Construction and functionalization of pyranone ring fused with pyran moiety: Design and synthesis of novel pyrano[4,3-b]pyran-5(4H)-ones as potential inhibitors of sirtuins

Article abstract of DOI:10.1016/j.bmcl.2013.05.014

Novel pyrano[4,3-b]pyran-5(4H)-one based small molecules were designed as potential inhibitors of sirtuins (i.e., yeast sir2, a homolog of human SIRT1). Elegant synthesis of these compounds was performed via a multi-step sequence consisting of MCR, Sandmeyer type iodination, Sonogashira type coupling followed by iodocyclization and then Pd-mediated various C-C bond forming reactions. The overall strategy involved the construction of a pyran ring followed by the fused pyranone moiety and subsequent functionalization at C-8 position of the resultant core pyrano[4,3-b]pyran-5(4H)-one framework. The crystal structure analysis of a representative iodolactonized product (6d) is presented. Some of the synthesized compounds showed promising inhibitory activities when tested against yeast sir2 in vitro. The compound 6g showed dose dependent inhibition (IC₅₀ = 78.05 μM) of yeast sir2 and good interactions with this protein in silico.

Full text of DOI:10.1016/j.bmcl.2013.05.014

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4195–4205
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Construction and functionalization of pyranone ring fused
with pyran moiety: Design and synthesis of novel
pyrano[4,3-b]pyran-5(4H)-ones as potential inhibitors of sirtuins
Ali Nakhi a, Md. Shafiqur Rahman a,b, Sivakumar Archana a,c, Ravada Kishore d, G. P. K. Seerapu a,
K. Lalith Kumar a, Devyani Haldar a, Manojit Pal a,
⇑
a Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
b Chemical Synthesis & Process Technologies, Department of Chemistry, University of Delhi, New Delhi 110 007, India
c Manipal College of Pharmaceutical Sciences, Manipal University, Manipal 576104, India
d School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel pyrano[4,3-b]pyran-5(4H)-one based small molecules were designed as potential inhibitors of sir-
tuins (i.e., yeast sir2, a homolog of human SIRT1). Elegant synthesis of these compounds was performed
via a multi-step sequence consisting of MCR, Sandmeyer type iodination, Sonogashira type coupling fol-
lowed by iodocyclization and then Pd-mediated various C–C bond forming reactions. The overall strategy
involved the construction of a pyran ring followed by the fused pyranone moiety and subsequent func-
tionalization at C-8 position of the resultant core pyrano[4,3-b]pyran-5(4H)-one framework. The crystal
structure analysis of a representative iodolactonized product (6d) is presented. Some of the synthesized
compounds showed promising inhibitory activities when tested against yeast sir2 in vitro. The compound
Received 21 March 2013
Revised 24 April 2013
Accepted 7 May 2013
Available online 15 May 2013
Keywords:
Pyranochromendione
Pyranopyranone
Iodocyclization
Sirtuins
6g showed dose dependent inhibition (IC50 = 78.05
l
M) of yeast sir2 and good interactions with this pro-
tein in silico.
Ó 2013 Elsevier Ltd. All rights reserved.
Naturally occurring
a
-pyrone and isocoumarin derivatives
The sirtuins are class III NAD-dependent deacetylases that cat-
alyze NAD+ dependent removal of acetyl group to generate deacet-
ylated proteins, nicotinamide, and O-acetyl-ADP-ribose. They play
important role in diverse biological processes such as transcrip-
tional silencing, regulation of apoptosis by deacetylation of p53,
fatty acid metabolism, cell cycle regulation, and aging.5 Among
the seven human sirtuins for example SIRT1–7, the SIRT1 has been
studied well which has several substrates such as p53, Ku70, NF-
have attracted particular attention especially in drug discovery
and pharmaceutical research because of their broad range of bio-
logical activities.1 An impressive number of reports are now
available in the literature that discloses a wide range of iso-
coumarin based small molecules of pharmacological importance
including the clinical candidate NM-3.2 Recently, we have re-
ported 1,8-dioxo-octahydroxanthenes3 (A, Fig. 1) and thie-
no[3,2-c]pyran-4-one based small molecules4 (B, Fig. 1) as
potential anticancer agents. While some of them showed prom-
ising growth inhibition when tested against a number of cancer
cells their mechanism of action was not clearly understood. As
part of our ongoing efforts on the identification of novel inhibi-
tors of sirtuins we became interested in evaluating the sirtuin
inhibitory properties of small molecules based on pyrano[4,3-
b]pyran-5(4H)-one (C, Fig. 1). The design of C was performed
by combining some of the structural features of both A and B
in a single molecular entity. We anticipated that compounds
based on C would show anticancer properties via inhibition of
sirtuins.
j
B, forkhead proteins etc.6 Studies have shown that sirtuins are
up-regulated in many cancers and inhibition of sirtuins allows
re-expression of silenced tumor suppressor genes, leading to re-
duced growth of cancer cells. Thus, inhibition of sirtuins is being
O
Ar
R
O
O
O
O
O
O
S
R2
O
O
Ar'/R'
R1
B
A
C
Ar'/R'
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail address: manojitpal@rediffmail.com (M. Pal).
Figure 1. Design of novel pyrano[4,3-b]pyran-5(4H)-one based inhibitors (C) of
sirtuins from the known cytotoxic agents A and B.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.014

