Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under ultrasound: Their evaluation as inhibitors of SIRT1

Article abstract of DOI:10.1016/j.bmcl.2012.08.017

An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A number of aurones were synthesized (within 5-30 min) and the molecular structure of a representative compound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C-C double bond present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC₅₀ = 1 μM) which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis.

Full text of DOI:10.1016/j.bmcl.2012.08.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6160–6165
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under
ultrasound: Their evaluation as inhibitors of SIRT1
Khanapur Manjulatha a, S. Srinivas b, Naveen Mulakayala a, D. Rambabu a, M. Prabhakar a,
a,
Kalle M. Arunasree a, Mallika Alvala a, M. V. Basaveswara Rao c, , Manojit Pal
⇑
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500 046, India
b Department of Chemistry, Acharya Nagarjuna University, Guntur 522510, AP, India
c Department of Chemistry, Krishna University, Machilipatnam 521001, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-
3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A num-
ber of aurones were synthesized (within 5–30 min) and the molecular structure of a representative com-
pound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C–C double bond
present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as
anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-
Received 23 May 2012
Revised 29 July 2012
Accepted 2 August 2012
Available online 9 August 2012
Keywords:
Aurones
SIRT1
hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC50 = 1 lM)
which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis.
Ó 2012 Elsevier Ltd. All rights reserved.
Cancer
Ultrasound
Protein acetylation by histone acetyltransferases (HATs) and
deacetylation by histone deacetylases (HDACs) regulate a wide
variety of cellular functions, including the recognition of DNA by
proteins, protein–protein interactions, and protein stability.1
HDACs are classified into four classes for example I, II, III and IV
that are often conserved from yeast to humans. The class III HDACs,
also known as sirtuins, belongs to the family of deacetylases that
require nicotinamide adenine dinucleotide (NAD) as a cofactor
for their catalytic activity. The mammalian sirtuin family consists
of seven members for example SIRT1-7. Each sirtuin is character-
ized by a conserved 275 amino acid catalytic core and unique addi-
tional N-terminal and/or C-terminal sequences of variable length.
Among the seven human sirtuins, SIRT1 has been studied well
cell survival.5 Findings from various studies suggest that SIRT1
inhibitors may have promising therapeutic value for the potential
treatment of cancer. Several small molecule inhibitors of sirtuins
(Fig. 1), such as nicotinamide, the 2-hydroxynaphthaldehyde
derivative sirtinol, the coumarin derivative splitomicin and its ana-
logue HR73, cambinol, tenovins, and the indole derivative EX5276,7
have been identified and shown to induce cell death in cancer cells.
Additionally, cambinol and tenovins have been tested in animal
models. However, except EX527 which is presently undergoing
Phase 1a clinical trial and being developed for the treatment of
Huntington’s disease no other SIRT1 inhibitors have progressed
into clinical trials as anticancer agents. Thus there is a need to
identify new chemical class of SIRT1 inhibitors that could be ben-
eficial for the treatment of cancer.
Naturally occurring aurones [or 2-benzylidenebenzofuran-
3(2H)-ones based upon D (Fig. 2)] that belong to a type of flavonoid
class posses a chalcone-like moiety part of which is closed into a 5-
membered ring through an oxygen atom, and an intact exocyclic
double bond with restricted rotations. These are biosynthesized
from chalcones in the presence of a key enzyme called aureusidin
synthase or obtained through the biosynthesis of other flavo-
noids.7c This class of compounds showed a range of pharmacolog-
ical activities8 including anti-cancer, anti-feedant and anti-
parasitic activities via modulating a variety of molecular targets
such as, G2/M cell-cycle arrest,9 arresting the cell cycle in G0/G1
phase and displayed apoptosis-inducing effect on Hep-2 cells,10
inhibition of Human Sphingosine Kinase,11 inhibition of P-gp
which has several substrates such as p53, Ku70, NF-
jB, forkhead
proteins, etc.2
The role of SIRT1 in cancer biology was first implicated when
p53 (also known as protein 53 or tumor protein 53 is a tumor sup-
pressor protein that in human is encoded by the TP53 gene)3a was
identified as a direct substrate of this member of sirtuin family.3b
Recent findings on direct interaction of SIRT1 with DNA methyl-
transferase 1 (DNMT1) further authenticated the role of SIRT1 in
cancer development.4 Moreover, study suggested that members
of forkhead transcription factor family that are regulated by SIRT1
lead to increased resistance to stress and apoptosis causing cancer
⇑
Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.017

K. Manjulatha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6160–6165
6161
H
N
CH3
O
O
O
N
O
N
NH2
N
H
OH
NH2
O
Nicotinamide
Splitomycin
EX527
Sirtinol
CH3
H
N
H
O
N
S
CH3
H3C
H3C
CH3
N
N
NH
H
N
H
N
CH3
OH
HO
O
O
S
N
H
O
Salermide
Tenovin-6
Cambinol
Figure 1. Examples of reported inhibitors of sirtuin.
