Synthesis and antiplasmodial evaluation of novel chromeno[2,3-b]chromene derivatives

Article abstract of DOI:10.1016/j.bmc.2011.12.037

5-Methoxyflavenes and 6-methoxyflavenes were found to undergo stereoselective acid-catalyzed rearrangement to generate a range of novel chromeno[2,3-b]chromene derivatives. When subjected to an in vitro antiplasmodial growth inhibition assay using Plasmod

Full text of DOI:10.1016/j.bmc.2011.12.037

Bioorganic & Medicinal Chemistry 20 (2012) 1527–1534
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiplasmodial evaluation of novel
chromeno[2,3-b]chromene derivatives
Ruth Devakaram a, David StC. Black a, Vanida Choomuenwai b, Rohan A. Davis b, Naresh Kumar a,
⇑
a School of Chemistry, The University of New South Wales, UNSW, Sydney, NSW 2052, Australia
b Eskitis Institute for Cell and Molecular Therapies, Griffith University, QLD 4111, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Methoxyflavenes and 6-methoxyflavenes were found to undergo stereoselective acid-catalyzed rear-
rangement to generate a range of novel chromeno[2,3-b]chromene derivatives. When subjected to an
in vitro antiplasmodial growth inhibition assay using Plasmodium falciparum (3D7 line) the chromene
Received 2 November 2011
Revised 12 December 2011
Accepted 18 December 2011
Available online 4 January 2012
analogues were shown to display IC50 values ranging from 6.8 to 39.8 lM.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dependensin
Dimerization
Flavenes
Acid-catalyzed
Chromenochromenes
Antiplasmodial
1. Introduction
procedures, and limited biological data. Therefore, it is highly
desirable to develop efficient synthetic methodologies, which
could generate not only the natural products themselves but also
synthetic analogues for pharmacological applications.
The typical methods reported for the synthesis of biflavonoids
include the Ulmann condensation of halogenoflavonoids,8 Suzuki
coupling reaction of halogenated flavanones with appropriate
boronic acid derivatives9 and Stille coupling of tributyltinflavones
with bromoflavones.10
Flavonoids are a broad class of polyphenolic secondary metab-
olites abundant in plants and in a variety of common foods such
as apples, onions, tea and red wine.1 Flavonoids possess a broad
range of pharmacological properties including antioxidant, anti-
cancer, antiviral and antiinflammatory properties and form the
largest class of phytoestrogens known so far.2
Biflavonoids are a series of naturally occurring compounds that
include flavone–flavone, flavanones–flavone and flavanones–flava-
none subunit linkages. More than 100 biflavonoids have been iden-
MeO
OMe
OMe
tified from plants since the isolation of gingetin in 1929.3,4
A
MeO
MeO
H
MeO
O
variety of biological activities for biflavonoids have been published,
including antiinflammatory, antimicrobial, antioxidant, and oth-
ers.4,5 Antiplasmodial activity has also been reported, for example
the two biflavonoids, sikokianins B and C were isolated from the
roots of Wikstroemia indica and displayed IC50 values of 0.54 and
H
H
H
O
0.56 lg/ml respectively, against a chloroquine-resistant strain of
Plasmodium falciparum.6 Similarly, a biflavanone was isolated as a
potent antiplasmodial natural product (IC50 value of 80 ng/ml)
from the extract of the outer bark of Ochna integerrima.7
Dependensin 1
Despite their promising biological activities, the development of
biflavonoids as therapeutic agents has been hampered by their low
abundance in nature, tedious extraction and purification
The natural product, dependensin 1 is a dimeric flavonoid iso-
lated from the root bark of the Tanzanian medicinal plant, Uvaria
dependens. The crude extract of this plant shows potent antimalar-
ial activity.11 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
⇑
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.2011.12.037

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R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
unable to verify it experimentally, obtaining a ring-opened com-
pound instead.11 However, our research group has reported the
successful synthesis of dependensin via the acid-catalyzed reaction
of 5,7,8-trimethoxyflavene.12 We have also previously shown that
the benzopyrano[4,3-b]benzopyran ring system present in depen-
densin could be synthesized via the acid-catalyzed dimerization
reaction of 4,7-diacetoxyflav-3-ene.12 But, similar acid-catalyzed
reactions of the corresponding 4,5-diacetoxy- and 4,6-diacetoxy-
flavenes produced a complex mixture of products.12
As part of our ongoing study on the synthesis of these dimeric
systems, we have investigated the acid catalyzed reactions of 5-
methoxy-, 6-methoxy- and 7-methoxyflavenes. Interestingly, we
found that the dimerization products obtained were dependent
on the position of the methoxy group in the flavene nucleus.
7-Methoxyflavenes and 5,7,8-trimethoxyflavenes underwent
acid-catalyzed dimerization as expected to yield analogues of
dependensin. Recently, we presented data on the development of
efficient synthetic methodologies to these dimeric structures pos-
sessing the benzopyrano[4,3-b]benzopyran ring system, along with
their preliminary antimalarial inhibition assay results.13
However, there are no reports in the literature for the synthesis
of such fused benzopyran ring systems via the generation of
carbocation intermediates mediated by acid-catalyzed reactions.
We report herein the facile one step methodology to the synthesis
of a series of highly functionalized chromeno[2,3-b]chromenes
from the acid-catalyzed reactions of 5-methoxyflavenes and 6-
methoxyflavenes. Both ring systems, containing the chromeno
[2,3-b]chromenes and the benzopyrano[4,3-b]benzopyrans present
in dependensin possess two benzopyran rings, but differ in the
position and manner in which they are fused together. Therefore,
it was anticipated that the new ring system might show interesting
antiplasmodial properties. This paper discusses the full character-
ization details for the novel chromeno[2,3-b]chromenes as well
as their biological activity in an in vitro antiplasmodial growth
inhibition assay.
2. Results and discussion
2.1. Synthesis of flavene precursors
However, it has been observed that the 5-methoxy- and 6-
methoxyflavenes underwent a completely different dimerization
reaction to yield a new range of dimeric structures namely the
chromeno[2,3-b]chromenes.14 It is postulated that these unique
scaffolds will serve as an ideal source for generating chemically di-
verse libraries for use in future drug discovery programs.
The literature cites examples of molecules from nature bearing
close resemblance to the chromeno[2,3-b]chromene ring system.
For example, albanols A 2 and albanol B 3, isolated from the root
bark of Morus alba, Moraceae (mulberry) were noteworthy, as
pharmacological tests on this species showed marked hypotensive
effect in rabbits.15 Similarly, a systematic study performed on
plants belonging to the Moraceae family showed the presence of
antimicrobial activity in the extract of Sorocea ilicifolia, from which
a number of phenolic components were isolated. One among them
was the ketalized Diels–Alder type adduct, named sorocein L 4.16
The strategy of reductive cyclization reported by Clark-Lewis
and Jemison20 was utilized for the direct conversion of 20-hydroxy-
chalcones 5a–i into the corresponding flav-3-enes 6a–i with the
use of NaBH4 in IPA (Scheme 1). This methodology was beneficial
as it could furnish the desired flavenes, the essential precursors
in fewer steps as well as generate improved overall yields. The fla-
venes 6a–i were obtained in yields ranging from 65% to 80%.
