dd,
J = 2.1, 6.4 Hz, 1H, H7), 3.78 and 3.79 (2s, 6H, 2 ꢀ CH3O), 5.04 (d,
J = 10.6 Hz, 1H, H6), 5.08 (d, J = 2.3 Hz, 1H, H12a), 6.04 (d,
115 °C; UV (MeOH): kmax 204 (
nm; IR (KBr): mmax 3454, 3010, 2928, 2919, 2843, 1608, 1594,
1511, 1469, 1443, 1410, 1250, 1178, 1088, 1033, 834, 777 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 2.48 (ddd, J = 2.1, 2.3, 10.9 Hz, 1H,
H6a), 3.15 (dd, J = 2.1, 6.0 Hz, 1H, H7), 3.78 (s, 6H, 2 ꢀ CH3O),
3.85 (s, 6H, 2 ꢀ CH3O), 5.01 (d, J = 10.9 Hz, 1H, H6), 5.09 (d,
J = 2.3 Hz, 1H, H12a), 5.99 (d, J = 15.8 Hz, 1H, Hb), 6.11 (dd, J = 6.0,
J = 15.8 Hz, 1H, Hb), 6.24 (dd, J = 6.4, 15.8 Hz, 1H, H ), 6.50–6.52
a
(m, 3H, H4, H9, H11), 6.59 (dd, J = 2.3, 8.3 Hz, 1H, H2), 6.85 (d,
J = 9.0 Hz, 1H, H8), 7.30–7.42 (m, 11H, H1, H20, H30, H40, H50, H60,
H200, H300, H400, H500, H600); 13C NMR (75.6 MHz, CDCl3): d 37.9 (C7),
41.4 (C6a), 55.2 (CH3O), 55.3 (CH3O), 67.1 (C12a), 76.8 (C6),
101.2 (C4), 101.8 (C11), 108.1 (C9), 108.3 (C2), 112.1 (C7a), 113.5
(C12b), 126.1 (C40), 127.3 (C400), 127.4 (C20, C60), 127.4 (C200, C600),
128.0 (Cb), 128.4 (C300, C500), 128.7 (C30, C50), 131.0 (C1), 131.8
15.8 Hz, 1H, H ), 6.49–6.53 (m, 3H, H4, H9, H11), 6.59 (dd,
a
J = 2.3, 8.3 Hz, 1H, H2), 6.79–6.86 (m, 3H, H8, H30, H50), 6.95 (d,
J = 8.7 Hz, 2H, H300, H500), 7.21 (d, J = 8.6 Hz, 2H, H20, H60), 7.26 (d,
J = 8.7 Hz, 2H, H200, H600), 7.32 (d, J = 8.3 Hz, 1H, H1); 13C NMR
(75.6 MHz, CDCl3): d 37.9 (C7), 41.5 (C6a), 55.2 (2 ꢀ CH3O), 55.3
(2 ꢀ CH3O), 67.2 (C12a), 76.5 (C6), 101.1 (C4), 101.2 (C11), 108.0
(C9), 108.2 (C2), 112.3 (C7a), 113.6 (C12b), 113.8 (C30, C50), 114.1
(C300, C500), 127.3 (C20, C60), 128.6 (Cb), 128.7 (C200, C600), 129.5
(C8), 133.2 (C ), 136.7 (C100), 138.6 (C10), 153.5 (C4a), 155.7
a
(C11a), 159.8 (C10), 161.5 (C3); HRMS (ESI) m/z calcd for
C
C
32H28O4Na (M+Na)+ 499.1888. Found 499.1847; Anal. Calcd for
32H28O4ꢂMeOH: C, 77.93; H, 6.34. Found: C, 77.77; H, 6.22.
(C100), 130.6 (C10), 131.1 (C1), 131.2 (C8), 133.0 (C ), 153.5 (C4a),
a
4.1.5.2. 6a,12a-Dihydro-3,10-dimethoxy-6-(40-bromophenyl)-7-
[(1E)-2-(400-bromophenylethenyl)]-6H,7H-[1]benzopyrano[4,3-b]-
[1]benzopyran (7b). White solid, yield: 71%; mp 128–130 °C; UV
155.8 (C11a), 159.0 (C40), 159.5 (C400), 159.7 (C10), 159.8 (C3);
MS (TOF-ESI) m/z calcd for
C
34H32O6 (M+1)+ 537.23. Found
537.22; Anal. Calcd for C34H32O6: C, 76.10; H, 6.01. Found: C,
76.26; H, 6.29.