R'
Z
R1
R4
R'
R
R'
COOH
b
R2
R3
Z
O
Z
OH
Z
O
O
R
O
O
R
a
c
O
NH
NH
NH
O
O
O
O
N
H
S
O
N
S
O
N
H
S
1
3a-h (Z = H)
6a-c (Z = OMe)
(Z = H)
2 (Z = H)
4 (Z = OMe)
5 (Z = OMe)
B
C
D
A
Scheme 1. Reagents and Conditions: (a) chloroacetic acid (1.1 equiv), NaH, DMF,
room temp, 12 h; (b) polyphosphoric acid, 80 °C, 8 h; (c) ArCHO, EDDA, CH3CN,
ultrasound or room temp.
Figure 2. Design of new aurone derivative D from the known inhibitor A.
related transport,12 high-affinity binding to cytosolic domain of P-
glycoprotein,13a modulation of ABCG213b activities etc. However,
anticancer activities of aurones due to the inhibition of SIRT1 have
not been reported earlier. Additionally, systematic structural
manipulations of the basic skeleton of a thiobarbiturate A, a prom-
Table 1
Synthesis of aurone derivatives 3 and 6a
Entry
Aurones
Time (min);% yieldb
Z
R1
R2
R3 R4
Ultrasound; No ultrasound;
Room temp Room temp
ising inhibitor of sirtuins (IC50 ꢀ10
l
M),14 lead to the aurone deriv-
ative D via B and C (Fig. 2). This prompted us to synthesize and
evaluate the SIRT1 inhibiting properties of D and subsequently
their potential as anticancer agents in vitro. Herein we report our
preliminary results of this study that is, 2-arylidenebenzofuran-
3-(2H)-ones as potential inhibitors of SIRT1.
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
Br
Br
H
H
H
CH3
H
H
H
H
Br
F
OCH3
H
H
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
5; 94
30; 85
OPrc
H
15; 93
12; 94
6; 95
30; 89
20; 96
8; 85
10; 95
20; 90
17; 91
120; 67
165; 86
110; 84
300; 86
265; 90
150; 63
175; 75
215; 58
180; 85
180; 78
H
OH
OH
OPrc
H
Commonly used methods for the synthesis of aurone deriva-
tives D generally involved the aldol condensation of benzofuran-
3(2H)-ones with a benzaldehyde. This reaction can be carried out
in the presence of alumina15a–c or HCl.15d Over the last three dec-
ades the ultrasound mediated reactions have become increasingly
popular and widely used methodologies in organic synthesis.15e–g
Compared to the traditional methods, these reactions are known
to be faster, convenient and high yielding process. Indeed, a variety
of organic reactions can be carried out within short reaction time
and under mild conditions in the presence of ultrasound.15h,i We
therefore decided to explore the ultrasound mediated reactions
for the preparation of the target aurone analogues required for
our study. To the best our knowledge the use ultrasound in the
synthesis of aurones has not been explored earlier.