2.2. Acid-catalyzed reactions of 6-methoxyflavenes
The 6-methoxyflavenes 6a–d were subjected to the dimeriza-
tion reaction in the presence of few drops of TFA or glacial AcOH
as catalysts in MeOH, which unexpectedly generated a series of
chromeno[2,3-b]chromenes 7a–d (Scheme 2), instead of analogues
of the natural product dependensin.
OH
OH
OH
OH
OH
OH
HO
O
H
O
HO
O
O
O
O
H
H
OH
OH
3
2
OH
OH
HO
OH
OH
O
O
OH
OH
4
The strategies employed for the synthesis of this ring system in-
clude the condensation of dimethylol ketones with 2-naphthol
using Amberlyst-15 as catalyst,17 reaction of phenolic Mannich
This was evident from the 1H NMR spectrum of compound 7b
which showed the presence of four aliphatic protons correspond-
ing to H11, H11a and H12 between d 2.84 and 3.46 ppm. The three
protons corresponding to H11a and H12 appeared as a singlet at d
2.84 ppm and the proton corresponding to H11 was seen as a dou-
blet of doublets at d 3.46 (J = 3.4, 9.8 Hz). The protons on the trans
bases with
a
-chloroacrylonitrile involving [4+2] cycloaddition18
and the Diels–Alder cylcoaddition reactions of o-quinone methides,
generated from 4H-1,2-benzoxazines with various dienophiles.19

R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
1529
R
of 6-methoxyflavenes 7a–d, with respect to the coupling patterns
of the aliphatic protons. The aliphatic protons in 7g corresponding
to H11, H11a and H12, were identified as doublet of doublet at d
2.59 (J = 11.7, 17.3 Hz) and a doublet at d 2.77 (J = 17.3 Hz) corre-
lating to H12, and doublet of doublets at d 2.91 (J = 6.4, 11.7 Hz)
OH
R
O
R1
NaBH4, IPA, r.t.
65-80%
R1
R
H
Br
Cl
Me
H
Br
Cl
R2
O
R2
6a-i
and
d 3.74 (J = 6.4, 9.0 Hz) correlating to H11a and H11,
5a-i
respectively.
chalcone
5a
R1
R2
H
H
H
flavene yields[%]
The presence of a diastereotopic methylene unit in 7g was evi-
dent in its COSY spectrum (correlation between the protons at
H12). In addition, analysis of the COSY NMR data led to the identi-
fication of the two 1,4-disubstituted and two trisubstituted ben-
zene rings. The presence of a cis fused ring system is evident
from a NOESY correlation between H11a and H200. The other impor-
tant NOESY correlations of interest were those from H12 to H11a
OMe
OMe
OMe
OMe
H
6a
6b
6c
6d
6e
6f
6g
6h
6i
72
65
68
74
77
70
70
80
79
5b
5c
5d
5e
H
OMe
OMe
OMe
OMe
OMe
5f
H
H
H
5g
5h
OMe
Me
and H to H20, giving evidence of the structure formed.
a
5i
H
Further assignments for 7g were determined on the basis of
HMQC and HMBC data. The presence of a methylene group at
H12 was also evident from its DEPT spectrum. HMBC correlations
of the methylene proton at d 2.59 to C5a, C11 and C12a and the
other methylene proton at d 2.77 to C5a, C11, C11a and C1 were
noteworthy, indicating the presence of two fused benzopyran
units. The aliphatic proton, H11a showed HMBC connections to
C11, C12a, C10 and C100 and the other aliphatic proton, H11 showed
HMBC connections to C5a, C6a, C10a, Cb and C10, thereby indicating
the attachment of the styryl group to position 11. This was further
Scheme 1. Synthesis of flavene precursors.
double bond, H and Hb appeared as a doublet of doublets and a
a
doublet at d 6.24 (J = 9.8, 15.8 Hz) and d 6.47 (J = 15.8 Hz) respec-
tively. The HMBC experiment showed two bond and three bond
proton to carbon couplings from the four aliphatic protons to C5a
at 100.4 ppm. DEPT-135 along with a broadband decoupled 13C
NMR spectrum indicated the presence of one CH2 group at C12
in the structure. Further, a single crystal of compound 7b was
obtained for X-ray crystallographic analysis14 confirming the struc-
ture indicated.
confirmed by the HMBC correlations from the vinylic protons, H
a
and Hb to both the benzopyran ring as well as to the 1,4-disubsti-
tuted benzene ring. Lastly, the attachment of the second 1,4-disub-
stituted benzene ring to C5a was evident from the HMBC
connections from H200 to C400 and C5a, in accordance with the as-
signed structure.
We have previously proposed a rationale14 for the observed
acid-catalyzed dimerization reaction. The reaction mechanism for
the formation of this ring system is postulated to involve the pro-
tonation of the flavene, followed by ring opening leading to the for-
mation of a highly stabilized benzylic carbocation intermediate. A
second molecule of the flav-3-ene can possibly undergo prototro-
pic rearrangement in situ to the corresponding flav-2-ene isomer
under acidic conditions. This can attack the carbocation intermedi-
ate giving rise to a second stable benzylic intermediate. The latter
on ring closure produces the required dimeric ring system.
By correlation of the 1H and 13C NMR spectra, the other dimeric
products of 6-methoxyflavenes 7a, 7c and 7d were assigned to con-
tain the same ring system. These dimeric structures 7a–d were
obtained in 70–74% yields.
Similar coupling patterns were observed in the other dimeric
structures 7e, 7f, 7h and 7i. The dimerization reaction mechanism
is expected to follow the same pathway taken by the 6-methoxy-
flavenes 6a–d.
The identity and purity of all compounds were confirmed by 1H
and 13C NMR spectroscopy, IR spectroscopy, mass spectrometry,
thin layer chromatography and elemental analyses.
2.4. Biological activity of the chromeno[2,3-b]chromene
derivatives
The new chromeno[2,3-b]chromene derivatives were all
screened for antiplasmodial activity using a chloroquine sensitive
P. falciparum line (3D7) and a standard in vitro growth inhibition
assay. Preliminary cytotoxicity data for 7a–i were all obtained
using NFF (neonatal foreskin fibroblasts) cells, which are routinely
used in toxicity studies to determine compound selectivity for the
malaria parasite. Table 1 shows the biological data of all tested
compounds.
2.3. Acid-catalyzed reactions of 5-methoxyflavenes
Similar acid-catalyzed reactions were attempted on the
5-methoxyflavenes 6e–i and they produced the chromeno
[2,3-b]chromenes 7e–i in 68–76% yields (Scheme 3), in parallel to
the 6-methoxyflavenes.
The 1H NMR spectra of these dimeric compounds 7e–i were
found to vary from those obtained previously via the dimerization
R
R
2"
TFA or AcOH
O
H
O
5a
O
MeOH
MeO 9
2 OMe
70-74%
MeO
11
12
H
α
β
6a-d
7a-d
7a
6a
6b
6c
6d
R = H
R = Br
R = Cl
R = H
2'
7b
7c
R = Br
R = Cl
R = Me
7d R = Me
R
Scheme 2. Synthesis of chromeno[2,3-b]chromenes 7a–d.

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R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
R
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 recorded in the
R
2''
O
TFA or AcOH,
MeOH
O
O
H
designated solvents on
a Bruker Avance DPX300 (300 MHz)
5a
spectrometer at the designated frequency and were internally ref-
erenced to the solvent peaks.