4196
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
Ar1/R1 (4)
O
O
Ar/R OEt
O
Ar/R OEt
tBuONO
NCCH2CO2Et
10%Pd/C,PPh3,CuI
Ar/R
CHO
O
O
+
Et3N,
DMAP, EtOH,
60 °C, 1-2 h
CuI, MeCN
0 oC, 1-2 hr
O
I
O
NH2
O
EtOH, 60 oC, 2-3 h
3
1
2
Sonogashira
Suzuki
Heck
O
Ar/R OEt
O
Ar/R O
O
O
Ar/R O
I2, CH2Cl2
rt, 2-4 h
Ar1/R1
O
O
O
Ar1/R1
O
Ar1/R1
O
Ar2/R2
I
5
6
7,8,9
Scheme 1. Synthesis of novel pyrano[4,3-b]chromendione and pyrano[4,3-b]pyran based novel small molecules.
Table 1
Preparation of 2-iodo substituted 5,6,7,8-tetrahydro-4H-chromene-3-carboxylate ester derivatives (3)a
O
Ar/R OEt
O
Ar/R OEt
tBuONO
O
O
CuI, MeCN
O
2
NH2
O
3
I
0
oC, 1-2 hr
Entry
1
2-Aminochromene derivatives (2)
Time (h)
Product (3)
Yield (%)
82
OH
OH
O
O
O
OEt
O
OEt
2a
1
1
3a
3b
O
O
O
NH2
O
I
OCH3
OCH3
O
O
OEt
OEt
2
3
4
2b
2c
2d
88
86
85
O
O
O
I
O
NH2
Cl
Cl
OEt
OEt
O
2
2
3c
O
O
F
I
O
F
NH2
O
O
OEt
O
O
O
OEt
3d
O
O
O
NH2
OEt
O
O
I
OEt
O
O
5
6
2e
2f
1
1
3e
3f
82
75
NH2
I
S
S
O
O
O
O
OEt
O
OEt
O
O
O
NH2
O
O
I
O
O
OEt
O
OEt
7
2g
1
3g
73
O
NH2
I

