Design, synthesis and structure-activity relationship of novel semi-synthetic flavonoids as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2014.06.007

Various flavonoid scaffold based derivatives viz furochalcones (3a-e, 6a-d and 9a-d), furoflavones (10a-d, 11a-d, 12a-d, 18a&b), flavones (21a-d), furoaurones (13a,b, 14a-d and 15a-d) and 7-styrylfurochromones (22a-d and 25a-e) were designed and synthesiz

Full text of DOI:10.1016/j.ejmech.2014.06.007

Accepted Manuscript
Design, Synthesis and Structure-Activity Relationship of Novel Semi-synthetic
Flavonoids as Antiproliferative Agents
F.A. Ragab , T.A.A. Yahya , Mona M. El-Naa , R.K. Arafa
PII:
S0223-5234(14)00525-X
10.1016/j.ejmech.2014.06.007
EJMECH 7049
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2014
Revised Date: 22 April 2014
Accepted Date: 4 June 2014
Please cite this article as: F.A. Ragab, T.A.A. Yahya, M.M. El-Naa, R.K. Arafa, Design, Synthesis and
Structure-Activity Relationship of Novel Semi-synthetic Flavonoids as Antiproliferative Agents, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.007.
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Graphical abstract
O
N
O
R
O
O
O
O
O
O
Molecular manipulation
sites
O
O
N(CH3)2
2
5e
MGꢀMID against 60 cancer cell lines
GI50 = 1.9 ꢁM
TGI = 5.24 ꢁM
LC50 = 60.25 ꢁM

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Highlights
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Novel semisynthetic flavonoid derivatives were prepared as antiproliferative agents
9a, 9b, 25c and 25e showed the lowest results for GI50, TGI and LC50
Kinase inhibitory ability against CDK2/cyclin E1, CDK4/cyclin D1 and GSK-3β
Mice-bearing EAC solid tumor was used to test the in vivo antitumor activity of 25e
25e caused decrease in tumor size, increase in GSK-3β and decrease in cyclin D1

ACCEPTED MANUSCRIPT
Design, Synthesis and Structure-Activity Relationship of Novel
Semi-synthetic Flavonoids as Antiproliferative Agents
†
‡
§
†,*
F. A. Ragab, T. A. A. Yahya, Mona M. El-Naa, R. K. Arafa
†
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, 11562
Cairo, Egypt
‡
Medicinal Chemistry Department, Faculty of Pharmacy, Sana`a University, Sana`a, Yemen
§
Pharmacology and Toxicology Department, Faculty of Pharmacy, October University for
Modern Sciences and Arts, Egypt
KEYWORDS: furochalcones, furoflavones, furoaurones, furostyrylfurochromones,
cytotoxicity, kinase inhibition.
Corresponding Author: Fax: 002023628426. E-mail: rkarafa@cu.edu.eg.
1

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ABSTRACT: Various flavonoid scaffold based derivatives viz furochalcones (3a-e, 6a-d and
9
a-d), furoflavones (10a-d, 11a-d, 12a-d, 18a&b), flavones (21a-d), furoaurones (13a,b,
4a-d and 15a-d) and 7-styrylfurochromones (22a-d and 25a-e) were designed and
1
synthesized. The novel compounds were evaluated for their antiproliferative activity against a
panel of 60 cancer cell lines comprising 9 types of tumors. Ten compounds belonging to the
major subgroups of flavonoids viz furochalcones (3a, 3d, 6b, 9a and 9b), furoflavones (12a
and 12c), furoaurones (15d), styrylfurochromones (25b and 25e) showed very promising
activity. These active compounds were also evaluated in vitro as kinase inhibitors against
CDK2/cyclin E1, CDK4/cyclin D1 and GSK-3β and the best inhibition was displayed against
GSK-3β with the allylfurochalcone derivative 9b exhibiting 80% decrease in GSK-3β
catalytic activity. On the other hand, the styrylfurochromone 25e interestingly showed a 13%
enhancement of GSK-3β catalytic power and a 12% reduction in CDK4/cyclin D1 activity.
Finally, the in vivo anti-tumor activity of 25e was evaluated against breast cancer induced in
mice. The results showed a profound anti-tumor effect of 25e that accompanies a significant
increase and decrease in the levels of GSK-3β and cyclin D1, respectively.
2

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1
. Introduction
Flavonoids represent an important class of naturally occurring compounds present in dietary
plants and herbal remedies [1]. The role of dietary flavonoids in cancer prevention is well
established and widely discussed. Many mechanisms of action have been identified including
cell cycle arrest, induction of apoptosis, inhibition of angiogenesis, antioxidant effect and
inhibition of some functional enzymes like cyclin-dependent kinases, tyrosine kinases,
aromatases, topoisomerases and glycogen phosphorylases [2-10]. Chemically, flavonoids are
classified into several subclasses among them are flavones (2-phenyl-4H-benzopyran-4-
ones), aurones (structural isomers of flavones; 2-benzylidene benzofuran-3(2H)-ones), 2-
styryl chromones (vinylogues of flavones) and o-hydroxychalcones (the flexible analogs of
aurones and flavones). Many natural products belonging to the flavonoid subclasses possess
anticancer activity like quercitin (I) and apigenin (II) (flavones), hamiltrone (III) (aurone),
hamilcone (IV) (chalcone) and hormothamnione (V) (styrylchromone) (Figure 1) [11-15]. In
addition, flavopiridol (L86–8275) (VI) (Figure 1), a semisynthetic flavones currently under
clinical development for management of chronic lymphocytic leukemia, exhibited cytotoxic
activity against several types of human cell lines associated with potent inhibition of cyclin-
dependent kinases (CDKs) CDK2/cyclin E1 and CDK4/cyclin D1 as well as glycogen
synthase kinase 3-beta (GSK-3β) [10,16]. While CDKs 2 and 4 are important regulators
progression through cell cycle, GSK-3β plays a profound regulatory rule in cell transduction
pathways responsible for various cellular functions including differentiation, growth,
proliferation, motility and survival. Dysregulation of CDKs, cyclins and GSK-3β expression
leads to many pathological transformations leading to neoplasia, tumorigenesis and cancer
progression [17,18]. With regards to GSK-3β, available evidences indicate that it may
function as a ‘‘tumor suppressor” for certain types of tumors, such as skin and mammary
tumors and as a ‘‘tumor promoter” for other types, such as colon and pancreatic cancers [19].
3

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On the other hand, the observed over-expression of cyclin D1 in a variety of tumor types,
including mammary carcinogenesis, suggests that tumor promotion is mediated by cyclin D1
dysregulation [20,21]. Furthermore, in mammary cancer, an association has been established
between active GSK-3β and cyclin D1 cellular levels. Being a direct target of GSK-3β, at
high levels of GSK-3β, cyclin D1 gets rapidly phosphorylated at Thr286 with subsequent
promotion of its proteasomal degradation leading to initiation of cellular apoptotic
mechanisms and halting tumor progression.
Since the anticancer activity of furoflavonoids has not been extensively studied, this
investigation deals with the design, synthesis and evaluation of antiproliferative activity of
some
semi-synthetic
furoflavonoids
including
furoflavones,
furoaurones,
furostyrylchromones and furochalcones derived from khellin and visnagin. It also deemed of
interest to study the correlation between introducing various substituents on the furoflavonoid
scaffold and the cytotoxic activity of these compounds. The design criteria behind the
preparation of these new derivatives focused on the introduction of variable functionalities on
different sites of the furoflavonoid backbone scaffold and depiction of structure activity
relationships through studying the subsequent effect of the adopted molecular manipulations
on the cytotoxic activity of this class of compounds. Initially, 8-bromofuroflavonoids were
synthesized as certain bromoflavones and bromochalcones were reported to possess cytotoxic
activity against various tumor cell lines [22,23]. Also, the 8-allylfuroflavonoid analogs were
prepared since the allyl group is considered as a precursor for acetic acid moiety where recent
data indicated that flavone acetic acid possesses a broad range of activity against many tumor
types [24-26]. On the other hand, alteration of furoflavonoid backbone scaffold between the
bioisosteres furoflavones, furoaurones, furostyrylchromones and furochalcones was carried
out to study the impact of ring size and/or cyclic versus non-cyclic alterations on the
biological behavior of these derivatives. Finally, 6-morpholinomethyl substituted
4