68-76%
10 11
1
H 12
α
MeO
OMe
OMe
6e-i
β
Low-resolution mass spectrometric analysis was carried out at
the Biomedical Mass Spectrometry Facility, UNSW, and the spectra
were recorded on Q-TOF Ultima API (Micro mass). High-resolution
mass spectra were performed at the Campbell Microanalytical Lab-
oratory, University of Otago, New Zealand. Infrared spectra were
recorded with a Thermo Nicolet 370 FTIR spectrometer. Ultravio-
let–visible spectra were recorded using a Varian Cary 100 Scan
spectrometer, and the absorption maxima together with the molar
7e-i
6e R = H
6f R = Br
6g R = Cl
7e
7f
R = H
3'
R = Br
7g R = Cl
R
6h
6i
R = OMe
R = Me
7h R = OMe
7i
R = Me
Scheme 3. Synthesis of chromeno[2,3-b]chromenes 7e–i.
absorptivity (e) are reported. Gravity column chromatography was
carried out using Grace Davison LC60A 40–63 micron silica gel
while vacuum column chromatography was carried out using
Grace Davison LC60A 6–35 micron silica gel. Reactions were mon-
itored using thin layer chromatography, performed on Merck DC
Table 1
Antiplasmodial activities and cytotoxicity data for compounds 7a–i
Compound
P. falciparum 3D7
NFFa cytotoxicity %
inhibition SD at 100 lM
IC50 SD (
l
M)
aluminium plates coated with silica gel GF254
.
7a
7b
7c
7d
7e
7f
7g
7h
10.2 1.2
17.2 3.0
35.6 3.7
8.9 0.2
17.3 0.3
39.8 3.6
8.3 2.7
8.2 0.4
6.8 2.7
3.9 ( 1.5)
51.4 19.5
51.0 11.9
40.4 22.3
40.0 15.8
48.6 12.9
38.0 16.4
83.6 15.3
54.6 15.6
60.1 21.4
4.1.1. General procedure for the synthesis of flavenes (6a–i)
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 unreacted
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.
7i
b
Dependensin
Chloroquine
46.9 ( 0.3)
c
0.03 ( 0.01)
a
b
c
NFF, neonatal foreskin fibroblast cells.
% Inhibition ( SD) at 50 M.
Chloroquine IC50 = 34.0 0.3
l
l
M.
Five compounds displayed antiplasmodial IC50 values <10
with 7i showing the most potent antiparasitic activity with an
IC50 of 6.8 M. All molecules displayed some toxicity towards the
NFF cells at 100 M. A comparison of the IC50 values of the chro-
lM,
l
l
meno[2,3-b]chromene derivatives with the natural product,
dependensin and previously reported analogues13 shows that the
scaffold present in 7a–i exhibits less antiplasmodial activity than
the dependensin scaffold. Therefore, the manner in which the
benzopyran rings are fused together is important for antiplasmo-
dial activity. Although compounds 7a–i show some selectivity to-
wards the malaria parasite, the low selectivity and moderate to
low antiplasmodial activity does not warrant further medicinal
chemistry efforts. However, these dimeric structures will be
screened in other biological assays in the future in order to identify
their potential use in other areas of drug discovery.
4.1.1.1. 6-Methoxyflav-3-ene (6a)21
Yield: 72%; UV (MeOH): kmax 204
(46379), 293 (14841) nm; IR (KBr): mmax 3425, 3018, 2959, 2926,
.
Colorless sticky residue,
(e
11257 cmꢁ1 Mꢁ1), 231
2852, 1615, 1493, 1465, 1431, 1269, 1203, 1159, 1039, 968,
807 cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.77 (s, 3H, CH3O), 5.84
;
(dd, J = 3.0, 10.9 Hz, 1H, H3), 5.88 (dd, J = 1.5, 3.0 Hz, 1H, H2), 6.52
(dd, J = 1.5, 10.9 Hz, 1H, H4), 6.60 (d, J = 3.0 Hz, 1H, H5), 6.68 (dd,
J = 3.0, 8.7 Hz, 1H, H7), 6.75 (d, J = 8.7 Hz, 1H, H8), 7.32–7.47 (m,
5H, H2, H3, H4, H5, H6); 13C NMR (75 MHz, CDCl3): d 55.7 (CH3O),
76.9 (C2), 111.7 (C5), 114.4 (C7), 116.5 (C8), 122.0 (C4a), 124.1
(C4), 125.8 (C3), 127.0 (C2, C6), 128.2 (C4), 128.6 (C3, C5), 140.6
(C1), 147.0 (C8a), 154.0 (C6).
3. Conclusion
A facile simple one-step methodology to the synthesis of a ser-
ies of novel chromeno[2,3-b]chromenes has been developed via
the acid-catalyzed dimerization reactions of 5-methoxyflavenes
and 6-methoxyflavenes. All synthetic chromene analogues 7a–i
displayed moderate to low antiplasmodial activity with IC50 values
4.1.1.2. 4-Bromo-6-methoxyflav-3-ene (6b). White solid, Yield:
65%; mp 98–100 °C; UV (MeOH): kmax 202 (e
34171 cmꢁ1 Mꢁ1),
232 (39101), 330 (4329) nm; IR (KBr): mmax 3441, 3006, 2958,
2931, 2831, 1608, 1580, 1490, 1461, 1427, 1271, 1219, 1152, 1028,
971, 821 cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.76 (s, 3H, CH3O),
;
ranging from 6.8 to 39.8 lM.
5.80 (dd, J = 3.0, 10.9 Hz, 1H, H3), 5.83 (dd, J = 1.5, 3.0 Hz, 1H, H2),
6.52 (dd, J = 1.5, 10.9 Hz, 1H, H4), 6.61 (d, J = 3.0 Hz, 1H, H5), 6.68
(dd, J = 3.0, 8.7 Hz, 1H, H7), 6.74 (d, J = 8.7 Hz, 1H, H8), 7.32 (d,
J = 8.3 Hz, 2H, H2, H6), 7.49 (d, J = 8.3 Hz, 2H, H3, H5); 13C NMR
(75 MHz, CDCl3): d 55.6 (CH3O), 76.0 (C2), 111.8 (C5), 114.6 (C7),
116.5 (C8), 121.8 (C4a), 122.2 (C4), 124.5 (C4), 125.1 (C3), 128.7
(C2, C6), 131.7 (C3, C5), 139.6 (C1), 146.7 (C8a), 154.1 (C6); MS
4. Experimental section
4.1. Materials and methods
All reagents and solvents were obtained from commercial
sources and purified if necessary. Melting points were measured
(TOF-ESI) m/z Calcd for
C
16H1379BrO2 (M+1)+ 317.02. Found

R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
1531
316.95; Anal. Calcd for C16H13BrO2: C, 60.59; H, 4.13. Found: C,
60.82; H, 4.26.