(MeOH): kmax 206 (
mmax 3431, 3018, 2953, 2911, 2833, 1610, 1587, 1504, 1443, 1267,
1197, 1160, 1130, 1112, 1034, 1009, 965, 826, 807 cmꢁ1 1H NMR
e
84,733 cmꢁ1 Mꢁ1), 267 (29,112) nm; IR (KBr):
4.1.5.5. 6a,12a-Dihydro-1,3,4,8,10,11-hexamethoxy-6-(40-bromo-
phenyl)-7-[(1E)-2-(400-bromophenylethenyl)]-6H,7H-[1]benzo-
pyrano[4,3-b][1]benzopyran (15a). White solid, yield: 68%; mp
;
(300 MHz, CDCl3): d 2.43 (ddd, J = 2.1, 2.3, 10.9 Hz, 1H, H6a), 3.13
(dd, J = 2.1, 6.4 Hz, 1H, H7), 3.78 and 3.79 (2s, 6H, 2 ꢀ CH3O),
5.03 (d, J = 10.9 Hz, 1H, H6), 5.05 (d, J = 2.3 Hz, 1H, H12a), 5.99 (d,
172–174 °C; UV (MeOH): kmax 208 (
(3702) nm; IR (KBr): mmax 3479, 2933, 2839, 1736, 1613, 1503,
1463, 1347, 1241, 1205, 1105, 1064, 961, 928, 809 cmꢁ1 1H
e
15,726 cmꢁ1 Mꢁ1), 263
J = 15.8 Hz, 1H, Hb), 6.22 (dd, J = 6.4, 15.8 Hz, 1H, H ), 6.52 (dd,
a
J = 2.3, 8.3 Hz, 1H, H9), 6.55 (d, J = 2.3 Hz, 2H, H4, H11), 6.61 (dd,
J = 2.3, 8.3 Hz, 1H, H2), 6.84 (d, J = 8.3 Hz, 1H, H8), 7.12 (d,
J = 8.6 Hz, 2H, H20, H60), 7.21 (d, J = 8.3 Hz, 2H, H300, H500), 7.32 (d,
J = 8.3 Hz, 1H, H1), 7.38 (d, J = 8.3 Hz, 2H, H200, H600), 7.55 (d,
J = 8.6 Hz, 2H, H30, H50); 13C NMR (75.6 MHz, CDCl3): d 38.0 (C7),
41.3 (C6a), 55.2 and 55.3 (2 ꢀ CH3O), 66.9 (C12a), 75.9 (C6),
101.2 (C4), 101.4 (C11), 108.2 (C9), 108.5 (C2), 111.5 (C7a), 113.4
(C12b), 121.2 (C40), 122.7 (C400), 127.7 (C200, C600), 129.0 (C20, C60),
130.8 (Cb), 130.9 (C8), 131.2 (C1), 131.5 (C30, C50), 131.9 (C300,
;
NMR (300 MHz, CDCl3): d 2.24 (ddd, J = 1.5, 2.3, 10.9 Hz, 1H,
H6a), 3.31 (dd, J = 1.5, 5.3 Hz, 1H, H7), 3.69, 3.73, 3.82, 3.86, 3.89
and 3.90 (6s, 18H, 6 ꢀ CH3O), 4.93 (d, J = 10.9 Hz, 1H, H6), 5.36
(d, J = 2.3 Hz, 1H, H12a), 5.95 (d, J = 15.8 Hz, 1H, Hb), 6.13 (dd,
J = 5.3, 15.8 Hz, 1H, H ), 6.16 (s, 1H, H2), 6.18 (s, 1H, H9), 7.15 (d,
a
J = 8.6 Hz, 2H, H20, H60), 7.18 (d, J = 8.3 Hz, 2H, H300, H500), 7.35 (d,
J = 8.3 Hz, 2H, H200, H600), 7.52 (d, J = 8.6 Hz, 2H, H30, H50); 13C
NMR (75.6 MHz, CDCl3): d 32.9 (C7), 40.9 (C6a), 55.7, 55.8, 56.0,
56.2, 60.8 and 60.9 (6 ꢀ CH3O), 61.4 (C12a), 75.9 (C6), 89.4 (C2),
89.5 (C9), 102.3 (C12b), 104.2 (C7a), 120.7 (C40), 122.4 (C400),
127.7 (Cb), 128.9 (C20, C60), 129.0 (C200, C600), 129.5 (C30, C50),
C500), 133.8 (C ), 135.5 (C100), 137.6 (C10), 153.4 (C4a), 155.4
a