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
4197
Table 1 (continued)
Entry
2-Aminochromene derivatives (2)
Time (h)
Product (3)
Yield (%)
F
F
OEt
O
OEt
OEt
OEt
8
2h
2i
2
3h
3i
68
70
O
O
O
H3C
O
O
I
H3C
O
O
NH2
OH
OH
OEt
OEt
OEt
OEt
O
9
2
O
O
O
H3C
I
H3C
NH2
a
All the reactions were carried out using the amine 2, tBuONO and CuI in MeCN at 0 °C.
Table 2
Effect of reaction conditions on Sonogashira coupling of 3a with phenylacetylene 4aa
OCH3
considered as a new approach for the discovery of novel anticancer
drugs. While a number of inhibitors, for example nicotinamide,
sirtinol, splitomicin, cambinol, tenovins, and EX5277 have been re-
ported none except EX527 (which is presently undergoing Phase
1a clinical trial for the treatment of Huntington’s disease) have
progressed into clinical trials as anticancer agents. This prompted
us to explore pyrano[4,3-b]pyran-5(4H)-one (C) as potential and
novel inhibitors of sirtuins. The design and selection of this class
of compounds was further supported by the docking studies (see
later for a discussion).
OCH3
O
OEt
O
OEt
4a
O
O
Pd catalyst, CuI, Et3N
EtOH, 60 oC, 2-3 h
O
O
I
5b
3b
The synthesis of our target compounds 6–9 (or C, Fig. 1) in-
volved a multi-step sequence consisting of a multi-component
reaction (MCR), Sandmeyer type iodination, Sonogashira type cou-
pling followed by iodocyclization and then Pd-mediated various C–
C bond forming reactions (Scheme 1).
Entry
Pd-catalysts
Base
Time (h)
Yieldb (%)
1
2
10% Pd/C–PPh3
Pd(PPh3)2Cl2
Et3N
Et3N
2
5
85
73
a
All the reactions were carried out using compound 3b (0.1 g, 0.2074 mmol),
phenyl acetylene 4a (0.02 mL, 0.2074 mmol), CuI (0.002 g, 0.0082 mmol), and Et3N
(0.85 mL, 0.6222 mmol), either 10% Pd/C (0.0020 mmol) PPh3 (0.001 g,
The starting material 2 was obtained through the MCR8 of 1,3-
diketones or b-keto esters, aldehydes and ethyl cyanoacetate in the
presence of catalytic amounts of 4-(N,N-dimethylamino)pyridine
&
0.0040 mmol), or Pd(PPh3)2Cl2, (0.0020 mmol) in ethanol (3 mL) under nitrogen.
b
Isolated yield.
Table 3
Pd/C-mediated preparation of 2-alkynyl substituted 5,6,7,8-tetrahydro-4H-chromene-3-carboxylate ester derivatives (5)a
Entry
2-Iodochromenone derivative (3)
Terminal alkyne (4)
Time (h)
Alkynyl ester (5)
OH
Yieldc (%)
O
O
O
OEt
O
1
3a
2
5a
81
4a
O
OCH3
OEt
2
3b
4a
1
5b
85
O
O
Cl
OEt
3
3c
4a
1
5c
70
O
O
(continued on next page)

4198
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
Table 3 (continued)
Entry
2-Iodochromenone derivative (3)
Terminal alkyne (4)
Time (h)
Alkynyl ester (5)
Yieldc (%)
F
O
OEt
4
3d
3d
3d
4a
1
5d
5e
5f
73
O
O
F
(CH2)5CH3
4b
O
OEt
5
2
68b
O
O
F
(CH2)5CH3
OH
4c
O
O
O
OEt
4c
6
1
70
O
O
O
O
OH
OEt
O
7
8
3e
3e
4a
2
2
5g
5h
77
OEt
4d
N
O
63b
4d
N
S
O
O
OEt
O
9
3f
4a
4a
1
2
5i
5j
82
85
O
O
O
OEt
O
10
3g
F
OEt
OEt
O
O
11
3h
4a
2
5k
74
H3C
O