2825, 1736, 1611, 1593, 1470, 1438, 1377, 1268, 1154, 1091,
1013, 829, 780, 750 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 3.76
;
(s, 3H, CH3O), 5.83 (dd, J = 3.8, 9.8 Hz, 1H, H3), 5.88 (dd, J = 1.9,
3.8 Hz, 1H, H2), 6.40 (d, J = 8.3 Hz, 1H, H6), 6.52 (d, J = 8.3 Hz, 1H,
H8), 6.79 (dd, J = 1.9, 9.8 Hz, 1H, H4), 7.04 (t, J = 8.3 Hz, 1H, H7),
7.39–7.43 (m, 4H, H2, H3, H5, H6); 13C NMR (75 MHz, CDCl3): d
55.6 (CH3O), 75.7 (C2), 103.5 (C6), 109.0 (C8), 110.7 (C4a), 119.1
(C4), 122.2 (C3), 128.4 (C2, C6), 128.7 (C3, C5), 129.4 (C7), 134.0
(C4), 139.2 (C1), 153.6 (C5), 155.3 (C8a); MS (TOF-ESI) m/z Calcd
for C16H1335ClO2Na (M+Na)+ 295.05. Found 295.03; Anal. Calcd
for C16H13ClO2.H2O: C, 66.10; H, 5.20. Found: C, 66.20; H, 5.45.
4.1.1.3. 4-Chloro-6-methoxyflav-3-ene (6c). White solid, Yield:
68%; mp 65–67 °C; UV (MeOH): kmax 204 (
229 (33355), 331 (2951) nm; IR (KBr): mmax 3456, 3006, 2950,
e
45814 cmꢁ1 Mꢁ1),
2937, 2833, 1610, 1576, 1491, 1468, 1433, 1271, 1215, 1148,
1042, 966, 834 cmꢁ1 1H NMR (300 MHz, CDCl3): d 3.76 (s, 3H,
;
CH3O), 5.80 (dd, J = 3.8, 10.9 Hz, 1H, H3), 5.84 (dd, J = 1.9, 3.8 Hz,
1H, H2), 6.52 (dd, J = 1.9, 10.9 Hz, 1H, H4), 6.59 (d, J = 2.6 Hz, 1H,
H5), 6.67 (dd, J = 2.6, 8.7 Hz, 1H, H7), 6.72 (d, J = 8.7 Hz, 1H, H8),
7.32 (d, J = 8.3 Hz, 2H, H2, H6), 7.35 (d, J = 8.3 Hz, 2H, H3, H5);
13C NMR (75 MHz, CDCl3): d 55.6 (CH3O), 76.0 (C2), 111.8 (C5),
114.6 (C7), 116.5 (C8), 121.8 (C4a), 124.5 (C4), 125.2 (C3), 128.3
(C2, C6), 128.7 (C3, C5), 134.1 (C4), 139.0 (C1), 146.7 (C8a), 154.1
(C6); MS (TOF-ESI) m/z Calcd for C16H1335ClO2 (M+1)+ 273.07.
Found 273.00; Anal. Calcd for C16H13ClO2.1/10H2O: C, 70.00; H,
4.85. Found: C, 69.97; H, 4.96.
4.1.1.8. 4,5-Dimethoxyflav-3-ene (6h). White solid, Yield: 80%;
mp 61–63 °C; UV (MeOH): kmax 203 (
e
21185 cmꢁ1 Mꢁ1), 229
(20204), 282 (7936) nm; IR (KBr): mmax 3428, 3013, 2965, 2940,
2843, 1601, 1584, 1466, 1426, 1271, 1240, 1204, 1103, 1029,
833, 747 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 3.70 (s, 3H,
;
CH3O), 3.76 (s, 3H, CH3O), 5.83 (dd, J = 3.8, 9.4 Hz, 1H, H3), 5.85
(dd, J = 1.9, 3.8 Hz, 1H, H2), 6.33 (d, J = 8.3 Hz, 1H, H6), 6.42 (d,
J = 8.3 Hz, 1H, H8), 6.78 (dd, J = 1.9, 9.4 Hz, 1H, H4), 6.89 (d,
J = 8.7 Hz, 2H, H3, H5), 7.03 (t, J = 8.3 Hz, 1H, H7), 7.29 (d,
J = 8.7 Hz, 2H, H2, H6); 13C NMR (75 MHz, CDCl3): d 55.2 (CH3O),
55.6 (CH3O), 76.3 (C2), 103.3 (C6), 109.1 (C8), 110.8 (C4a), 113.9
(C3, C5), 118.7 (C4), 122.9 (C3), 128.7 (C2, C6), 129.2 (C7), 132.8
(C1), 153.8 (C5), 155.3 (C8a), 159.6 (C4); HRMS (ESI) m/z Calcd
for C17H16O3Na (M+Na)+ 291.0999. Found 291.1011; Anal. Calcd
for C17H16O3: C, 76.10; H, 6.01. Found: C, 76.11; H, 6.04.
4.1.1.4. 6-Methoxy-4-methylflav-3-ene (6d). Colorless sticky
residue, Yield: 74%; UV (MeOH): kmax 204 (
e
81758 cmꢁ1 Mꢁ1),
255 (27838), 290 (16061) nm; IR (KBr): mmax 3435, 3011, 2999,
2921, 2833, 1610, 1583, 1495, 1464, 1430, 1263, 1201, 1153,
1036, 971, 804 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.35 (s, 3H,
;
CH3), 3.76 (s, 3H, CH3O), 5.82 (dd, J = 3.4. 9.4 Hz, 1H, H3), 5.86
(dd, J = 1.1, 3.4 Hz, 1H, H2), 6.51 (dd, J = 1.1, 9.4 Hz, 1H, H4), 6.59
(d, J = 2.6 Hz, 1H, H5), 6.67 (dd, J = 2.6, 8.7 Hz, 1H, H7), 6.72 (d,
J = 8.7 Hz, 1H, H8), 7.17 (d, J = 8.3 Hz, 2H, H3, H5), 7.34 (d,
J = 8.3 Hz, 2H, H2, H6); 13C NMR (75 MHz, CDCl3): d 21.1 (CH3)
55.6 (CH3O), 76.5 (C2), 111.6 (C5), 114.4 (C7), 116.5 (C8), 122.0
(C4a), 124.1 (C4), 125.9 (C3), 127.0 (C2, C6), 129.2 (C3, C5), 137.6
(C4), 138.1 (C1), 147.0 (C8a), 153.9 (C6); (TOF-ESI) m/z Calcd for
4.1.1.9. 5-Methoxy-4-methylflav-3-ene (6i). Colorless sticky res-
idue, Yield: 79%; UV (MeOH): kmax 205 (e
27890 cmꢁ1 Mꢁ1), 224
(16588), 275 (6161) nm; IR (KBr): mmax 3454, 3002, 2978, 2924,
2838, 1605, 1583, 1468, 1439, 1268, 1235, 1098, 1018, 818,
C
C
17H16O2K (M+K)+ 291.12. Found 291.08; Anal. Calcd for
17H16O2.1.6H2O: C, 72.63; H, 6.88. Found: C, 72.55; H, 6.69.