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furoflavonoids were also prepared since previous studies showed that Mannich bases can
enhance the cytotoxicity [27,28]. The synthesized compounds were subjected to screening
against a panel of 60 cell lines representing 9 cancer types viz leukemia, lung, colon, CNS,
melanoma, ovarian, renal, prostate and breast. Literature search showed that flavonoid based
pharmaceutically active molecules mainly elicit their anticancer activity through inhibition of
kinases specially cyclin-dependent kinases and GSK-3β [20,21]. On this basis, the new
flavonoids were screened for their potential kinase inhibitory activity against CDK2/cyclin
E1, CDK4/cyclin D1 and GSK-3β which gave interesting results. Also, the in vivo effect of
2
5e, the most active compound in this study, on mammary tumor induced in mice along with
evaluation of its role as a modulator of GSK-3β and cyclin D1 was performed. To our
knowledge this is the first report of antiproliferative semi-synthetic flavonoids displaying this
mechanism of action.
Figure 1
2
. Results And Discussion
2
.1. Chemistry
Schemes I-VI depict the pathways for the synthesis of the intermediates and target
compounds. The key starting furochalcones 3a-e, which are important intermediates for the
synthesis of flavonoids, were obtained from the natural furochromone 1 through hydrolytic
alkaline cleavage of the γ-pyrone ring to furnish the acylated hydroxybenzofuran 2, followed
by Claisen-Schmidt condensation with the appropriate aromatic aldehydes [29,30]. Similarly,
the bromochalcones 6a-d were obtained from the semisynthetic bromofurochromone 4 in two
consecutive steps involving alkaline hydrolysis with 15% KOH [31] to give the acylated
bromohydroxybenzofuran 5 followed by a Claisen-Schmidt condensation with different
aromatic aldehydes [32]. On the other hand, the allylfurochalcones 9a-d were prepared from
5

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2
by O-allylation with allyl bromide to give the allyl ether 7 [33] followed by Claisen
rearrangement through refluxing with N,N-diethylaniline to give 8 that upon condensation
with aromatic aldehydes produced the target compounds (Scheme 1).
Synthesis of the methoxylated furoflavones 10a-d, 11a-d and 12a-d is illustrated in
scheme II where the target compounds were obtained from the furochalcones 3a-e, 6a-d and
9
a-d, respectively. This was achieved through oxidative cyclization with freshly sublimed
SeO in n-butanol. It is noteworthy that in the case of allylchalcones 9a-d, SeO led to
2
2
oxidization of the allyl group to an acrylaldehyde moiety along with the oxidative cyclization
to furnish derivatives 12a-d [34]. Spectral analysis of 12a-d were in accordance with the
-1
proposed structures where their I.R. spectra showed a band at around 1700 cm
1
corresponding to the aldehydic carbonyl group and their HNMR showed a peak around 9.5
ppm attributed to the aldehydic proton. On the other hand, subjecting the chalcones 3d, 3e,
6
1
a-d and 9a-d to oxidative cyclization using 30% H O and 20% NaOH, the furoaurones 13a,
2 2
3b, 14a-d and 15a-d were obtained, respectively, predominantly without any trace of their
structural furoflavone isomers. This can be mechanistically attributed, as depicted in Figure
2
5
, to the presence of the 4-methoxy substituent which directs ring closure to formation of the
-membered ring (aurones) rather than the 6-membered ring (flavones) favoring bond angles
that would allow less steric hindrance between the O-substituent (OCH ) and the carbonyl
3
group. However, in absence of the OCH substituent, previous reports showed that
3
hydroxychalcones were oxidized to 3-hydroxyflavones [35]. The structures of the aurones
were elucidated through I.R. which showed the presence of bands at the range of 1692-
-1
-1
1
697cm corresponding to the carbonyl group which appears at 1640-1650 cm in case of
flavones. Aurones also gave a negative FeCl test which confirms the failure of formation of
3
1
3
-hydroxyfuroflavones. Furthermore,
HNMR of the aurones, unlike the 3-
6

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hydroxyfuroflavone derivatives, showed peaks at around 6.7 ppm attributed to the vinyl
proton with absence peaks signifying the presence of the hydroxyl group.
Figure 2
Scheme III describes the synthesis of the phenolic furoflavone analogues 18a&b.
This was achieved starting by demethoxylation of furoflavones 10a&b using HBr to give the
desmethyl counterparts16a&b [36]. Then, the latter were subjected to O-allylation to give the
allyl ether analogues 17a&b. Finally, Claisen rearrangement of derivatives 17 gave the allyl
hydroxyfuroflavones 18a&b [33].
Additionally, the methoxylated allylflavones 21a-d were synthesized from 10a-d in
three steps as shown in scheme IV. The first step involves oxidative cleavage of the furan
ring with K Cr O in H SO [36] to give the hydroxy aldehydes 19a-d which were then O-
2
2
7
2
4
allylated to yield derivatives 20a-d. Finally, Claisen rearrangement gave the target allyl
flavones 21a-d [33].
Scheme V depicts the synthesis of 8-bromo-7-styrylfurochromones 22a-d from 9-
bromovisnagin 4 through condensation with various aromatic aldehydes using sodium
ethoxide as a basic catalyst [37].
Finally, the 6-morpholinomethyl-7-styryl furochromones 25a-e were obtained by
subjecting ω-acetyl khellinone 23 [38] to Mannich reaction using morpholine hydrochloride
and formalin which affected cyclization giving the 7-methyl-6-morpholinomethylfurochrome
2
4 [39] that was then subjected to Claisen-Schmidt condensation with different aromatic
aldehydes in ethanolic solution of sodium ethoxide to furnish the target compounds (Scheme
VI).
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2
.2. Biological Screening
In vitro cytotoxicity studies on tumoral cells. Forty three of the synthesized flavonoids were
selected by the national cancer institute (NCI, Bethesda, MD, USA) for the in vitro disease-
oriented human cell screening panel assay for their cytotoxic activity. A primary single-dose
(10 ꢀM) assay was performed by incubating each test compound with the full panel of the 60
tested cell lines. A 48 h continuous drug exposure protocol was used and a sulphorhodamine
B (SRB) protein assay was employed to estimate cell viability or growth [40]. The data
obtained are a mean-graph of the percentage growth inhibition (GI %) caused by the tested
compounds (Table S1).
Furthermore, ten flavonoids, which satisfied pre-determined threshold inhibition
criteria, were chosen to progress to a five dose (0.01-100 µM) screening assay and data
obtained were used to create a log concentration percent growth inhibition curve. The
selected compounds belong to the major subgroups of flavonoids viz furochalcones (3a, 3d,
6
b, 9a and 9b), furoflavones (12a and 12c), furoaurones (15d), styrylfurochromones (25b
and 25e). During the course of this screening, the response parameters corresponding to
compound concentration leading to 50% decrease in net cell growth (GI ), compound
5
0
concentration resulting in total growth inhibition (TGI) and compound concentration
affecting net 50% loss in initial cells at the end of the 48h incubation period (LC ) were
5
0
calculated for each cell line. Subpanel and full panel mean-graph mid-point values (MG-
MID) were also determined which represent the average GI , TGI or LC values of all cell
5
0
50
lines in the full panel.
Table 1 illustrates the GI (µM) of the ten tested compounds. With regards to the
5
0
sensitivity of the individual cell lines, all the ten compounds except 3d showed an obvious
activity (GI < 2 µM) against one or more of the tested cell lines.
5
0
8