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
4199
Table 3 (continued)
Entry
2-Iodochromenone derivative (3)
Terminal alkyne (4)
Time (h)
Alkynyl ester (5)
Yieldc (%)
F
(CH2)4CH3
OEt
OEt
4e
12
3h
3h
2
5l
66
O
O
H3C
O
O
O
(CH2)4CH3
F
(CH2)9CH3
OEt
OEt
4f
13
2
5m
65b
O
O
H3C
(CH2)9CH3
OH
OEt
OEt
O
O
14
3i
4a
1
5n
85
H3C
OH
OEt
OEt
O
O
15
3i
4c
2
5o
80
H3C
O
OH
a
Reactions were carried out compound 3 (0.2074 mmol), terminal alkyne (4) (0.2074 mmol), 10% Pd/C (0.0020 mmol), PPh3 (0.0040 mmol), CuI (0.0082 mmol), and Et3N
(0.6222 mmol) in EtOH (5.0 mL) at 60 °C.
b
Pd(PPh3)2Cl2 was used instead of 10% Pd/C–PPh3.
Isolated yield.
c
Table 4
I2 mediated synthesis of 4-iodo-7,8-dihydropyrano[4,3-b]chromenedione and 8-iodo-4,5-dihydropyrano[4,3-b]pyran (6)a
O
Ar/R OEt
O
Ar/R O
I2, CH2Cl2
rt, 2-4 h
Ar1/R1
O
O
Ar1/R1
O
O
I
5
6
Entry
Alkynyl ester (5)
Time (h)
Product (6)
Yieldb (%)
OH
O
O
1
5a
2
6a
93
O
O
I
OCH3
O
O
I
2
5b
2
6b
97
O
O
(continued on next page)

4200
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
Table 4 (continued)
Entry
Alkynyl ester (5)
Time (h)
Product (6)
Cl
Yieldb (%)
O
O
O
O
I
3
4
5
6
5c
2
6c
6d
6e
6f
80
O
O
O
O
F
O
I
5d
5e
5f
2
4
4
88
58
54
O
F
O
I
O
F
(CH2)9CH3
O
O
O
O
O
O
O
OH
O
O
O
I
O
7
8
5g
5h
2
4
6g
6h
85
53
I
O
I
N
S
O
O
O
9
5i
5j
2
2
O
O
6i
6j
85
85
O
O
I
O
O
10
I
F
O
O
11
5k
4
6k
74
O
O
H3C
O
I

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
4201
Table 4 (continued)
Entry
Alkynyl ester (5)
Time (h)
Product (6)
Yieldb (%)
F
O
O
O
12
13
14
5l
4
6l
60
65
75
70
O
O
H3C
O
O
O
O
(CH2)5CH3
I
F
O
O
O
5m
5n
5o
4
4
4
6m
6n
6o
H3C
(CH2)9CH3
I
OH
O
O
O
O
H3C
I
OH
O
O
O
15
O
OH
H3C
I
a
All reactions were carried out by using alkynes 5 (0.9677 mmol) and I2 (0.9677 mmol) in CH2Cl2 (3 mL).
Isolated yields.
b
(DMAP) in ethanol. Compound 2 was then converted to the corre-
sponding 2-iodo derivative 3 under a modified Sandmeyer condi-
tions the results of which are summarized in Table 1.
Having prepared the iodo derivatives 6 we then focused on fur-
ther structural elaboration of these compounds by using various
Pd-catalyzed C–C bond forming reactions such as Sonogashira, Su-
zuki, and Heck coupling reactions. Thus, the compound 6a and 6d
was reacted with a terminal alkyne in the presence of 10% Pd/C–
PPh3–CuI as catalysts and Et3N as a base in EtOH to afford the cor-
responding 4-alkynyl substituted 7,8-dihydropyrano[4,3-b]chro-
mene-1,9-(6H,10H)-dione derivative 7a and 7b (Table 5).
Similarly, Suzuki coupling of compound 6a and 6k with an appro-
priate arylboronic acid provided 8a and 8b in good yield (Table 5).
The compound 3 was then coupled with various terminal al-
kynes 4 under Pd–Cu catalysis to give the desired internal alkyne
5. Initially, we examined the coupling of 3b with a terminal alkyne
4a under two reaction conditions (Table 2). Since the use of Pd/C
has been explored as an inexpensive, easily separable and recycla-
ble catalyst for the alkynylation of aryl and heteroaryl halides9
hence the initial coupling reaction of 3b with 4a was carried out
in the presence of 10% Pd/C, PPh3, CuI and Et3N in ethanol. To
our satisfaction, the desired product 5b was isolated in 85% yield
(entry 1, Table 2). Similarly, the use of Pd(PPh3)2Cl2 in the presence
of CuI was also found to be effective though the product yield was
marginally low (entry 2, Table 2). Thus, a series of internal alkyne 5
were prepared in good to acceptable yields (Table 3) using either
10% Pd/C–PPh3–CuI or Pd(PPh3)2Cl2–CuI (e.g., entries 5, 8 and 13,
Table 3) as catalyst system.
In recent years, I2 or ICl-mediated intramolecular electrophilic
cyclization of the alkynes in an exo-dig or endo-dig fashion has be-
come convenient and economical method for the straightforward
access of various iodo heterocycles.10 The 2-alkynyl substituted
5,6,7,8-tetrahydro-4H-chromene-3-carboxylate ester derivatives
(5) thus prepared were then subjected to I2-mediated electrophilic
cyclization which provided the pyrano[4,3-b]pyran-5(4H)-one core
that is 4-iodo-7,8-dihydropyrano[4,3-b]chromenedione or 8-iodo-
4,5-dihydropyrano[4,3-b]pyran (6) exclusively via a regioselective
6-endo-dig ring closure (Table 4).
All the iodo compounds (6) prepared were characterized by
spectral data and the molecular structure of a representative com-
pound 6d was determined unambiguously by X-ray crystallo-
graphic analysis (Fig. 2).11
Figure 2. X-ray crystal structure of 6d (ORTEP diagram). Thermal ellipsoidal
diagram is drawn at 30% probability (hydrogen atoms are omitted for clarity).