745 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.39 (s, 3H, CH3), 3.86 (s,
;
3H, CH3O), 5.79 (dd, J = 3.4, 9.8 Hz, 1H, H3), 5.87 (dd, J = 1.9,
3.4 Hz, 1H, H2), 6.46 (d, J = 8.3 Hz, 1H, H6), 6.50 (d, J = 8.3 Hz, 1H,
H8), 6.94 (dd, J = 1.9, 9.8 Hz, 1H, H4), 7.09 (t, J = 8.3 Hz, 1H, H7),
7.21 (d, J = 8.6 Hz, 2H, H3, H5), 7.27 (d, J = 8.6 Hz, 2H, H2, H6);
13C NMR (75 MHz, CDCl3): d 21.1 (CH3), 55.6 (CH3O), 76.5 (C2),
103.3 (C6), 109.1 (C8), 110.8 (C4a), 118.7 (C4), 123.0 (C3), 127.1
(C2, C6), 129.2 (C3, C5), 129.7 (C7), 137.8 (C4), 138.0 (C1), 153.9
(C5), 155.3 (C8a); MS (TOF-ESI) m/z Calcd for C17H16O2 (M+H)+
253.12. Found 253.12; Anal. Calcd for C17H16O2.H2O: C, 75.53; H,
6.71. Found: C, 75.77; H, 6.38.
4.1.1.5. 5-Methoxyflav-3-ene (6e)22
89–91 °C; UV (MeOH): kmax 204 (
13733 cmꢁ1 Mꢁ1), 228 (12076),
283 (4156) nm; IR (KBr): max 3418, 3013, 2961, 2938, 2840, 1601,
. White solid, Yield: 77%; mp
e
m
1582, 1466, 1438, 1272, 1246, 1202, 1107, 1033, 837, 752 cmꢁ1; 1H
NMR (300 MHz, CDCl3): d 3.84 (s, 3H, CH3O), 5.77 (dd, J = 3.4,
10.4 Hz, 1H, H3), 5.86 (dd, J = 1.9, 3.4 Hz, 1H, H2), 6.45 (d,
J = 8.3 Hz, 2H, H6, H8), 6.90 (dd, J = 1.9, 10.4 Hz, 1H, H4), 7.06 (t,
J = 8.3 Hz, 1H, H7), 7.28–7.42 (m, 5H, H2, H3, H4, H5, H6); 13C
NMR (75 MHz, CDCl3): d 55.6 (CH3O), 76.6 (C2), 103.4 (C6), 109.1
(C8), 110.8 (C4a), 118.7 (C4), 122.8 (C3), 127.0 (C2, C6), 128.2
(C4), 128.5 (C3, C5), 129.2 (C7), 140.8 (C1), 153.9 (C5), 155.3 (C8a).
4.1.2. General procedure for acid-catalyzed dimerization
reactions (7a–i)
To a solution of the appropriate flavene (1.0 equiv) in MeOH
(20 mL) was added 10 drops of acid (TFA or AcOH) and the solution
was heated at 60–70 °C for 12 h. The solvent was partially removed
under 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.
4.1.1.6. 4-Bromo-5-methoxyflav-3-ene (6f). White solid, Yield:
70%; mp 56–58 °C; UV (MeOH): kmax 203 (
226 (63329), 283 (18772) nm; IR (KBr): mmax 3423, 3007, 2963,
e
66797 cmꢁ1 Mꢁ1),
2934, 2838, 1601, 1580, 1467, 1439, 1272, 1244, 1196, 1098,
1012, 899, 742 cmꢁ1 1H NMR (300 MHz, DMSO-d6): d 3.76 (s,
;
3H, CH3O), 5.84 (dd, J = 3.4, 9.8 Hz, 1H, H3), 5.88 (dd, J = 1.9,
3.4 Hz, 1H, H2), 6.39 (d, J = 8.3 Hz, 1H, H6), 6.53 (d, J = 8.3 Hz, 1H,
H8), 6.78 (dd, J = 1.9, 9.8 Hz, 1H, H4), 7.06 (t, J = 8.3 Hz, 1H, H7),
7.34 (d, J = 8.6 Hz, 2H, H2, H6), 7.54 (d, J = 8.6 Hz, 2H, H3, H5);
13C NMR (75 MHz, CDCl3): d 55.6 (CH3O), 75.7 (C2), 103.5 (C6),
109.0 (C8), 110.7 (C4a), 119.2 (C4), 122.1 (C3), 122.3 (C4), 128.7
(C2, C6), 129.4 (C7), 131.6 (C3, C5), 139.7 (C1), 153.6 (C5), 155.3
(C8a); MS (TOF-ESI) m/z Calcd for C16H1379BrO2 (M+1)+ 317.02.
Found 316.99; Anal. Calcd for C16H13BrO2: C, 60.59; H, 4.13. Found:
C, 60.78; H, 4.24.
4.1.2.1. (E)-2,9-Dimethoxy-5a-phenyl-11-styryl-11a,12-dihydro-
5aH ,11H-chromeno[2,3-b]chromene (7a). White solid, Yield:
70%; mp 204–206 °C; UV (MeOH): kmax 204 (e
45414 cmꢁ1 Mꢁ1),
231 (14449), 256 (16881), 293 (8914) nm; IR (KBr): mmax 3440,
3003, 2933, 2837, 1612, 1498, 1464, 1430, 1273, 1231, 1203,
1153, 1035, 972, 952, 823, 802 cmꢁ1 1H NMR (300 MHz, CDCl3):
;
4.1.1.7. 4-Chloro-5-methoxyflav-3-ene (6g). Yellow sticky resi-
due, Yield: 70%; UV (MeOH): kmax 204 (
(58394), 275 (18204) nm; IR (KBr): mmax 3439, 3043, 2983, 2945,
d 2.88 (s, 3H, H11a, H12), 3.49 (dd, J = 3.0, 9.0 Hz, 1H, H11), 3.69
(s, 3H, CH3O), 3.73 (s, 3H, CH3O), 6.28 (dd, J = 9.0, 15.8 Hz, 1H,
H ), 6.51 (d, J = 15.8 Hz, 1H, Hb), 6.63 (d, J = 3.0 Hz, 2H, H1, H10),
a
e
79277 cmꢁ1 Mꢁ1), 222

1532
R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
6.72 (dd, J = 3.0, 8.7 Hz, 1H, H3), 6.80 (dd, J = 3.0, 8.7 Hz, 1H, H8),
6.88 (d, J = 8.7 Hz, 1H, H4), 6.98 (d, J = 8.7 Hz, 1H, H7), 7.28–7.52
(m, 10H, H2, H3, H4, H5, H6, H2, H3, H4, H5, H6); 13C NMR
(75 MHz, CDCl3): d 23.2 (C12), 37.9 (C11a), 42.4 (C11), 55.5
(CH3O), 55.6 (CH3O), 100.9 (C5a), 113.6 (C1), 113.8 (C10), 113.9
(C3), 114.3 (C8), 117.1 (C7), 117.5 (C4), 121.6 (C12a), 122.6
(C10a), 125.9 (ArCH), 126.3 (ArCH), 126.8 (ArCH), 127.7 (ArCH),
(dd, J = 3.0, 9.4 Hz, 1H, H11), 3.69 (s, 3H, CH3O), 3.73 (s, 3H, CH3O),
6.22 (dd, J = 9.4, 15.8 Hz, 1H, H ), 6.43 (d, J = 15.8 Hz, 1H, Hb), 6.53
a
(d, J = 3.0 Hz, 1H, H10), 6.60 (d, J = 3.0 Hz, 1H, H1), 6.72 (dd, J = 3.0,
9.0 Hz, 1H, H3), 6.78 (dd, J = 3.0, 8.7 Hz, 1H, H8), 6.87 (d, J = 9.0 Hz,
1H, H4), 6.97 (d, J = 8.7 Hz, 1H, H7), 7.14 (d, J = 8.3 Hz, 2H, H3, H5),
7.18 (d, J = 8.3 Hz, 2H, H3, H5), 7.29 (d, J = 8.3 Hz, 2H, H2, H6), 7.42
(d, J = 8.3 Hz, 2H, H2, H6); 13C NMR (75 MHz, CDCl3): d 20.9 (CH3),
21.2 (CH3), 23.9 (C12), 37.8 (C11a), 42.4 (C11), 55.5 (CH3O), 55.6
(CH3O), 100.9 (C5a), 113.5 (C1), 113.7 (C10), 113.9 (C3), 114.2 (C8),
117.1 (C7), 117.5 (C4), 121.6 (C12a), 122.7 (C10a), 125.8 (C3, C5),
127.9 (ArCH), 128.6 (ArCH), 128.8 (C ), 133.7 (Cb), 136.7 (C1),
a
140.5 (C1), 145.7 (C4a), 146.3 (C6a), 154.1 (C9), 154.2 (C2); HRMS
(ESI) m/z Calcd for
C
32H28O4Na (M+Na)+ 499.1888. Found
499.1860; Anal. Calcd for C32H28O4.1/2EtOH: C, 79.33; H, 6.25.