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On leukemia cell lines, compound 12c showed the highest activity against RPMI-
8
226 with GI 1.37 µM; 12a, 12c, 25b and 25e displayed activity against CCRF-CEM with
5
0
GI in the range of 1.09-1.85 µM. Compound 25e proved to be potent toward all the tested
5
0
leukemia subpanels with GI range of 1.17-1.85 µM.
5
0
On non-small cell lung cancer cell lines, compounds 3a, 6b, 9a, 9b, 25b, 25c and 25e
exhibited distinctive activity against HOP-92 subpanel with GI range of 0.24-1.69 µM.
5
0
Also, 9a, 9b and 25c showed remarkable activity against NCI-H226 with GI range of 1.48-
5
0
1
.52 µM. Furthermore, compound 25e exhibited good cytotoxic activity against NCI-H322M
and NCI-H522 with GI50 1.65 and 1.83 µM, respectively and compound 25b displayed
cytotoxicity against NCI-H522 with GI 1.77 µM.
5
0
Regarding colon cancer cell lines, compound 6b and 25e were the most potent against
Colo-205 with GI 1.76 and 1.66 µM, respectively. Compounds 9a and 9b showed good
5
0
activity against HCC-2998 with GI values of 1.73 and 1.79 µM, respectively. Compound
5
0
1
2a and 12c were highly active against HCT-116 with GI 1.7 and 1.06 µM, respectively.
50
Finally, the rest of the examined cell lines were best inhibited by only one of the tested
compounds where 12c inhibited HCT-15 with GI 1.73 µM, 9b inhibited HT29 with a GI
50
5
0
of 1.89 µM and 25e inhibited KM12 with a GI of 1.73 µM.
5
0
On CNS cancer cell lines, compounds 9a and 9b revealed superior activity against
SF-539 with a GI of 1.71 and 1.79 µM, respectively. Compounds 6b, 9a, 15dand 25e were
5
0
all active against SNB-75 with a GI range of 1.39-1.8 µM. Lastly, SF-295 growth was
5
0
inhibited by 25e with GI of 1.82 µM.
5
0
With reference to melanoma cancer cell lines, derivatives 3a, 9a, 9b, 12a and 12c
showed potent activity against LOXIMVI with GI range of 1.08-1.69 µM. On the other
5
0
hand, 25b and 25e were active against SK-MEL-2 with GI50 of 1.65 and 1.39 µM,
9

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respectively. Derivatives 12c, 25b and 25e were effective against both SK-MEL-5 with a
GI range of 1.09-1.69 µM and UACC-62 with a GI range of 1.77-1.9 µM.
5
0
50
Regarding ovarian cancer, the subpanel IGROV-1 was sensitive towards 25b and 25e
with GI values 1.94 and 1.79 µM, respectively. While OVCAR-3 was responsive to 6b
5
0
(
GI 1.91 µM), 9b (GI 1.97 µM) and 25e (GI 1.76 µM), NCI/ADR-RES was sensitive to
50 50 50
9
a with IC of 1.88 µM. Finally, 6b affected SK-OV-3 displaying a relatively low GI of
50 50
1
.11 µM.
Concerning the renal cancer subpanel cell lines, the flavonoid 25e was active against
7
86-O, CAKI-1, SKF393 and UO-31 with IC50 values 1.93, 1.89, 1.81 and 1.43 µM,
respectively. Also, compounds 9a affected A498 cell line at a GI of 1.74 µM. Lastly,
5
0
RXF393 was responsive to 9a, 9b and 25e (GI range 1.72-1.85 µM).
5
0
As for prostate cancer, compound 25e was active against the two subpanels PC-3 and
DU-145 with GI50 1.69 and 1.67 µM, respectively.
On breast cancer, compound 25e was active against the three subpanels MCF-7,
HF478T and MDA-MB-468 with GI50 range of 1.68-1.88 µM. Finally, compound 12c
displayed activity towards MCF-7 (GI 1.63 µM); while 25b was effective against HS-578T
5
0
(
GI 1.9 µM).
50
2
.3. SAR findings
Table 2 displays the median growth inhibitory concentration (Med-GI , µM) of the ten most
5
0
active flavonoids in this study. Concerning the major subgroups of the synthesized
flavonoids, the styrylfurochromones represented by compounds 25b and 25e, showed
superior activity against all the tested cell lines with GI range of 1.53-4.30 µM. The most
5
0
active member in this series was 25e with GI range of 1.53-2.31 µM.
5
0
1
0

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Furthermore, chalcones represent the second subgroup of flavonoids displaying good
cytotoxic activity specially derivatives 3a, 6b, 9a and 9b. The most active members were 9a
and 9b carrying a 7-allyl substituent with GI50 range of 2.25-4.3 µM. Compound 9b,
possessing a 4-chloro substituent on ring B of the chalcone, showed increased activity against
colon, melanoma, ovarian and prostate cell lines compared to its dechloro analogue 9a.
Replacement of the 7-allyl group in 9b with a bromo substituent furnished compound 6b
which in comparison to 9b displayed superior activity towards leukemia and prostate cancer
cell lines and nearly equal activity against colon and ovarian cell lines. On the other hand,
comparison of the chalcones 9a with the 7-deallyl analog 3a revealed that the presence of the
allyl group markedly increased the activity against all tested cell lines (GI range of 2.25-4.3
5
0
µM for 9a versus 3.55-7.45 µM for 3a). Finally, the presence of the 2-methoxy group in ring
B as represented by 3d markedly decreased the cytotoxic activity (GI range of 16.6-40.4
5
0
µM) compared to the demethoxy congener 3a.
Regarding the flavone subgroup, the most active members of which are 12a and 12c,
a clear cytotoxic activity was displayed against all tested cell lines with GI ranging between
5
0
1
.96 and 13.01 µM. It was evident that the presence of the 4-methoxy group in the isolated
phenyl ring in 12c increased the activity against all tested cell lines except non-small cell
lung cancer which slightly decreased. Comparing the chalcone 9a (GI range of 2.25-4.3
5
0
µM) with its cyclized flavone congener 12a (GI range of 2.33-13.01µM) revealed that the
5
0
flexible analog 9a was much more active against all tested cell lines except for leukemia
which showed nearly equal sensitivity.
Lastly in this regards, concerning the aurones subgroup, the most active member in
this series was found to be 15d which elicited a remarkable activity against all tested cell
lines (GI range of 3.42-6.69 µM).
5
0
1
1

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Relating to the broad spectrum antitumor activity, the results revealed that all ten
tested compounds exhibited variable effective degree of GI , TGI and LC (MG-MID)
5
0
50
(
Table 3). Compound 25e showed the highest activity at the two levels GI and TGI with
50
values 1.9 and 5.24 µM, respectively. Yet, 25e showed an LC of 60.25 µM which was
5
0
lower than that of derivatives 9a and 9b the most active at this level, displaying values of
7.15 and 41.68 µM, respectively. It is worth mentioning though that the most active
3
derivative in this study 25e exhibited high LC50 (< 10 µM) against some subpanels, for
example, leukemia HL-60(TB) (9.7 µM); small cell lung cancer NCI-H460 (8.48 µM); colon
cancer Colo-205 (5.87 µM), HT-29 (6.68 µM); CNS cancer SNB-75 (5.75 µM) and
melanoma MALME-3M (8.65 µM), SK-MEL-2 (5.89 µM), SK-MEL-5 (4.99 µM) and
UACC-62 (7.61 µM) (data not shown).
Finally, compound 3d was the least active at the three levels with GI , TGI and LC
50
5
0
(MG-MID) of 38.9, 89.12 and 100 µM, respectively.
2
.4. Kinase inhibition assay
The profiling data for the ten most cytotoxic compounds in this study against the 3 kinases
CDK2, CDK4 and GSK-3β as potential molecular targets was performed. Profiling results
for CDK2/Cyclin E1 protein kinase showed weak inhibition of the target by compounds 3d,
6
b, and 12a compared to control (Table 4).
Regarding the profiling data against CDK4/Cyclin D1 protein kinase target, weak
inhibition of the target activity was observed by the flavonoids 3a, 6b, 15d, 25b and 25e
compared to control with percentage inhibition of enzyme activity between 6 to 15% (Table
4
).
Finally, profiling data against GSK-3β protein kinase target showed weak to potent
inhibition of the target activity by eight of the ten tested compounds and to our surprise weak
1
2

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stimulation was observed by the two derivatives belonging to the styrylfurochromone class
(25b and 25e) (Table 4). Compound 9b showed the highest inhibition of GSK-3β activity at
2
0 ꢀM concentration of the compound where the GSK-3β activity was inhibited by 80%
compared to control. The compounds 12a and 15d also showed good inhibition of GSK-3β
activity by 58% and 56%, respectively, compared to control. Derivatives 3a, 3d, 6b, 9a and
1
2c showed variable degrees of enzyme inhibition with percentages ranging from 11 to 38%.
To our surprise and in contrast to the other tested flavonoid derivatives, the
styrylfurochromones 25b and 25c both caused stimulation of enzyme activity by 5 and 13%,
respectively. Special interest was given to 25e, the most potent compound in this study (MG-
MID GI and TGI of 1.9 and 5.24 µM, respectively). We decided to go further with in vivo
5
0
studies with 25e in an attempt to correlate the observed high in vitro antiproliferative activity
with the unexpected stimulation of GSK3-β activity.
2
.5. Effect of 25e on the growth of Ehrlich solid tumor
Treatment of mice with 25e (3 mg/kg; i.p.) and (6 mg/kg; i.p.) daily for 5 days significantly
inhibited the growth of Ehrlich solid tumor, the tumor size was 62.1% and 57.1% of that of
control mice, respectively (Figure 3). While the final tumor size in mice receiving 25e (3
mg/kg; i.p.) for 5 days was non-significantly changed when compared to the initial volume,
the reduction in mice receiving 25e (6 mg/kg; i.p.) for the same period was significant when
compared to the initial volume. Our results showed that treatment of mice-bearing Ehrlich
solid tumor with 25e resulted in significant decrease in the tumor size in a dose-dependent
manner when compared to the control group.
Figure 3
1
3