4202
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
Table 5
Functionalization of compounds (6) via Pd mediated C–C bond forming Sonogashiraa, Suzukib and Heckc reactions
O
Ar
O
O
Ar
O
O
O
Pd catalyst
Base, Solvent
O
O
R/Ar'
7-9
I
6
Entry
Iodo compound (6)
R = alkyne/acrylate Ar1 = aryl boronic acid
Time (h)
Product (7–9)
Yieldd (%)
OH
O
(CH2)3CH3
O
4g
O
7aa
1
6a
2
73
O
(CH2)3CH3
F
O
O
O
7ba
2
3
4
6d
6a
6k
4g
2
1
1
64
70
67
O
(CH2)3CH3
OH
B(OH)2
O
O
O
8ab
O
B(OH)2
OH
F
OEt
O
O
O
8bb
O
OH
OH
O
O
O
O
O
9ac
5
6a
1
71
O
O
O
a
Reaction were carried out using 10% Pd/C (0.0041 mmol), PPh3 (0.0165 mmol), CuI (0.0041 mmol), Et3N (0.82 mmol), and a terminal alkyne (0.7348 mmol) in EtOH (3 mL)
at 60 °C.
b
Reactions were carried out by using Pd(OAc)2 (0.0041 mmol, 5 mol %), K2CO3 (0.82 mmol) an appropriate boronic acid (0.61 mmol) in DMF (3 mL) at 80 °C.
Reaction was carried out by using Pd(OAc)2 (0.0049 mmol, 5 mol %), K2CO3 (0.82 mmol), ethyl acrylate (0.82 mmol) in DMF (3 mL) at 80 °C.
Isolated yields.
c
d
The compound 6a afforded the corresponding alkene 9a when cou-
pled with ethyl acrylate under Heck conditions (Table 5). A Pd-
mediated reductive deiodination of compound 6b, 6d, and 6k
afforded 10a–10c in good yields (Table 6).
we tested some of the synthesized compounds (6–8) in vitro by
using yeast cell based reporter silencing assay. Compounds were
tested without separating their individual steroisomers at the con-
centration of 50 lM for their ability to inhibit yeast sirtuin family
The identification of novel inhibitors of sirtuin being the goal of
NAD-dependent histone deacetylase (HDAC) sir2 protein (a yeast
the present work and due to our continuing interest in this area12
homologue of mammalian SIRT1). Splitomicin,13 a known inhibitor