Found: C, 79.23; H, 6.29.
126.5 (C2, C6), 128.8 (C ), 129.0 (C2, C6), 129.2 (C3, C5), 133.9 (Cb),
a
134.0 (C1), 137.4 (C4), 137.5 (C4), 138.6 (C1), 145.7 (C4a), 146.3
(C6a), 153.9 (C9), 154.1 (C2); HRMS (ESI) m/z Calcd for C34H32O4Na
(M+Na)+ 527.2201. Found 527.2187; Anal. Calcd for C34H32O4.EtOH:
C, 78.52; H, 6.96. Found: C, 78.71; H, 6.66.
4.1.2.2. (E)-5a-(4-Bromophenyl)-11-(4-bromostyryl)-2,9-dimethoxy-
11a,12-dihydro-5aH, 11H-chromeno[2,3-b]chromene (7b). White
solid, Yield: 73%; mp 212–214 °C; UV (MeOH): kmax 203 (
e
61384 cmꢁ1 Mꢁ1), 220 (35949), 230 (28486), 263 (34167), 291
(17870), 299 (12616) nm; IR (KBr): mmax 3444, 3006, 2929, 2833,
1615, 1489, 1464, 1427, 1275, 1230, 1200, 1154, 1041, 971, 951,
4.1.2.5. (E)-1,10-Dimethoxy-5a-phenyl-11-styryl-11a,12-dihy-
dro-5aH ,11H-chromeno[2,3-b]chromene (7e). White solid,
Yield: 75%; mp 127–129 °C; UV (MeOH): kmax 206 (e 48225 cm
803 cmꢁ1
;
1H NMR (300 MHz, CDCl3): d 2.84 (s, 3H, H11a, H12),
ꢁ1 Mꢁ1), 258 (10664) nm; IR (KBr): mmax 3418, 3026, 2933, 2836,
3.46 (dd, J = 3.4, 9.8 Hz, 1H, H11), 3.70 (s, 3H, CH3O), 3.73 (s, 3H,
1594, 1489, 1469, 1438, 1349, 1266, 1208, 1096, 1029, 961, 830,
CH3O), 6.24 (dd, J = 9.8, 15.8 Hz, 1H, H ), 6.47 (d, J = 15.8 Hz, 1H,
780 cmꢁ1 1H NMR (300 MHz, CDCl3):
; d 2.71 (dd, J = 12.4,
a
Hb), 6.55 (d, J = 2.3 Hz, 1H, H10), 6.60 (d, J = 3.0 Hz, 1H, H1), 6.72
(dd, J = 3.0, 8.6 Hz, 1H, H3), 6.80 (dd, J = 2.3, 8.3 Hz, 1H, H8), 6.87
(d, J = 8.6 Hz, 1H, H4), 6.96 (d, J = 8.3 Hz, 1H, H7), 7.24 (d,
J = 8.6 Hz, 2H, H2, H6), 7.37 (d, J = 8.3 Hz, 2H, H3, H5), 7.41 (d,
J = 8.3 Hz, 2H, H2, H6), 7.44 (d, J = 8.6 Hz, 2H, H3, H5); 13C NMR
(75 MHz, CDCl3): d 23.6 (C12), 37.8 (C11a), 42.3 (C11), 55.5
(CH3O), 55.6 (CH3O), 100.4 (C5a), 113.6 (C1), 113.7 (C10), 114.0
(C3), 114.4 (C8), 117.2 (C7), 117.5 (C4), 121.3 (C4), 121.6 (C12a),
17.3 Hz, 1H, H12), 2.79 (d, J = 17.3 Hz, 1H, H12), 2.99 (dd, J = 6.4,
12.4 Hz, 1H, H11a), 3.64 (s, 3H, CH3O), 3.71 (s, 3H, CH3O), 3.78
(dd, J = 6.4, 9.0 Hz, 1H, H11), 6.19 (dd, J = 9.0, 15.4 Hz, 1H, H ),
a
6.33 (d, J = 15.4 Hz, 1H, Hb), 6.39 (d, J = 8.3 Hz, 1H, H2), 6.51 (d,
J = 8.3 Hz, 1H, H9), 6.69 (d, J = 8.3 Hz, 1H, H4), 6.72 (d, J = 8.3 Hz,
1H, H7), 7.06 (t, J = 8.3 Hz, 2H, H3, H8), 7.10–7.56 (m, 10H, H2,
H3, H4, H5, H6, H2, H3, H4, H5, H6); 13C NMR (75 MHz, CDCl3): d
20.3 (C12), 37.0 (C11a), 38.8 (C11), 55.3 (CH3O), 55.6 (CH3O),
100.5 (C5a), 103.0 (C2), 104.0 (C9), 109.1 (C4), 109.7 (C7), 110.7
(C12a), 111.3 (C10a), 125.7 (C4), 125.9 (C2, C6), 126.0 (C3), 126.6
122.1 (C10a), 123.1(C4), 125.8 (C2, C6), 127.8 (C2, C6), 128.5 (C ),
a
131.6 (C3, C5), 131.7 (C3, C5), 133.2 (Cb), 135.4 (C1), 139.5 (C1),
145.5 (C4a), 145.9 (C6a), 154.2 (C9), 154.3 (C2); HRMS (ESI) m/z
Calcd for C32H2679Br2O4Na (M+Na)+ 655.0098. Found 655.0086);
Anal. Calcd for C32H26Br2O4: C, 60.59; H, 4.13. Found: C, 60.32; H,
4.13.
(C8), 127.9 (C ), 128.1 (C4), 128.3 (C3, C5), 128.5 (C2, C6), 129.7
a
(C3, C5), 131.2 (Cb), 136.9 (C1), 140.5 (C1), 152.8 (C4a), 153.7
(C6a), 157.4 (C1), 158.7 (C10); HRMS (ESI) m/z Calcd for
C
C
32H28O4Na (M+Na)+ 499.1888. Found 499.1878; Anal. Calcd for
32H28O4.1/4EtOH: C, 79.98; H, 6.09. Found: C, 80.18; H, 6.32.