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2
.6. Effect of 25e on Cyclin D1 and GSK-3β in tumor tissue
Treatment of mice with 25e at a low dose (3 mg/kg; i.p.) and high dose (6 mg/kg; i.p.) daily
for 5 days significantly declined the tumor level of cyclin D1 by 40.6% and 59.1%, (Figure 4)
while the level of GSK-3β increased by 101.5% and 197.2%, respectively compared to
untreated group (Figure 5). It can be concluded that the tumor suppressor effect of 25e on
Ehrlich solid tumor is mediated, in part, through increase in GSK-3β and decrease in cyclin
D1 levels. These results are in consistence with the studies of Dong et al. [41] and Altiok et
al. [42] that attributed the antitumor of new antitumor compounds against human breast
cancer to their ability to promote a decline in cyclin D1 level accompanied by increased level
of GSK-3β which phosphorylates cyclin D1 leading eventually to its degradation.
Figures 4 and 5
3
. Conclusion
In this study a library of 50 semisynthetic flavonoids viz furochalcones, furoflavones,
furoaurones and styrylfurochromones was synthesized to investigate their antiproliferative
activity. Results of the in vitro screening performed against a panel of 60 cell lines belonging
to nine types of cancer revealed that ten compounds possessed promising broad spectrum in
vitro antiproliferative activity. Four compounds, 9a, 9b, 25c and 25e, showed the lowest
results for GI , TGI and LC (MG-MID). The ten most active compounds were also tested
5
0
50
for their potential kinase inhibitory activity against the 3 protein kinases CDK2/cyclin E1,
CDK4/cyclin D1 and GSK-3β and showed variable degrees of enzyme inhibition with the
exception of 25c and 25e which unexpectedly caused stimulation of GSK-3β activity.
Furthermore, the in vivo antitumor activity of 25e was assessed against mice-bearing Ehrlich
solid tumor and resulted in significant decrease in the tumor size in a dose-dependent manner
accompanied by significant increase in GSK-3β and decrease in cyclin D1 levels. These
results suggest that the effect of 25e on tumor tissue is likely related to its ability to reduce
1
4

ACCEPTED MANUSCRIPT
cyclin D1 level through activation of GSK-3β activity that phosphorylates cyclin D1 thus
promoting its proteolysis. Further investigation into the exact mechanism of action of these
flavonoid derivatives and specially the styrylfurochromone ones is underway.
4
. Experimental
4
.1. Chemistry
Melting points are uncorrected and determined in one end open capillary tubes using Gallen
Kamp melting point apparatus MFB-595-010M (GallenKamp, London, England).
Microanalysis was carried out at the microanalytical unit, Faculty of Science, Cairo
University. All compounds were within ± 0.4 % of the theoretical values. IR spectra were
-1
determined using KBr discs (cm ) on Shimadzu Infrared Spectrometer IR-435 (Shimadzu,
Kyoto, Japan), Perkin-Elmer FT-IR 1650 (Perkin-Elmer, Waltham, Massachusetts 02451,
TM
1
USA) and Mattson Genesis II FTIR Spectrometer (Mattson, Madison, WI, USA). H-NMR
(
DMSO-d , D O) δ ppm spectra were determined using Joel NMR Varian Gemini 200 A
6
2
Spectrometer (Joel, Tokyo, Japan) and Varian Mercury VX-300 MHz NMR Spectrometer
(Varian, Oxford, England). Mass spectra were recorded using Hewlett Packard Varian
(Varian, Polo, USA) and Shimadzu Gas Chromatograph Mass spectrometer-QP 1000 EX
(Shimadzu, Kyoto, Japan). TLC were carried out using Art.DC-Plastikfolien, Kieselgel 60
F254 sheets (Merck, Darm-stadt, Germany), the developing solvents were CCl4/
CH COOC H (9:1) or (4:1) and the spots were visualized by Vilber Lourmat 77202 (Vilber,
3
2
5
Marne La Vallee, France).
4
.1.1. General procedure for the synthesis of 1-(6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-
(substituted)phenylprop-2-en-1-ones (3a-e)
1
5

ACCEPTED MANUSCRIPT
To a mixture of (2) (1 g, 4.6 mmol) and 30% sodium hydroxide solution (4 ml); a solution of
the appropriate aromatic aldehydes (5.5 mmol) in alcohol (5 ml) was added, the resulting red
solution was allowed to stand for 24h at room temperature, diluted with water to 100 ml, then
acidified with glacial acetic acid. The formed precipitate was collected by filtration, dried and
recrystallized from ethanol [43].
Compounds 3a-c were obtained as previously reported in the literature [43].
4
.1.1.1.
3d)
C H O (324.33), Yield % 93, m.p.134-145°C; IR (KBr, cm ): 3421 (OH), 1624 (C=O).
1-(6-Hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(2-methoxyphenyl)prop-2-en-1-one
(
-1
19
16
5
+
1
MS (m/z) rel.intensity: 324 [M , 65.76], 77 (100). H-NMR (DMSO-d ) δ ppm: 3.86 (s, 3H,
6
7
OCH ), 4.06 (s, 3H, OCH ) 6.82 (s, 1H, C -H), 7.20 (d, J = 15.9 Hz, 1H, CH=CHCO), 7.21
3
3
(d, J = 2.1 Hz, 1H, furan), 7.64-7.72 (m, 3H, 2H benz + 1H CH=CHCO), 7.74 (d, J = 8.3 Hz,
2
H, benz), 7.88 (d, J = 2.1 Hz, 1H, furan), 10.55 (s,1H, OH, exch. with D O). Anal.Calcd. for
2
C H O : C, 70.36; H, 4.97. Found: C, 70.75; H, 4.76.
19
16
5
4
.1.1.2. 1-(6-Hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(4-methylphenyl)prop-2-en-1-one (3e)
-1
C H O (308.33), Yield %93, mp.115-117°C; IR (KBr, cm ): 3430 (OH), 1628 (C=O), MS
19
16
4
+
(
m/z) rel.intensity: 308 [M , 100]. Anal.Calcd. for C H O : C, 74.01; H, 5.23. Found: C,
19 16 4
7
4.55; H, 4.94.
4
.1.2. General procedure for the synthesis of 1-(7-bromo-6-hydroxy-4-methoxy-1-
benzofuran-5-yl)-3-substituted-phenyl prop-2-en-1-ones (6a-d)
These derivatives were prepared employing the procedure adopted for derivatives 3 starting
with the bromoacetyl derivative 5 [32].
1
6