The key starting materials that is, benzofuran-3(2H)-one (2) and
7-methoxybenzofuran-3(2H)-one (5) required for our synthesis
were prepared from the corresponding phenols (Scheme 1). Thus,
phenol and 2-methoxy phenol were O-alkylated with chloroacetic
acid in the presence of sodium hydride to yield the desired phe-
noxyacetic acid intermediate 1 and 4 respectively, which on intra-
molecular Friedel–Crafts type cyclization16a provided the desired
compound 2 and 5. Subsequent aldol type condensation of com-
pound 2 or 5 with suitably substituted benzaldehyde derivatives
in the presence of ethylenediamine diacetate (EDDA) in acetoni-
trile provided the corresponding product 3 or 6 (Table 1). These
reactions were carried out at room temperature under ultrasound
3g
3h
6a OCH3 Br
6b OCH3
6c OCH3
OPrc
10
11
H
H
OPrc
Oallyl 10; 93
a
All the reactions were carried out using compound 2 or 5 (0.6 mmol) and an
appropriate aryl aldehyde (0.6 mmol) in the presence of EDDA (10 mol %) at room
temperature.
b
Isolated yield.
Pr = Prenyl (or 3-methyl-2-butenyl).
c
irradiation to increase the product yield at the same time decreas-
ing the reaction time. In order to justify the use of ultrasound these
reactions were also carried out in the absence of ultrasound at
room temperature and results are presented in Table 1. A variety
of aurones were prepared by using a range of aryl aldehydes con-
taining mono or di substituents selected from F, Br, Me, OH,
OMe, O-Allyl and O-Prenyl (Table 1). The reactions were completed
within 5–30 min under ultrasound irradiation depending on the
nature of aryl aldehydes employed affording the desired products
in good to excellent yields. The durations of these reactions how-
ever were increased to 30–300 min when performed in the ab-
sence of ultrasound. Additionally, a 10–30% decrease in product
yields was observed in many of these cases. The ultrasound medi-
ated reaction therefore was found to be advantageous in the pres-
ent synthesis of aurones. While most of the benzaldehydes used

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K. Manjulatha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6160–6165
Figure 3. ORTEP representation of the compound 3f (thermal ellipsoids are drawn at 50% probability level).
Table 2
Table 2 (continued)
In vitro assay of aurone derivatives 3 and 6
Entry Aurones
% inhibition @ 10 l
Ma
Entry Aurones
% inhibition @ 10 l
Ma
Enzymatic assay
SIRT1
Cell based assay
Enzymatic assay
Cell based assay
MDA-MB-231 MCF-7
SIRT1
MDA-MB-231 MCF-7
O
1
2
Control
Suramin
O
0
82.3
0.23
26.0
0.15
32.0
Br
O
OH
Br
11
12.0
20.3
O
75.3
54.3
47.8
O
3
98.5
46.0
20.2
44.1
21.0
O
6a
O
3a
O
O
Br
12
13
O
35.4
25.1
32.5
22.0
O
4
75.3
71.0
O
O
6b
3b
O
O
5
6
7
30.8
16.5
29.8
34.5
20.1
32.0
O
O
O
O
Br
3c
O
O
O
O
6c
a
50.5
49.0
Data represent the mean values of three independent determinations.
F
3d
OH
OH
O
O
O
3e
8
48.9
31.8
38.3
36.7
3f
O
O
O
9
33.0
18.1
30.0
29.6
O
Figure 4. Western blot showing inhibition of SIRT1 by compound 3a in MDA-MB-
231 cells resulted in increase of Ac-p53 levels in a dose dependent manner.
3g
O
10
32.5
are commercially available some were prepared via alkylation for
example, allylation and prenylation of salicylaldehyde and 5-
bromo salicylaldehyde using allyl bromide and prenyl bromide
(1-bromo-3-methyl-2-butene).16b,16c
O
3h

K. Manjulatha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6160–6165
6163
Table 3
All the compounds (3a–h and 6a–c) prepared were well charac-
terized by spectral (NMR, IR and MS) data.17a It is evident from the
spectral data that a single geometric isomer (Z) was obtained in all
the cases. The Z-isomer is known to be thermodynamically more
stable than E-isomer and the Z-aurones could be photoisomerized
Docking scores obtained after docking compounds 3 and 6 into SIRT1 protein
Compound
Docking score
3a
Suramin
À4.74
À3.33
Figure 5. Docking of compound 3a into the active site of SIRT1.