4.1.2.3. (E)-5a-(4-Chlorophenyl)-11-(4-chlorostyryl)-2,9-dimethoxy-
11a,12-dihydro-5aH,11H-chromeno[2,3-b]chromene
solid, Yield: 74%; mp 180–182 °C; UV (MeOH): kmax 203 (
cmꢁ1 Mꢁ1), 221 (6487), 268 (18721) nm; IR (KBr):
max 3434, 3003,
2942, 2833, 1614, 1492, 1464, 1426, 1278, 1231, 1197, 1152, 1038,
976, 953, 820, 809 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.84 (s, 3H,
H11a, H12), 3.46 (dd, J = 3.4, 9.0 Hz, 1H, H11), 3.70 (s, 3H, CH3O),
(7c). White
4.1.2.6.
(E)-5a-(4-Bromophenyl)-11-(4-bromostyryl)-1,10-di-
e
11232
methoxy-11a,12-dihydro-5aH, 11H-chromeno[2,3-b]chromene
(7f). White solid, Yield: 68%; mp 118–120 °C; UV (MeOH): kmax
m
204 (
3005, 2932, 2830, 1593, 1487, 1468, 1350, 1269, 1210, 1088,
1054, 978, 832, 770 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.59 (dd,
e
103747 cmꢁ1 Mꢁ1), 265 (11329) nm; IR (KBr): mmax 3459,
;
;
3.73 (s, 3H, CH3O), 6.23 (dd, J = 9.0, 15.8 Hz, 1H, H ), 6.48 (d,
J = 12.0, 17.7 Hz, 1H, H12), 2.77 (d, J = 17.7 Hz, 1H, H12), 2.94 (dd,
J = 6.4, 12.0 Hz, 1H, H11a), 3.61 (s, 3H, CH3O), 3.70 (s, 3H, CH3O),
3.72 (dd, J = 6.4, 9.0 Hz, 1H, H11), 6.05 (dd, J = 9.0, 15.8 Hz, 1H,
a
J = 15.8 Hz, 1H, Hb), 6.56 (d, J = 3.0 Hz, 1H, H10), 6.60 (d, J = 3.0 Hz,
1H, H1), 6.72 (dd, J = 3.0, 8.7 Hz, 1H, H3), 6.80 (dd, J = 3.0, 9.0 Hz,
1H, H8), 6.87 (d, J = 8.7 Hz, 1H, H4), 6.97 (d, J = 9.0 Hz, 1H, H7), 7.25
(d, J = 8.3 Hz, 2H, H2, H6), 7.28 (d, J = 8.3 Hz, 2H, H3, H5), 7.34 (d,
J = 8.6 Hz, 2H, H3, H5), 7.45 (d, J = 8.6 Hz, 2H, H2, H6); 13C NMR
(75 MHz, CDCl3): d 23.6 (C12), 37.8 (C11a), 42.3 (C11), 55.5 (CH3O),
55.6 (CH3O), 100.4 (C5a), 113.6 (C1), 113.7 (C10), 114.0 (C3), 114.3
(C8), 117.2 (C4), 117.5 (C7), 121.3 (C12a), 122.2 (C10a), 127.5 (C3,
H ), 6.30 (d, J = 15.8 Hz, 1H, Hb), 6.35 (d, J = 8.3 Hz, 1H, H2), 6.49
a
(d, J = 8.3 Hz, 1H, H9), 6.64 (d, J = 8.3 Hz, 2H, H4, H7), 6.79 (t,
J = 8.3 Hz, 2H, H3, H8), 7.06 (d, J = 8.3 Hz, 2H, H2, H6), 7.16 (d,
J = 8.3 Hz, 2H, H3, H5), 7.27 (d, J = 8.3 Hz, 2H, H2, H6), 7.49 (d,
J = 8.3 Hz, 2H, H3, H5); 13C NMR (75 MHz, CDCl3): d 21.5 (C12),
36.7 (C11a), 38.8 (C11), 55.3 (2ꢀCH3O), 99.7 (C5a), 103.4 (C2),
104.3 (C9), 107.5 (C4), 108.8 (C7), 109.4 (C12a), 110.8 (C10a),
119.9 (C4), 122.8 (C4), 126.5 (C3), 127.8 (C2, C6), 128.0 (C2, C6),
C5), 128.0 (C ), 128.3 (C2, C6), 128.7 (C3, C5), 128.8 (C2, C6), 133.1
a
(Cb), 133.4 (C4), 134.8 (C4), 135.0 (C1), 138.9 (C1), 145.5 (C4a),
145.9 (C6a), 154.2 (C9), 154.3 (C2); HRMS (ESI) m/z Calcd for
C
for C32H26Cl2O4.EtOH: C, 69.04; H, 5.45. Found: C, 69.27; H, 5.14.
128.6 (C ), 128.9 (C8), 130.4 (C3, C5), 131.0 (C3, C5), 131.9 (Cb),
a
32H2635Cl2O4Na (M+Na)+ 567.1108. Found 567.1095; Anal. Calcd
135.9 (C1), 139.8 (C1), 152.3 (C4a), 153.8 (C6a), 157.8 (C1), 158.4
(C10); MS (TOF-ESI) m/z Calcd for C32H2679Br2O4 (M+1)+ 633.03.
Found 633.02; Anal. Calcd for C32H26Br2O4: C, 60.59; H, 4.13.
Found: C, 60.32; H, 4.36.
4.1.2.4. (E)-2,9-Dimethoxy-11-(4-methylstyryl)-5a-p-tolyl-11a,12-
dihydro-5aH ,11H-chromeno[2,3-b]chromene (7d). White solid,
Yield: 72%; mp 217–219 °C; UV (MeOH): kmax 204 (
e
31109 cm
4.1.2.7. (E)-5a-(4-Chlorophenyl)-11-(4-chlorostyryl)-1,10-dime-
thoxy-11a,12-dihydro-5aH,11H-chromeno[2,3-b]chromene
(7g). White solid, Yield: 72%; mp 120–122 °C; UV (MeOH): kmax
ꢁ1 Mꢁ1), 229 (15067), 261 (11049), 290 (5107) nm; IR (KBr): mmax
3435, 2998, 2922, 2832, 1614, 1492, 1464, 1427, 1272, 1232, 1200,
1153, 1039, 971, 951, 820, 803 cmꢁ1; 1H NMR (300 MHz, CDCl3): d
2.32 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.85 (s, 3H, H11a, H12), 3.50
205 (
e
56444 cmꢁ1 Mꢁ1), 264 (15800) nm; IR (KBr): mmax 3420,
3008, 2930, 2837, 1594, 1491, 1469, 1439, 1347, 1267, 1210,

R. Devakaram et al. / Bioorg. Med. Chem. 20 (2012) 1527–1534
1533
1094, 1052, 967, 831, 772 cmꢁ1; 1H NMR (300 MHz, CDCl3): d 2.59
4.2. Biological experiments
(dd, J = 11.7, 17.3 Hz, 1H, H12), 2.77 (d, J = 17.3 Hz, 1H, H12), 2.91
(dd, J = 6.4, 11.7 Hz, 1H, H11a), 3.61 (s, 3H, CH3O), 3.70 (s, 3H,
CH3O), 3.74 (dd, J = 6.4, 9.0 Hz, 1H, H11), 6.04 (dd, J = 9.0,
4.2.1. Method for in vitro antiplasmodial growth inhibition
assay
15.8 Hz, 1H, H ), 6.29 (d, J = 15.8 Hz, 1H, Hb), 6.32 (d, J = 8.3 Hz,
The P. falciparum growth inhibition assays were carried using an
isotopic microtest, as previously described.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 cul-
ture plates containing serial dilutions of control (chloroquine) or
test compounds. Following 48 h incubation under standard P. falci-
a
1H, H2), 6.49 (d, J = 8.3 Hz, 1H, H9), 6.65 (d, J = 8.3 Hz, 2H, H4,
H7), 6.85 (t, J = 8.3 Hz, 2H, H3, H8), 7.11 (d, J = 8.3 Hz, 2H, H2,
H6), 7.18 (d, J = 8.3 Hz, 2H, H3, H5), 7.29 (d, J = 8.7 Hz, 2H, H3,
H5), 7.44 (d, J = 8.7 Hz, 2H, H2, H6); 13C NMR (75 MHz, CDCl3): d
20.8 (C12), 36.4 (C11a), 38.5 (C11), 55.3 (CH3O), 55.6 (CH3O),
100.0 (C5a), 103.1 (C2), 104.0 (C9), 108.9 (C7), 109.7 (C4), 110.5
(C12a), 111.0 (C10a), 127.1 (C3), 127.3 (C3, C5), 127.5 (C3, C5),
parum culture conditions, 0.5 l
Ci [3H]-hypoxanthine was added to
each well after which the plates were cultured for a further 24 h.