(C11a), 160.0 (C10), 161.6 (C3); MS (TOF-ESI) m/z calcd for
C
32H26Br2O4 (M+1)+ 633.03 (Br79). Found 632.98 (Br79); Anal. Calcd
130.2 (C ), 131.2 (C11), 131.6 (C4), 132.9 (C300, C500), 136.1 (C100),
for C32H26Br2O4: C, 60.59; H, 4.13. Found: C, 60.37; H, 4.16.
a

R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
5205
137.8 (C10), 149.1 (C3), 149.9 (C10), 153.2 (C1), 153.9 (C8), 153.9
(C11a), 154.5 (C4a); MS (TOF-ESI) m/z calcd for C36H34Br2O8Na
(M+Na)+ 775.05 (Br79). Found 775.02 (Br79); Anal. Calcd for
126.0 (C20, C60), 127.1 (Cb), 127.2 (C30, C50), 129.0 (C200, C600),
131.3 (C300, C500), 131.5 (C ), 134.5 (C4), 135.8 (C11), 136.0 (C100),
a
136.7 (C10), 137.9 (C40), 138.0 (C400), 149.5 (C3), 149.9 (C10),
152.2 (C1), 153.4 (C8), 154.0 (C11a), 154.6 (C4a); HRMS (ESI) m/z
calcd for C38H40O8Na (M + Na)+ 647.2623. Found 647.2615; Anal.
Calcd for C38H40O8: C, 73.06; H, 6.45. Found: C, 72.80; H, 6.70.
C36H34Br2O8: C, 57.31; H, 4.54. Found: C, 57.58; H, 4.82.
4.1.5.6. 6a,12a-Dihydro-1,3,4,8,10,11-hexamethoxy-6-(40-chloro-
phenyl)-7-[(1E)-2-(400-chlorophenylethenyl)]-6H,7H-[1]benzopy-
rano[4,3-b][1]benzopyran (15b). White solid, yield: 70%; mp
4.2. Biological experiments
166–168 °C; UV (MeOH): kmax 210
(38,495) nm; IR (KBr): mmax 3476, 2934, 2839, 1726, 1610, 1502,
1456, 1348, 1242, 1205, 1115, 1061, 974, 938, 812 cmꢁ1 1H NMR
(e
98,869 cmꢁ1 Mꢁ1), 261
4.2.1. Method for antimalarial growth inhibition assays
;
P. falciparum growth inhibition assays were carried using an
isotopic microtest, as previously described (Andrews et al.,
2000).23 Briefly, ring-stage P. falciparum 3D7 infected erythrocytes
(0.5% parasitemia and 2.5% hematocrit) were seeded into triplicate
wells of 96 well tissue culture plates containing serial dilutions of
control (chloroquine) or test compounds. Following 48 h incuba-
(300 MHz, CDCl3): d 2.24 (ddd, J = 1.1, 2.3, 11.3 Hz, 1H, H6a), 3.31
(dd, J = 1.1, 6.0 Hz, 1H, H7), 3.69, 3.73, 3.82, 3.86, 3.88 and 3.90 (6s,
18H, 6 ꢀ CH3O), 4.94 (d, J = 11.3 Hz, 1H, H6), 5.37 (d, J = 2.3 Hz, 1H,
H12a), 5.96 (d, J = 15.8 Hz, 1H, Hb), 6.14 (dd, J = 6.0, 15.8 Hz, 1H,
H ), 6.18 (2s, 2H, H2, H9), 7.20 (d, J = 8.6 Hz, 2H, H20, H60), 7.23 (d,
a
J = 8.6 Hz, 2H, H30, H50), 7.26 (d, J = 8.7 Hz, 2H, H300, H500), 7.37 (d,
J = 8.7 Hz, 2H, H200, H600); 13C NMR (75.6 MHz, CDCl3): d 32.9 (C7),