6164
K. Manjulatha et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6160–6165
to E-isomers.17b,c While the geometry of the double bond could be
established on the basis of chemical shift (d) value of the vinylic
proton as well as carbon observed in the corresponding 1H and
13C NMR spectra17–19 we however used X-ray single crystal study
of a representative compounds to establish its molecular structure
unambiguously. Thus the structure of compound 3f was examined
by X-ray diffraction study (Fig. 3) which confirmed the presence of
an exocyclic C–C double bond with Z-geometry.20
in the enzyme assay. The binding pose of 3a and interacting amino
acids within 5 Å are shown in Fig. 5. The two amino acid residues
for example, Asp105 and Asn103 played key role in the interaction
of 3a with the SIRT1 protein whereas the bromo substituent of 3a
participated in a hydrophobic interaction.
In conclusion, various aurone derivatives have been explored as
new and potential inhibitors of SIRT1. Synthesis of these com-
pounds were carried out by reacting benzofuran-3(2H)-one with
a range of benzaldehydes in the presence of a mild base for exam-
ple, EDDA under ultrasound. To the best of our knowledge ultra-
sound mediated synthesis of this class of compounds was not
known in the literature earlier. The single crystal X-ray diffraction
study was used to confirm the molecular structure of a representa-
tive compound unambiguously along with the Z-geometry of the
C–C double bond present within the molecule. Some of the com-
pounds synthesized showed SIRT1 inhibiting properties in vitro.
Some of them also showed anti proliferative properties against
two cancer cell lines for example, MDAMB-231 and MCF-7. Com-
pound 3a was identified as a potent inhibitor of SIRT1 which
showed a dose dependent increase in the acetylation of p53 result-
ing in induction of apoptosis. Overall, our study suggests that the
aurone framework presented here could be an attractive template
for the identification of novel and potent inhibitors of SIRT1.
All the synthesized compounds were initially tested for SIRT1
inhibitory potential21 at 10
lM using the Fluor-de-Lys peptide as
a substrate according to a reported biochemical enzymatic assay
method (see ESI). Suramin, a known inhibitor of sirtuin was used
as a reference compound in this assay. A number of compounds
tested were found to be active in this assay (>50% inhibition, Ta-
ble 1) the compound 3a being the best among them. The presence
of a bromo substituent on the arylidene ring was found to be ben-
eficial in terms of activity for example, 3a–c and 6a (>70% inhibi-
tion, Table 1). The presence of
a phenolic hydroxyl group
increased the activity further for example, 3a (Table 1). Since over
expression of SIRT1 has been observed in several types of cancer
and that inhibition of SIRT1 by small molecules demonstrated inhi-
bition of cancer cell proliferation21a hence all the compounds were
evaluated for their anti proliferative properties against two differ-
ent cancer cell lines for example, MDA-MB-231 and MCF-7. Both of
these cell lines are metastatic breast cancer cell lines. The cytotox-
icity of these compounds were evaluated at the concentration of
Acknowledgment
10 lM by using an MTT [(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
The authors thank management of ILS for encouragement and
support.
tetrazolium bromide] assay as reported earlier.22 It is evident from
the MTT assay results (Table 2) that compounds 3a, 3c, 3e, 3f, 3j
and 6b showed better or comparable anti proliferative properties
than suramin. Notably, in spite of promising SIRT1 inhibitory prop-
erties compound 3b and 6a did not show significant anti prolifer-
ative properties in the present assay perhaps due to their poor
permeability through the cell membrane. Nevertheless, the com-
pound 3a was identified as a promising agent that showed superior
anti proliferative properties than suramin. This prompted us to
evaluate the SIRT1 inhibitory potential of compound 3a further.
To our delight, compound 3a was found to be a potent inhibitor
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.017.