ACCEPTED MANUSCRIPT
4
.1.2.1. 1-(7-Bromo-6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-phenylprop-2-en-1-one (6a)
-1
C H BrO (373.2), Yield % 85, m.p. 138-140 °C; IR (KBr, cm ): 3414 (OH), 1630 (C=O).
18
13
4
+
+
1
MS (m/z) rel.intensity: 374 [M + 2, 11.8], 372 [M , 12.3], 103 (100). H-NMR (DMSO-d ) δ
6
ppm: 4.03 (s, 3H, OCH ), 7.44-7.56 (m, 7H, 5H benz + 1H CH=CHCO + 1H furan), 7.73 (d,
3
J = 2.1 Hz, 1H, furan), 8.01 (d, J = 15.87 Hz, 1H, CH=CHCO), 11.30 (s, 1H, OH, exch. with
D O). Anal.Calcd. for C H BrO : C, 57.93; H, 3.51. Found: C, 58.11; H, 3.52.
2
18 13
4
4
.1.2.2. 1-(7-Bromo-6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(4-chlorophenyl)prop-2-en-
1
-one (6b)
-1
C H BrClO (407.64), Yield % 83, m.p. 173-175°C; IR (KBr, cm ): 3398 (OH), 1626
18
12
4
+
+
+
(
(
(
C=O). MS (m/z) rel.intensity: 408 [M + 4, 16.2], 406 [M + 2, 10.8], 404 [M , 5.3], 53
1
100). H-NMR (DMSO-d ) δ ppm: 4.08 (s, 3H, OCH ), 7.34 (d, J = 2.1 Hz, 1H, furan), 7.39
6
3
d, J = 15.8 Hz, 1H, CH=CHCO), 7.46-7.57 (m, 3H, 2H benz +1H CH=CHCO), 7.88 (d, J=
.4Hz, 2H, benz), 7.97 (d, J = 2.1Hz, 1H, furan) 11.25 (s,1H, OH, exch. with D O).
8
2
Anal.Calcd. for C H BrClO : C, 53.03; H, 2.97. Found: C, 53.13; H, 2.51.
1
8
12
4
4
.1.2.3. 1-(7-Bromo-6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(4-methoxy-phenyl) prop-2-
en-1-one (6c)
C H BrO (403.22), Yield % 87, m.p. 168-170°C; IR (KBr, cm ): 3444 (OH), 1630 (C=O).
-1
19
15
5
+
+
1
MS (m/z) rel.intensity: 404 [M + 2, 16.03], 402 [M , 19.02], 269(100). H-NMR (DMSO-d )
6
δ ppm: 3.81 (s, 3H, OCH ), 4.09 (s, 3H, OCH ), 7.30 (d, J = 15.8 Hz, 1H, CH=CHCO), 7.36
3
3
(d, J = 2.1 Hz, 1H, furan), 7.42-7.48 (m, 3H, 2H benz +1H CH=CHCO), 7.49 (d, J = 8.4 Hz,
2
H, benz), 8.0 (d, J = 2.1 Hz, 1H, furan), 11.27 (s, 1H, OH, exch. with D O). Anal.Calcd. for
2
C H BrO : C, 56.59; H, 3.75. Found: C, 56.62; H, 4.15.
19
15
5
4
.1.2.4. 1-(7-Bromo-6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(2-methoxy-phenyl)prop-2-
en-1-one (6d)
1
7

ACCEPTED MANUSCRIPT
-1
C H BrO (403.22), Yield % 83, m.p. 160-162°C; IR (KBr, cm ): 3430 (OH), 1624(C=O).
19
15
5
+
+
1
Ms (m/z) rel.intensity: 404 [M + 2, 4.1], 402 [M , 3.8], 111 (100). H-NMR (DMSO-d ) δ
6
ppm: 3.83 (s, 3H, OCH ), 4.08 (s, 3H, OCH ) 7.33 (d, J = 15.84 Hz, 1H, CH=CHCO), 7.40
3
3
(d, J = 2.1 Hz, 1H, furan), 7.61-7.69 (m, 3H, 2H, benz +1H, CH=CHCO), 7.75 (d, J = 8.3 Hz,
2
H, benz), 8.17 (d, J = 2.1 Hz, 1H, furan), 11.20 (s,1H, OH, exch. with D O). Anal.Calcd. for
2
C H BrO : C, 56.59; H, 3.75. Found: C, 56.66; H, 4.0.
19
15
5
4
5
.1.3. General procedure for the synthesis of 1-(7-allyl-6-hydroxy-4-methoxy-1-benzofuran-
-yl)-3-(substituted)phenyl-prop-2-en-1-ones (9a-d)
These derivatives were prepared employing the procedure adopted for derivatives 3 starting
with the allylfuroacetophenone derivative 8 [36]. Derivative 9a was obtained as previously
reported [36].
4
.1.3.1. 1-(7-Allyl-6-hydroxy-4-methoxy-1-benzofuran-5-yl)-3-(4-chlorophenyl)prop-2-en-1-
one (9b)
C H ClO (368.08), Yield % 94, m.p. 115-117°C; IR (KBr, cm ): 3420 (OH), 1624 (C=O).
-1
21
17
4
+
1
MS (m/z) rel.intensity: 368 [M , 20.96], 102 (100). H-NMR (DMSO-d ) δ ppm: 3.56 (d, J=
6
5
.4Hz, 2H, -CH CH=CH ), 4.12 (s, 3H, OCH ), 5.02 (dd, 1H, -CH CH=CH , J = 1.80 Hz,
2 2 3 2 2 gem
J = 10.17 Hz), 5.10 (dd, 1H, -CH CH=CH , J = 1.80 Hz, Jtrans = 17.13 Hz), 6.01-6.31 (m,
cis
2
2
gem
1
H, -CH CH=CH ), 7.28-7.95 (m, 8H, 4H benz + 2H furan + 2H α,β-H protons), 12.00 (s,
2 2
1
H, OH, exch. with D O). Anal.Calcd. for C H ClO : C, 68.36; H, 4.65. Found: C, 68.40;
2 21 17 4
H, 4.50.
4
.1.3.2. 1-(7-Allyl-6-hydroxy-4-methoxybenzofuran-5-yl)-3-(2-methoxyphenyl)prop-2-en-1-
one (9d)
1
8

ACCEPTED MANUSCRIPT
-1
C H O (364.13), Yield %82, m.p. 122-124°C; IR (KBr, cm ): 3320 (OH), 1639(C=O). MS
22
20
5
+
1
(
m/z) rel.intensity: 364 [M , 51.41], 91 (100). H-NMR (DMSO-d ) δ ppm: 3.62 (d, J=
6
5
.4Hz, 2H, -CH CH=CH ) , 3.88 (s, 3H, OCH ), 4.11 (s, 3H, OCH ), 4.96 (dd, 1H, -
2 2 3 3
CH CH=CH , J = 1.80 Hz, J = 10.2 Hz), 4.97 (dd, 1H, -CH CH=CH , J = 1.80 Hz,
2
2
gem
cis
2
2
gem
Jtrans = 17.2 Hz), 6.2-6.6 (m, 1H, -CH CH=CH ), 7.03-7.86 (m, 8H, 4H benz + 2H furan + 2H
2
2
α,β-protons), 12.11 (s, 1H, OH exch. with D O). Anal.Calcd. for C H O : C, 72.51; H, 5.53.
2
22 20
5
Found: C, 72.76; H, 5.60.
4
.1.4. General procedure for the synthesis of 4-methoxy-7-(substituted)phenyl-furo[3,2-
g]chromen-5-ones (10a-d)
A mixture of freshly sublimed selenium dioxide (9 mmol) and the appropriate chalcone (3a-
d) (3.4 mmol) in n-butanol (15 ml) was heated under reflux for 24h after-which the obtained
turbid solution was filtered while hot to remove the selenium metal. The filtrate was
concentrated and left to cool to produce a precipitate which was filtered, dried and
recrystallized from ethanol [34].
Derivatives 10a-c were obtained as previously reported [34].
4
.1.4.1. 4-Methoxy-7-(2-methoxyphenyl)-5H-furo[3,2-g]chromen-5-one (10d)
-1
C H O (322.31), Yield %79, m.p. 165-167°C; IR (KBr, cm ): 3421 (OH), 1643 (C=O).
19
14
5
+
1
MS (m/z) rel.intensity: 322 [M , 85.54], 293 (100). H-NMR (CDCl ) δ ppm: 3.96 (s, 3H,
3
9
OCH ), 4.21 (s, 3H, OCH ), 7.03-7.13 (m, 4H, 1H C -H + 1H furan + 2H benz), 7.37 (s, 1H,
3
3
6
C -H), 7.45 (t, J = 8.4 Hz, 1H, benz), 7.61 (d, J = 2.1 Hz, 1H, furan), 7.93 (d, J = 8.4 Hz, 1H,
benz). Anal.Calcd. for C H O : C, 70.80; H, 4.38. Found: C, 70.64; H, 4.29.
1
9
14
5
1
9