41.0 (C6a), 55.7, 56.0, 56.3, 56.4, 60.8 and 60.9 (6 ꢀ CH3O), 61.4
(C12a), 75.9 (C6), 89.4 (C2), 89.5 (C9), 102.4 (C12b), 104.3 (C7a),
127.4 (C30, C50), 127.8 (Cb), 128.5 (C20, C60), 128.5 (C300, C500), 128.7
tion under standard P. falciparum culture conditions, 0.5 l
Ci [3H]-
hypoxanthine was added to each well after which the plates
cultured for a further 24 h. Cells were harvested onto 1450 MicroB-
eta filter mats (Wallac) and 3H incorporation was determined using
a 1450 MicroBeta liquid scintillation counter. Percentage inhibition
of growth was compared to matched DMSO controls (0.5%). IC50
values were calculated using linear interpolation of inhibition
(C200, C600), 130.6 (C ), 131.3 (C11), 131.5 (C4), 132.8 (C40), 134.2
a
(C400), 135.6 (C100), 137.3 (C10), 148.0 (C3), 149.1 (C10), 152.2 (C1),
153.3 (C8), 154.0 (C11a), 154.8 (C4a); MS (TOF-ESI) m/z calcd for
curves (Huber and Koella 1993).24 The mean IC50
( SD) is shown
C
C
36H34Cl2O8Na (M+Na)+ 687.15. Found 687.06; Anal. Calcd for
36H34Cl2O8: C, 64.97; H, 5.15. Found: C, 65.13; H, 5.26.
for three independent experiments, each carried out in triplicate.
4.2.2. Method for in vitro cytotoxicity assays25
4.1.5.7. 6a,12a-Dihydro-1,3,4,8,10,11-hexamethoxy-6-(40-meth-
oxyphenyl)-7-[(1E)-2-(400-methoxyphenylethenyl)]-6H,7H-[1]-
benzopyrano[4,3-b][1]benzopyran (15c). White solid, yield:
Neonatal foreskin fibroblast (NFF) cells were cultured in RPMI
1640 (Life Technologies, Inc., Rockville, MD) supplemented with
10% FCS (CSL Biosciences, Parkville, Victoria, Australia), 1% strepto-
mycin (Life Technologies, Inc., Rockville, MD; complete medium) at
37 °C and 5% CO2. Cells were maintained in log phase growth and
then seeded (3000/well) into 96 well tissue culture plates (Corning,
USA) and were grown for 24 h before treatment. Compounds were
dissolved in 100% DMSO and diluted in complete medium; the
DMSO concentration in the medium did not exceed 1%. Control
cells were treated with the equivalent dose of DMSO. Three days
after treatment initiation, the cells were washed with PBS and
fixed in methylated spirits and total protein was determined using
sulforhodamine B as described previously (Skehan et al., 1990).
Compounds were tested in triplicate in two independent
experiments.