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l
pound 3a to inhibit SIRT1 was also evaluated in MDA-MB-231 cells
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grid coordinates of X:À12.9111, Y:À27.7633, Z:À33.29672. Molec-
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good docking score (the predicted strength of the non-covalent
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20. Crystal data of 3f: Molecular formula = C16H12O3, Formula weight = 252.26,
Crystal system = Monoclinic, space group = P21/n, a = 11.857 (14) Å, b = 5.396
17. (a) Spectral data for selected compounds; Compound 3a: yellow solid; mp 240–
242 °C; 1H NMR (400 MHz, CDCl3): d 10.47 (bs, 1H), 8.28 (d, J = 2.8 Hz, 1H),
7.71–7.78 (m, 2H), 7.43 (d, J = 8 Hz, 1H) 7.28 (s, 1H), 7.24–7.20 (m, 2H), 6.92 (d,
J = 8.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): d 183.6, 165.4, 156.8, 146.3,
137.8, 134.2, 132.8, 124.4, 124.2, 121.0, 120.9, 118.0, 113.4, 110.7, 104.8; IR
(KBr) vmax 3200, 3050, 1694, 1641, 1287, 1261, 888 cmÀ1; MS (ES mass): m/z
316.9 (M+1, 100%); HRMS: calcd for C15H10BrO3:317.9813, found 317.9814.
Compound 3c: white solid; mp 173–174 °C; 1H NMR (400 MHz, CDCl3 + DMSO-
d6): d 7.72–7.79 (m, 3H), 7.61–7.69 (m, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.34 (d,
J = 8.4 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 6.82 (s, 1H); 13C NMR (100 MHz,
CDCl3 + DMSO-d6): 184.5, 166.0, 146.0, 137.0, 132.7, 132.2, 132.1, 131.2, 131.0,
124.7, 124.2, 123.6, 122.9, 121.5, 112.9; IR (KBr) vmax 3050, 1715, 1655, 1602,
1298, 949 cmÀ1; MS (ES mass): m/z 301.0 (M+1, 100%); HRMS: calcd for
(6) Å, c = 18.744 (2) Å, V = 1190.8 (2)Å3, T = 296 K, Z = 4, Dc = 1.407 Mg mÀ3
,
l
(Mo-K
a
) = 0.10 mmÀ1
,
8297 reflections measured, 1707 independent
(I)], R1_obs = 0.063, Goodness
reflections, 1247 observed reflections [I>2.0
r
of fit = 1.07. Crystallographic data (excluding structure factors) for 3f have
been deposited with the Cambridge Crystallographic Data Center as
supplementary publication number CCDC 836065.
21. (a) Kalle, A. M.; Mallika, A.; Badiger, J.; Alinakhi; Talukdar, P.; Sachchidanand
Biochem. Biophys. Res. Commun. 2010, 401, 13; (b) Layek, M.; Kumar, Y. S.;
Islam, A.; Karavarapu, R.; Sengupta, A.; Halder, D.; Mukkanti, K.; Pal, M. Med.
Chem. Commun. 2011, 2, 478; (c) Mulakayala, N.; Rambabu, D.; Raja, M. R.;
Chaitanya, M.; Kumar, C. S.; Kalle, A. M.; Krishna, G. R.; Reddy, C. M.; Rao, M. V.
B.; Pal, M. Bioorg. Med. Chem. 2012, 20, 759.
22. Plumb, J. A. Methods Mol. Med. 2004, 88, 165.
C
15H10BrO2: 300.9864, found 300.9876. Compound 3e: white solid; mp 231–
23. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
232 °C; 1HNMR (400 MHz, CDCl3 + DMSO-d6): d 9.72 (s, 1H), 8.19 (d, J = 1.6 Hz,
1H), 7.82–7.73 (m, 2H), 7.54 (s, 1H), 7.22 (d, J = 7.6 Hz, 2H), 7.23 (t, J = 7.6 Hz,
1H), 6.53 (d, J = 7.6 Hz, 2H), 3.81 (s, 3H); 13C NMR (100 MHz, CDCl3 + DMSO-
d6): d 182.4, 164.0, 161.4, 158.4, 143.9, 135.1, 131.7, 122.9, 122.7, 121.9, 120.8,
111.7, 111.3, 105.5, 99.7, 54.0; IR (KBr) vmax 3378, 3023, 1701, 1645, 1298, 915
cmÀ1; MS (ES mass): m/z 268.9 (M+1, 100%); HRMS: calcd for C16H13O4:
24. Autiero, I.; Costantini, S.; Colonna, G. PLoS ONE 2009, 4, 7350.
25. Jacobson, M. P.; Pincus, D. L.; Rapp, C. S.; Day, T. J. F.; Honig, B.; Shaw, D. E.;
Friesner, R. A. Proteins 2004, 55, 351.
26. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177.