ACCEPTED MANUSCRIPT
4
.1.5. General procedure for the synthesis of 9-bromo-4-methoxy-7-(substituted)phenyl-
furo[3,2-g]chromen-5-ones (11a-d)
The target compounds were obtained from chalcones 6a-d employing the procedure
described for synthesis of derivatives 9.
4
.1.5.1. 9-Bromo-4-methoxy-7-phenyl-5H-furo[3,2-g]chromen-5-one (11a)
-1
C H BrO (371.18), Yield % 62, m.p. 228-230°C; IR (KBr, cm ): 1640 (C=O). MS (m/z)
18
11
4
+
+
1
rel.intensity: 372 [M + 2, 98.4], 370 [M , 100]. H-NMR (DMSO-d ) δ ppm: 4.02 (s, 3H,
6
6
OCH ), 6.90 (s, 1H, C -H), 7.41 (d, J = 2.1 Hz, 1H, furan), 7.56-7.59 (m, 3H, benz), 8.11 (d,
3
J = 8.1 Hz, 2H, benz) 8.16 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd. for C H BrO : C, 58.24;
1
8
11
4
H, 2.99. Found: C, 58.10; H, 3.0.
.1.5.2. 9-Bromo-7-(4-chlorophenyl)-4-methoxy-5H-furo[3,2-g]chromen-5-one (11b)
4
-1
C H BrClO (405.63), Yield % 63, m.p. 278-280°C; IR (KBr, cm ): 1647 (C=O). MS (m/z)
18
10
4
+
+
+
1
rel.intensity: 408 [M + 4, 1.2], 406 [M + 2, 1.1], 404 [M , 0.5], 77 (100). H-NMR (DMSO-
6
d ) δ ppm: 4.10 (s, 3H, OCH ), 6.93 (s, 1H, C -H), 7.42 (d, J = 2.1 Hz, 1H, furan), 7.50 (d, J
6
3
=
8.2 Hz, 2H, benz), 8.12-8.32 (m, 3H, 2H benz + 1H furan). Anal.Calcd. for C H BrClO :
1
8
10
4
C, 53.03; H, 2.48. Found: C, 52.70; H, 2.57.
.1.5.3. 9-Bromo-4-methoxy-7-(4-methoxyphenyl)-5H-furo[3,2-g]chromen-5-one
4
(11c)
-1
C H BrO (401.21), Yield % 60, m.p. 261-263°C; IR (KBr, cm ): 1645 (C=O). MS (m/z)
19
13
5
+
+
1
rel.intensity: 402 [M +2, 69.24], 400 [M , 77.38], 132 (100). H-NMR (DMSO-d ) δ ppm:
6
6
3
.85 (s, 3H, OCH ), 4.08 (s, 3H, OCH ), 6.81 (s, 1H, C -H), 7.12 (d, J = 8.2 Hz, 2H, benz),
3
3
7
.41 (d, J = 2.1 Hz, 1H, furan), 8.10 (d, J = 8.2 Hz, 2H, benz), 8.17 (d, J = 2.1 Hz, 1H, furan).
Anal.Calcd. for C H BrO : C, 56.88; H, 3.27. Found: C, 56.79; H, 3.57.
1
9
13
5
4
.1.5.4. 9-Bromo-4-methoxy-7-(2-methoxyphenyl)-5H-furo[3,2-g]chromen-5-one (11d)
2
0

ACCEPTED MANUSCRIPT
-1
C H BrO (401.21), Yield % 61, m.p. 235-237°C; IR (KBr, cm ): 1640 (C=O). MS (m/z)
19
13
5
+
+
1
rel.intensity: 402 [M + 2, 44.3], 400 [M , 42.6], 106 (100). H-NMR (DMSO-d ) δ ppm:
6
6
3
.96 (s, 3H, OCH ), 4.11 (s, 3H, OCH ), 6.92 (s, 1H, C -H), 7.19 (t, J = 8.2 Hz, 1H, benz),
3
3
7
.27 (d, J = 8.2 Hz, 1H, benz), 7.43 (d, J = 2.1 Hz, 1H, furan), 7.58 (t, J = 8.2 Hz, 1H, benz),
8
.07 (d, J = 8.2 Hz, 1H, benz), 8.17 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd. for C H BrO :
1
9
13
5
C, 56.88; H, 3.27. Found: C, 56.70; H, 3.10.
4
.1.6. General procedure for the synthesis of 3-(4-methoxy-5-oxo-7-(substituted)phenyl-5H-
furo[3,2-g]chromen-9-yl)acrylaldehydes (12a-d)
A mixture of freshly sublimed selenium dioxide (9 mmol) and the appropriate chalcone (9a-
d) (3.4 mmol) in n-butanol (15 ml) was heated under reflux for 24h. The obtained turbid
solution was filtered while hot to remove the selenium metal. The filtrate was concentrated
and left to cool to produce a precipitate which was filtered, dried and recrystallized from
ethanol.
4
.1.6.1. 3-(4-Methoxy-5-oxo-7-phenyl-5H-furo[3,2-g]chromen-9-yl)acrylaldehyde (12a)
-1
C H O (346.33), Yield % 65, m.p. 225-227°C; IR (KBr, cm ): 1722 (CHO) 1654 (C=O).
21
14
5
1
6
H-NMR (DMSO-d ) δ ppm: 4.21 (s, 3H, OCH ), 6.87 (s, 1H, C -H), 7.45 (dd, J = 7.8 Hz,
6
3
1
H, -CH=CH-CHO), 7.59 (d, J = 2.1 Hz, 1H, furan), 7.62 (d, J = 8.2 Hz, 2H, benz), 8.07-
.21 (m, 3H, benz), 8.24 (d, 1H, J = 7.8 Hz, -CH=CH-CHO), 8.11 (d, J = 2.1 Hz, 1H, furan),
.88 (d, J = 7.8 Hz, 1H, -CH=CH-CHO). Anal.Calcd. for C H O : C, 72.83; H, 4.07.
8
9
21
14
5
Found: C, 72.79; H, 4.45.
4
.1.6.2. 3-[7-(4-Chlorophenyl)-4-methoxy-5-oxo-5H-furo[3,2-g]chromen-9-yl]acrylaldehyde
(12b)
2
1

ACCEPTED MANUSCRIPT
-1
C H ClO (380.78), Yield % 62, m.p. 202-203°C; IR (KBr, cm ): 1716 (CHO) 1645
21
13
5
1
6
(
C=O). H-NMR (DMSO-d ) δ ppm: 4.21 (s, 3H, OCH ), 6.88 (s, 1H, C -H), 7.14 (d, J= 8.2
6
3
Hz, 2H, benz), 7.23 (dd, J = 7.8 Hz, 1H, -CH=CH-CHO), 7.61 (d, J = 2.1 Hz, 1H, furan),
8
.08 (d, J = 8.2 Hz, 2H, benz), 8.13 (d, J = 7.8 Hz, 1H, -CH=CH-CHO), 8.23 (d, J = 2.1 Hz,
H, furan), 9.89 (d, J = 7.8 Hz, 1H, -CH=CH-CHO). Anal.Calcd. for C H ClO : C, 66.24;
1
2
1
13
5
H, 3.44. Found: C, 66.63; H, 3.75.
4
.1.6.3.
aldehyde (12c)
C H O (376.36), Yield %60, m.p. 218-220°C; IR (KBr, cm ): 1720 (CHO), 1639 (C=O).
3-[4-Methoxy-7-(4-methoxyphenyl)-5-oxo-5H-furo[3,2-g]chromen-9-yl]acryl-
-1
22
16
6
1
6
H-NMR (DMSO-d ) δ ppm: 3.86 (s, 3H, OCH ), 4.19 (s, 3H, OCH ), 6.73 (s, 1H, C -H),
6
3
3
7
.08 (d, J = 8.2 Hz, 2H, benz), 7.17 (dd, J = 7.8 Hz, 1H, -CH=CH-CHO), 7.42 (d, J = 2.1
Hz, 1H, furan), 7.99 (d, J = 8.2 Hz, 2H, benz), 8.13 (d, J = 7.8 Hz, 1H, -CH=CH-CHO), 8.17
d, J = 2.1 Hz, 1H, furan), 9.85 (d, J = 7.8 Hz, 1H, -CH=CH-CHO). Anal.Calcd. for
C H O : C, 70.21; H, 4.29. Found: C, 70.59; H, 4.61.
(
22
16
6
4
.1.6.4.
aldehyde (12d)
C H O (376.36), Yield %62, m.p. 175-177°C; IR (KBr, cm ): 1725 (CHO), 1650 (C=O).
3-[4-Methoxy-7-(2-methoxyphenyl)-5-oxo-5H-furo[3,2-g]chromen-9-yl]acryl-
-1
22
16
6
1
6
H-NMR (DMSO-d ) δ ppm: 3.83 (s, 3H, OCH ), 4.13 (s, 3H, OCH ), 6.56 (s, 1H, C -H),
6
3
3
7
.00 (d, J = 8.2 Hz, 2H, benz), 7.04 (dd, J = 7.8 Hz, 1H, -CH=CH-CHO), 7.32 (d, J = 2.1 Hz,
H, furan), 7.98 (d, J = 8.2 Hz, 2H, benz), 8.04 (d, J = 7.8 Hz, 1H, -CH=CH-CHO), 8.06 (d, J
1
=
2.1 Hz, 1H, furan), 9.73 (d, J = 7.8Hz, 1H, -CH=CH-CHO). Anal.Calcd. for C H O : C,
22 16 6
7
0.21; H, 4.29. Found: C, 69.93; H, 3.98.
2
2