71%; mp 188–190 °C; UV (MeOH): kmax 210 (
265 (18,123) nm; IR (KBr): mmax 3442, 2934, 2838, 1735, 1609,
e
68,254 cmꢁ1 Mꢁ1),
1510, 1464, 1349, 1246, 1204, 1112, 1060, 974, 928, 820 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 2.26 (ddd, J = 1.1, 2.3, 11.3 Hz, 1H,
H6a), 3.34 (dd, J = 1.1, 5.3 Hz, 1H, H7), 3.67, 3.73, 3.76, 3.83, 3.85,
3.86, 3.89 and 3.90 (8s, 24H, 8 ꢀ CH3O), 4.91 (d, J = 11.3 Hz, 1H,
H6), 5.39 (d, J = 2.3 Hz, 1H, H12a), 5.95 (d, J = 15.8 Hz, 1H, Hb),
6.05 (dd, J = 5.3, 15.8 Hz, 1H, H ), 6.14 (s, 1H, H2), 6.17 (s, 1H,
a
H9), 6.78 (d, J = 8.7 Hz, 2H, H30, H50), 6.92 (d, J = 8.6 Hz, 2H, H300,
H500), 7.20 (d, J = 8.7 Hz, 2H, H20, H60), 7.23 (d, J = 8.6 Hz, 2H, H200,
H600); 13C NMR (75.6 MHz, CDCl3): d 32.9 (C7), 41.1 (C6a), 55.1,
55.8, 56.0, 56.2, 56.3, 56.4, 60.8 and 60.9 (8 ꢀ CH3O), 61.6 (C12a),
76.2 (C6), 89.2 (C2), 89.6 (C9), 103.3 (C12b), 104.6 (C7a), 113.7
(C30, C50), 113.8 (C300, C500), 127.2 (C20, C60), 127.6 (Cb), 128.5 (C200,
Acknowledgments
C600), 130.1 (C100), 130.4 (C10), 130.4 (C ), 131.3 (C4), 131.5 (C11),
a
147.1 (C3), 149.5 (C10), 151.9 (C1), 152.2 (C8), 152.9 (C11a),
We thank the University of New South Wales, and the Austra-
lian Research Council (ARC) for Linkage Project (LP0455373) fund-
ing to N.K. and D.B., and Future Fellowship funding to K.T.A., and
the Australian Red Cross Blood Service for providing human blood
and sera for P. falciparum culture.
153.5 (C4a), 158.9 (C40), 159.7 (C400); MS (TOF-ESI) m/z calcd for
C
C
38H40O10Na (M+Na)+ 679.25. Found 679.10; Anal. Calcd for
38H40O10: C, 69.50; H, 6.14. Found: C, 69.69; H, 6.41.
4.1.5.8.
6a,12a-Dihydro-1,3,4,8,10,11-hexamethoxy-6-tolyl-7-
[(1E)-2-tolylethenyl]-6H,7H-[1]benzopyrano[4,3-b][1]benzopy-
References and notes
ran (15d). White solid, yield: 73%; mp 167–169 °C; UV (MeOH):
kmax 210 (
3477, 2932, 2839, 1726, 1610, 1504, 1456, 1348, 1243, 1205,
1119, 1061, 961, 939, 807 cmꢁ1 1H NMR (300 MHz, CDCl3): d
e
38,692 cmꢁ1 Mꢁ1), 263 (8242) nm; IR (KBr): mmax
1. Nianhuan, Y.; Aiming, S.; Xiaobing, W.; Seth, D.; Kit, S. L. J. Comb. Chem. 2007, 9,
668.
2. Huang, W. H.; Chien, P. Y.; Yang, C. H.; Lee, A. R. Chem. Pharm. Bull. 2003, 51,
339.
;
2.26 (ddd, J = 1.1, 2.3, 10.9 Hz, 1H, H6a), 2.31 and 2.39 (2s, 6H,
2 ꢀ CH3), 3.37 (dd, J = 1.1, 5.6 Hz, 1H, H7), 3.68, 3.75, 3.82, 3.83,
3.85 and 3.86 (6s, 18H, 6 ꢀ CH3O), 4.93 (d, J = 10.9 Hz, 1H, H6),
5.39 (d, J = 2.3 Hz, 1H, H12a), 5.96 (d, J = 15.8 Hz, 1H, Hb), 6.14
3. Mohammed, R.; Muhammad, R.; Umesh, R. D. Chem. Biodivers. 2007, 4, 2495.
4. Nkunya, M. H. H.; Waibel, R.; Achenbach, H. Phytochemistry 1993, 34, 853.
5. Guerin, P. J.; Olliaro, P.; Nosten, F.; Druilhe, P.; Laxminarayan, R.; Blinka, F.;
Kilama, W. L.; Ford, N.; White, N. J. Lancet Infect. Dis. 2002, 2, 564.