Cells were harvested onto 1450 MicroBeta filter mats (Wallac)
and 3H incorporation was determined using a 1450 MicroBeta li-
quid scintillation counter. Percentage inhibition of growth was
compared to matched DMSO controls (0.5%). IC50 values were cal-
culated using linear interpolation of inhibition curves.24 The mean
IC50 is shown for two independent experiments, each carried out in
triplicate.
128.1 (C8), 128.4 (C ), 128.6 (C2, C6), 128.9 (C2, C6), 131.3 (Cb),
a
132.1 (C4), 134.6 (C4), 136.2 (C1), 139.2 (C1), 152.3 (C4a), 153.7
(C6a), 157.3 (C1), 158.4 (C10); HRMS (ESI) m/z Calcd for
C
32H2635Cl2O4Na (M+Na)+ 567.1108. Found 567.1079; Anal. Calcd
for C32H26Cl2O4.1/4H2O: C, 69.89; H, 4.86. Found: C, 70.08; H, 4.87.
4.1.2.8. (E)-1,10-Dimethoxy-5a-(4-methoxyphenyl)-11-(4-meth-
oxystyryl)- 11a,12-dihydro-5aH,11H-chromeno[2,3-b]chromene
(7h). White solid, Yield: 76%; mp 134–136 °C; UV (MeOH): kmax
4.2.2. Method for in vitro cytotoxicity assay25
204 (
3001, 2933, 2835, 1594, 1489, 1468, 1439, 1348, 1251, 1215,
1089, 1034, 981, 834, 777 cmꢁ1 1H NMR (300 MHz, CDCl3): d
e
36939 cmꢁ1 Mꢁ1), 267 (9405) nm; IR (KBr): mmax 3429,
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 (Corn-
ing, USA) and were grown for 24 h before treatment. Compounds
were dissolved in 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.25 Compounds were
tested in triplicate in three independent experiments.
;
2.70 (dd, J = 12.1, 17.1 Hz, 1H, H12), 2.81 (d, J = 17.1 Hz, 1H, H12),
2.91 (dd, J = 6.4, 12.1 Hz, 1H, H11a), 3.61 (s, 3H, CH3O), 3.68 (s,
3H, CH3O), 3.77 (s, 3H, CH3O), 3.79 (s, 3H, CH3O), 3.80 (dd, J = 6.4,
9.8 Hz, 1H, H11), 6.04 (dd, J = 9.8, 16.2 Hz, 1H, H ), 6.31 (d,
a
J = 16.2 Hz, 1H, Hb), 6.36 (d, J = 8.3 Hz, 1H, H2), 6.47 (d, J = 8.3 Hz,
1H, H9), 6.60–6.86 (m, 4H, H3, H4, H7, H8), 6.94 (d, J = 8.7 Hz,
2H, H3, H5), 7.15 (d, J = 9.0 Hz, 2H, H3, H5), 7.37 (d, J = 8.7 Hz,
2H, H2, H6), 7.44 (d, J = 9.0 Hz, 2H, H2, H6); 13C NMR (75 MHz,
CDCl3): d 22.9 (C12), 38.8 (C11a), 39.8 (C11), 55.1 (CH3O), 55.2
(CH3O), 55.4 (2ꢀCH3O), 99.6 (C5a), 102.8 (C2), 103.6 (C9), 109.5
(C4), 109.7 (C7), 113.5 (C12a), 113.6 (C10a), 126.9 (C3, C5), 127.0
(C3), 127.2 (C3, C5), 127.8 (C8), 128.3 (C ), 128.8 (C2, C6), 129.9
a
(C2, C6), 131.8 (Cb), 136.3 (C1), 139.6 (C1), 152.8 (C4a), 152.9
(C6a), 157.6 (C1), 158.5 (C10), 158.9 (C4), 159.7 (C4); HRMS (ESI)
m/z Calcd for C34H32O6Na (M+Na)+ 559.2099. Found 559.2071;
Anal. Calcd for C34H32O6: C, 76.10; H, 6.01. Found: C, 76.22; H, 6.29.
Acknowledgments
We thank the University of New South Wales, and the Austra-
lian Research Council (ARC) for funding to N.K. and D.B. Katherine
Andrews from Griffith University is acknowledged for assistance
with the P. falciparum assays. The Australian Red Cross Blood
Service are acknowledged for providing human blood and sera
for P. falciparum culture.
4.1.2.9. (E)-1,10-Dimethoxy-11-(4-methylstyryl)-5a-p-tolyl-11a,
12-dihydro-5aH,11H-chromeno[2,3-b]chromene (7i). White so-
lid, Yield: 74%; mp 186–188 °C; UV (MeOH): kmax 204
(e
50304 cmꢁ1 Mꢁ1), 261 (11142) nm; IR (KBr): mmax 3432, 3023,
2933, 2837, 1593, 1485, 1468, 1437, 1350, 1268, 1208, 1087,
References and notes
1055, 976, 825, 770 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.24 (s,
;
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3.61 (dd, J = 6.3, 8.3 Hz, 1H, H11), 3.66 (s, 3H, CH3O), 3.78 (s, 3H,
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a
Hb), 6.47 (d, J = 8.3 Hz, 2H, H2, H9), 6.61 (d, J = 8.3 Hz, 1H, H4),
6.70 (d, J = 8.3 Hz, 1H, H7), 6.87 (t, J = 8.3 Hz, 2H, H3, H8), 7.00 (d,
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(C2, C6), 129.3 (C3, C5), 130.9 (Cb), 133.9 (C1), 136.2 (C4), 137.5
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(C10); HRMS (ESI) m/z Calcd for C34H32O4Na (M+Na)+ 527.2201.
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6.80. Found: C, 77.30; H, 6.54.

1534
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