ACCEPTED MANUSCRIPT
4
.1.7. General procedure for the synthesis of 2-(substituted)benzylidene-4-methoxyfuro[3,2-
f][1]benzofuran-3(2H)-ones (13a & 13b)
To a mixture of the appropriate chalcone (3d or 3e) (10 mmol), ethanol (30 ml) and 20%
aqueous solution of sodium hydroxide (30 ml), 30% hydrogen peroxide solution (6 ml) was
added with continuous stirring at room temperature. The reaction mixture was allowed to
stand overnight and acidified with acetic acid. The solid separated was filtered, washed with
water and crystallized from benzene.
4
.1.7.1. 4-Methoxy-2-(2-methoxybenzylidene)furo[3,2-f][1]benzofuran-3(2H)-one (13a)
-1
C H O (322.31), Yield % 80, m.p. 197-199°C; IR (KBr, cm ): 1693 (C=O). MS (m/z)
19
14
5
+
1
rel.intensity: 322 [M , 53.3], 290 (100). H-NMR (CDCl ) δ ppm: 3.90 (s, 3H, OCH ), 4.41
3
3
8
(
s, 3H, OCH ), 7.04 (s, 1H, C -H), 6.96 (s, 1H, vinyl proton), 7.33-7.51 (m, 5H, 4H benz +
3
1
H furan), 8.26 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd. for C H O : C, 70.80; H, 4.38.
19 14 5
Found: C, 70.59; H, 4.41.
4
.1.7.2. 4-Methoxy-2-(4-methylbenzylidene)furo[3,2-f][1]benzofuran-3(2H)-one (13b)
-1
C H O (306.31), Yield % 80, m.p. 197-199°C; IR (KBr, cm ): 1693 (C=O). MS (m/z)
19
14
4
+
1
rel.intensity: 306 [M , 100]. H-NMR (CDCl ) δ ppm: 2.36 (s, 3H, CH ), 4.33 (s, 3H,
3
3
7
OCH ), 6.73 (s, 1H, C -H), 7.30 (s, 1H, vinyl proton), 7.31-7.35 (m, 3H, 2H benz + 1H
3
furan), 7.86 (d, J = 8.4 Hz, 2H, benz), 7.97 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd. for
C H O : C, 74.50; H, 4.61. Found: C, 74.29; H, 4.27.
19
14
4
4
.1.8. General procedure for the synthesis of 8-bromo-2-(substituted)benzylidene-4-
methoxyfuro[3,2-f][1]-benzofuran-3(2H)-ones (14a-d)
These compounds were obtained starting with chalcones 6a-d adopting the procedure
described for preparation of derivatives 13.
2
3

ACCEPTED MANUSCRIPT
4
.1.8.1.
2-Benzylidene-8-bromo-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
(14a)
-1
C H BrO (371.18), Yield % 82, m.p. 213-215°C; IR (KBr, cm ): 1697 (C=O). MS (m/z)
18
11
4
+
+
1
rel.intensity: 372 [M +2, 25.8], 370 [M , 24.6], 90 (100). H-NMR (DMSO-d ) δ ppm: 4.34
6
(
s, 3H, OCH ), 6.84 (s, 1H, vinyl), 7.45 (t, J = 8.2 Hz, 1H, benz), 7.45-7.54 (m, 3H, 2H, benz
3
+
1H, furan), 7.98 (d, J = 8.2 Hz, 2H, benz), 8.06 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd for
C H BrO : C, 58.24; H, 2.99. Found: C, 58.41; H, 2.95.
18
11
4
4
.1.8.2.
14b)
C H BrClO (405.63), Yield % 86, m.p. 223-225 °C; IR (KBr, cm ): 1692 (C=O). MS
8-Bromo-2-[4-chlorobenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
(
-1
18
10
4
+
+
+
1
(
m/z) rel.intensity: 408 [M + 4, 23.1], 406 [M + 2, 76.9], 404 [M , 60.3] 99 (100). H-
NMR (DMSO-d ) δ ppm: 4.40 (s, 3H, OCH ), 6.94 (s, 1H, vinyl), 7.54 (d, J = 2.1 Hz, 1H,
6
3
furan), 7.65 (d, J = 8.6 Hz, 2H, benz), 8.05 (d, J = 8.6 Hz, 2H, benz), 8.18 (d, J = 2.1 Hz, 1H,
furan). Anal.Calcd for C H BrClO : C, 53.03; H, 2.48. Found: C, 53.15; H, 2.48.
1
8
10
4
4
.1.8.3. 8-Bromo-2-[4-methoxybenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
14c)
C H BrO (401.21), Yield % 87, mp.233-235 °C; IR (KBr, cm ): 1690 (C=O). MS (m/z)
(
-1
19
13
5
+
+
1
rel.intensity: 402 [M + 2, 97], 400 [M , 100]. H-NMR (DMSO-d ) δ ppm: 3.83 (s, 3H,
6
OCH ), 4.31 (s, 3H, OCH ), 6.78 (s, 1H, vinyl), 7.05 (d, J = 8.4 Hz, 2H, benz), 7.38 (d, J =
3
3
2
.1 Hz, 1H, furan), 7.91 (d, J = 8.4 Hz, 2H, benz), 8.04 (d, J = 2.1 Hz, 1H, furan).
Anal.Calcd. for C H BrO : C, 56.88; H, 3.27. Found: C, 57.10; H, 3.12.
1
9
13
5
4
.1.8.4. 8-Bromo-2-[2-methoxybenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
14d)
C H BrO (401.21), Yield % 81, m.p. 280-230 °C; IR (KBr, cm ): 1697 (C=O). MS (m/z)
(
-1
19
13
5
+
+
1
rel.intensity: 402 [M +2, 23.5], 400 [M , 22.5], 89 (100). H-NMR (DMSO-d ) δ ppm: 3.91
6
2
4

ACCEPTED MANUSCRIPT
(
s, 3H, OCH ), 4.34 (s, 3H, OCH ), 7.12-7.16 (m, 3H, 1H, vinyl + 2H, benz), 7.44-7.48 (m,
3
3
2
H, 1H, benz + 1H, furan), 8.09 (d, J = 2.1 Hz, 1H, furan), 8.19 (d, J = 8.2 Hz, 1H, benz).
Anal.Calcd for C H BrO : C, 56.88; H, 3.27. Found: C, 57.20; H, 3.45.
1
9
13
5
4
.1.9. General procedure for the synthesis of 8-allyl-2-(substituted)benzylidene-4-
methoxyfuro[3,2-f][1]benzofuran-3(2H)-ones (15a-d)
To a mixture of the appropriate chalcone (9a-d) (10 mmol), ethanol (30 ml) and 20% aqueous
solution of sodium hydroxide (30 ml) at room temperature, was added 30% hydrogen
peroxide solution (6 ml) with continuous stirring. The reaction mixture was allowed to stand
overnight and acidified with dilute acetic acid. The solid separated was filtered, washed with
water and crystallized from ethanol.
4
.1.9.1. 8-Allyl-2-[benzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one (15a)
-1
C H O (332.1), Yield % 82, m.p. 163-165°C; IR (KBr, cm ): 1697 (C=O). MS (m/z)
21
16
4
+
1
rel.intensity: 332 [M , 100]. H-NMR (DMSO-d ) δ ppm: 3.72 (d, J = 5.4 Hz, 2H, -CH –
6
2
CH=CH ), 4.31 (s, 3H, OCH ), 5.14 (dd, 1H, -CH –CH=CH , J = 1.9 Hz, J = 10.10 Hz),
2
3
2
2
gem
cis
5
.23 (dd, 1H, -CH –CH=CH , J = 1.9 Hz, Jtrans = 16.8 Hz), 6.23 (m, 1H, -CH –CH=CH ),
2 2 gem 2 2
6
.76 (s, 1H, vinyl proton), 7.42 (d, J = 8.4 Hz, 2H, benz), 7.56 (d, J = 2.1 Hz, 1H, furan),
7
.94-7.98 (m, 4H, 3H benz + 1H furan). Anal.Calcd. for C H O : C, 75.89; H, 4.85. Found:
2
1
16
4
C, 76.00; H, 5.03.
4
.1.9.2. 8-Allyl-2-[4-chlorobenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one (15b)
-1
C H ClO (366.79), Yield % 85, m.p. 170-172°C; IR (KBr, cm ): 1692 (C=O). MS (m/z)
21
15
4
+
1
rel.intensity: 367 [M + 1, 0.74], 53 (100). H-NMR (DMSO-d ) δ ppm: 3.68 (d, J = 5.4 Hz,
6
2
1
H, -CH –CH=CH ), 4.23 (s, 3H, OCH ), 5.10 (dd, 1H, -CH –CH=CH , J = 2.0 Hz, J =
2 2 3 2 2 gem cis
0.2 Hz), 5.13 (dd, 1H, -CH –CH=CH , J = 2.0 Hz, Jtrans = 17.1 Hz), 6.11 (m, 1H, -CH2–
2
2
gem
2
5