6. Pradel, G.; Schlitzer, M. Curr. Mol. Med. 2010, 10, 335.
7. Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning, J.; Lwin, K. M.;
Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.; Silamut, K.; Imwong, M.;
Chotivanich, K.; Lim, P.; Herdman, T.; An, S. S.; Yeung, S.; Singhasivanon, P.;
Day, N. P. J.; Lindegardh, D. M. N.; Socheat, D.; White, N. J. N. Engl. J. Med. 2009,
361, 455.
(dd, J = 5.6, 15.8 Hz, 1H, H ), 6.15 and 6.17 (2s, 2H, H2, H9), 7.05
a
(d, J = 8.3 Hz, 2H, H30, H50), 7.13 (d, J = 8.6 Hz, 2H, H300, H500,), 7.15
(d, J = 8.3 Hz, 2H, H20, H60), 7.19 (d, J = 8.6 Hz, 2H, H200, H600); 13C
NMR (75.6 MHz, CDCl3): d 21.0 and 21.2 (2 ꢀ CH3), 32.9 (C7),
40.9 (C6a), 55.7, 55.9, 56.2, 56.4, 60.8 and 60.9 (6 ꢀ CH3O), 61.5
(C12a), 76.5 (C6), 89.2 (C2), 89.6 (C9), 103.2 (C12b), 104.5 (C7a),
8. Deodhar, M.; Black, D. StC.; Kumar, N. Tetrahedron 2007, 63, 5227.
9. Devakaram, R.; Black, D. StC.; Kumar, N. Tetrahedron Lett. 2010, 51, 3636.

5206
R. Devakaram et al. / Bioorg. Med. Chem. 19 (2011) 5199–5206
10. Sylvie, M.; Branko, D.; Somchit, P.; Patrick, E.; Ivan, G.; Adrian, G.; Filomena, M.;
Manuel, G.; Denis, G.; Andrew, K. Bioorg. Med. Chem. 2006, 14, 1599.
11. Pradeep, D. L.; Sachin, S. S.; Kiran, N.; Beena, N. Tetrahedron Lett. 2005, 46, 1573.
12. Christelle, P.; Catherine, F.; Jean-Philippe, B.; Hubert, L.; Albert-Jose, C.
Tetrahedron 2000, 56, 6047.
13. Clark-Lewis, J. W.; Jemison, R. W. Aust. J. Chem. 1968, 21, 2247.
14. Crystallographic data for the structure of compound 6b have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 820413. The X-ray crystal structure was obtained by
Don Craig, Crystallography Laboratory, Analytical Centre, The University of
New South Wales, Sydney, Australia.
15. Crystallographic data for the structure of compound 10c have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 820412. The X-ray crystal structure was obtained by
Mohan Bhadbhade, Crystallography Laboratory, Analytical Centre, The
University of New South Wales, Sydney, Australia.
16. Kang, W.; Li, G.; Hao, X. Acta Bot. Sin. 2003, 45, 1003.
17. Ohkatsu, Y.; Satoh, T. J. Jpn. Petrol. Inst. 2008, 51, 298.
18. Forghieri, M.; Laggner, C.; Paoli, P.; Langer, T.; Manao, G.; Camici, G.; Bondioli,
L.; Prati, F.; Costantino, L. Bioorg. Med. Chem. 2009, 17, 2658.
19. Chimenti, F.; Fioravanti, R.; Bolasco, A.; Chimenti, P.; Secci, D.; Rossi, F.; Yanez,
M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52, 2818.
20. Singh, Om. V.; Muthukrishnan, M.; Sunderavadivelu, M. Indian J. Chem., Sect B
2005, 44B, 2575.
21. Robinson, T. J. Chem. Soc. 1918, 113, 876.
22. Krohn, K.; Ahmed, I.; John, M. Synthesis 2009, 5, 779.
23. Andrews, K. T.; Walduck, A.; Kelso, M. J.; Fairlie, D. P.; Saul, A.; Parsons, P. G. Int.
J. Parasitol. 2000, 30, 761.
24. Huber, W.; Koella, J. C. Acta Trop. 1993, 55, 257.
25. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1107.