ACCEPTED MANUSCRIPT
CH=CH ), 6.75 (s, 1H, vinyl proton), 7.3 (d, J= 8.4Hz, 2H, benz), 7.56 (d, J= 2.1Hz, 1H,
2
furan), 7.99-8.00 (m, 3H, 2H benz + 1H furan). Anal.Calcd. for C H ClO : C, 68.76; H,
2
1
15
4
4
.12. Found: C, 69.03; H, 4.14.
.1.9.3. 8-Allyl-2-[4-methoxybenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
4
(15c)
-1
C H O (362.38), Yield % 80, m.p. 150-152°C; IR (KBr, cm ): 1695(C=O). MS (m/z)
22
18
5
+
1
rel.intensity: 362 [M , 100]. H-NMR (DMSO-d ) δ ppm: 3.68 (d, J = 5.4 Hz, 2H, -CH –
6
2
CH=CH ), 3.83 (s, 3H, OCH ), 4.28 (s, 3H, OCH ), 5.10 (dd, 1H, -CH –CH=CH , J = 2.1
2
3
3
2
2
gem
Hz, J = 10.2 Hz), 5.15 (dd, 1H,-CH –CH=CH , J = 2.1 Hz, Jtrans= 17.3 Hz), 5.97-6.15 (m,
cis
2
2
gem
1
H, -CH –CH=CH ), 6.74 (s, 1H, vinyl proton), 7.08 (d, J=8.4Hz, 2H, benz), 7.30 (d, J = 2.1
2 2
Hz, 1H, furan), 7.92 (d, J = 8.4 Hz, 2H, benz), 8.00 (d, J = 2.1 Hz, 1H, furan). Anal.Calcd.
for C H O : C, 72.92; H, 5.01. Found: C, 72.81; H, 4.91.
2
2
18
5
4
.1.9.4.
15d)
C H O (362.38), Yield % 78, m.p. 128-130°C; IR (KBr, cm ): 1693 (C=O). H-NMR
8-Allyl-2-[2-methoxybenzylidene]-4-methoxyfuro[3,2-f][1]benzofuran-3(2H)-one
(
-1
1
22
18
5
(
DMSO-d ) δ ppm: 3.67 (d, J = 5.4 Hz, 2H, -CH –CH=CH ), 3.90 (s, 3H, OCH ), 4.30 (s,
6
2
2
3
3
H, OCH ), 5.15 (dd, 1H,-CH CH=CH , J
= 2.1 Hz, Jcis = 10.2 Hz), 5.20 (dd, 1H,-
3
2
2
gem
CH CH=CH , J = 2.1 Hz, Jtrans = 17.2 Hz), 6.05-6.20 (m, 1H, -CH CH=CH ), 7.04 (s, 1H,
2
2
gem
2
2
vinyl proton), 7.12-7.29 (m, J = 8.4 Hz, 2H, benz), 7.39 (d, J = 2.1 Hz, 1H, furan), 7.42 (t, J
8.4 Hz, 1H, benz), 7.99 (d, J = 2.1 Hz, 1H, furan), 8.18 (d, J = 8.4 Hz, 1H, benz).
Anal.Calcd. for C H O : C, 72.92; H, 5.01. Found: C, 72.85; H, 4.92.
=
2
2
18
5
4
.1.10. General procedure for the synthesis of 9-allyl-4-hydroxy-7-substituted phenyl-5H-
furo[3,2-g]chromen-5-ones (18a & 18b)
2
6

ACCEPTED MANUSCRIPT
A mixture of (16a or 16b) [44] (10 mmol), allyl bromide (1.45 g, 12 mmol) and anhydrous
potassium carbonate (3 g) in acetone (70 ml) was refluxed for 24h, then filtered while hot and
the residue was washed repeatedly with small portions of hot acetone. Evaporating the
combined filtrate gave an oily residue of the allyloxy derivatives (17a or 17b). The latter,
without purification, was refluxed for 2h with N,N-diethylaniline (20 ml), then allowed to
stand at room temperature for 5h. The reaction mixture was poured onto a mixture of crushed
ice (100 g) and conc. HCl (20 ml) with good stirring. The resulting precipitate was collected
by filtration, washed with water, dried and recrystallized from ethanol.
4
.1.10.1. 9-Allyl-4-hydroxy-7-phenyl-5H-furo[3,2-g]chromen-5-one (18a)
-1
C H O (318.32), Yield %70, m.p. 149-151°C; IR (KBr, cm ): 3421 (OH), 1643 (C=O).
20
14
4
+
1
MS (m/z): 318 [M , 97.60], 261 (100). H-NMR (CDCl ) δ ppm: 3.82 (d, J = 5.6 Hz, 2H, -
3
CH –CH=CH ), 5.12 (dd, 1H, -CH –CH=CH , J = 2.2 Hz, J = 9.9 Hz), 5.16 (dd, 1H, -
2
2
2
2
gem
cis
CH –CH=CH , J = 2.2 Hz, Jtrans = 16.5 Hz), 6.16 (m, 1H, -CH –CH=CH ), 6.65 (s,
2
2
gem
2
2
6
1
H,C -H), 7.24 (d, J = 8.4 Hz, 2H, benz), 7.73-7.79 (m, 4H, 3H benz + 1H furan), 7.89 (d, J
=
2.1 Hz, 1H, furan), 13.55 (s, 1H, OH, exch. with D O). Anal.Calcd. for C H O : C, 75.46;
2 20 14 4
H, 4.43. Found: C, 74.86; H, 4.69.
4
.1.10.2.
9-Allyl-7-(4-chlorophenyl)-4-hydroxy-5H-furo[3,2-g]chromen-5-one
(18b)
-1
C H ClO (352.77), Yield % 72, m.p. 160-162°C; IR (KBr, cm ): 3420 (OH), 1639 (C=O).
20
13
4
+
1
MS (m/z): 352 [M , 100]. H-NMR (CDCl ) δ ppm: 3.85 (d, J = 5.6 Hz, 2H, -CH –
3
2
CH=CH ), 5.09 (dd, 1H, -CH –CH=CH , J = 2.2 Hz, J = 9.9 Hz), 5.12 (dd, 1H, -CH –
2
2
2
gem
cis
2
6
CH=CH , J = 2.2 Hz, Jtrans = 16.5 Hz), 6.12 (m, 1H, -CH –CH=CH ), 6.69 (s, 1H,C -H),
2
gem
2
2
7
1
3
.27 (d, J = 8.4 Hz, 2H, benz), 7.63-7.69 (m, 3H, 2H benz + 1H furan), 7.86 (d, J = 2.1 Hz,
H, furan), 13.52 (s, 1H, OH, exch. with D O). Anal.Calcd. for C H ClO : C, 68.09; H,
2
20 13
4
.71. Found: C, 67.86; H, 3.43.
2
7