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
4203
Table 6
Pd-mediated reductive deiodination of compound 6b, 6d, and 6k.a
Table 7
Yeast based in vitro assay for sir2 inhibition by compounds 6–8a
Entry
Compound
% of inhibition
O
Ar
O
O
Ar
O
Pd(OAc)2
Et3N, HCOOH
1
2
3
4
5
6
7
8
9
6b
6d
6f
6g
6i
32.38
49.53
25.30
43.90
43.20
15.30
0.50
1.01
3.30
1.51
O
O
DMF, 80 oC,1h
Ar'/R
Ar'/R
O
O
6
I
10
6j
Entry Compound (6) Product (10)
OCH3
Yieldb (%)
6k
7a
8a
8b
10
Splitomicin was used as a reference compound.
O
O
a
Data represent the mean values of three independent determinations.
1
6b
10a 83
O
O
F
O
O
2
3
6d
6k
10b 72
O
O
(CH2)9CH3
F
OEt
O
10c 80
O
O
O
a
All reactions were carried out by using compound 6 (0.3616), formic acid
(0.0361 mmol), Pd(OAc)2 (0.0036 mmol) and Et3N (0.3616 mmol) in DMF (3 ml) at
Figure 3. Dose dependent % inhibition of sir2 by the compound 6g.
80 °C for 1 h.
b
Isolated yields.
To understand the binding mode of the compound 6g with
yeast sir2 protein in silico docking was performed by using the
crystal structure coordinating with NAD-dependent protein deace-
tylase (PDB ID: 2HJH).14 Since two enantiomers are possible for
compound 6g hence the docking studies was performed by using
both R- and S-isomer individually. The phenyl group at C-3 posi-
tion of S-isomer was accommodated well into the hydrophobic
pocket of the active site consisting of Arg 497, Gly 264, and Asp
273 residues and the dimethyl group at C-7 interacted with Lys
475 and Ser 473 (Figs. 4 and 5). While similar interactions were
also observed in case of R-isomer (Fig. 4) the S-isomer however
showed better score than the R-antipode. The dock score of indi-
vidual isomer along with the contributing factors are listed in Ta-
ble 8. It is evident from the dock score that the (S)-isomer
showed better interactions with sir2 protein than its (R)-antipode
indicating that further SAR studies need to be performed around
the (S)-isomer. Overall, the compound 6g is of further interest as
a novel small molecule based inhibitor of sirtuins.
of sirtuin, was used as a reference compound. In this assay a yeast
strain (TEL::URA3 strain (MAT ura3-52 lys2-801 ade2-101
a
trp 63 his3 200 leu3 200 leu2-D1 TEL adh4::URA) was used in
D
D
D
which, a reporter gene URA3 was inserted in the silenced telomeric
region where it is silenced by yeast sir2 protein (Scheme 2). Inhibi-
tion of sir2 protein by an inhibitor would allow the URA3 gene to
be expressed thereby resulting in death of the yeast cell in pres-
ence of 5-FOA through the formation of toxic 5-fluorouracil. The
results of our in vitro assay are summarized in Table 7. Among
the compounds tested, 6b, 6d, 6g and 6i (entries 1, 2, 4, and 5, Ta-
ble 7) showed inhibitory activities against yeast sir2 at 50 lM
whereas 6k, 7a, 8a and 8b were inactive. In a dose response study
the compound 6g showed dose dependent inhibition of sir2 with
an IC50 = 78.05 lM (Fig. 3).
URA3
Sir2
In conclusion, a series of novel pyrano[4,3-b]pyran-5(4H)-one
based small molecules were designed as potential inhibitors of
sir2. These compounds were obtained in good yields via an elegant
multi-step method consisting of MCR (involving aldehydes, ethyl
cyanoacetate and 1,3-diketone/b-keto ester), Sandmeyer type
iodination, Sonogashira type coupling followed by iodocyclization
and then Pd-mediated various C–C bond forming reactions. The
crystal structure analysis of a representative iodolactonized prod-
uct (6d) is presented. Some of the compounds synthesized showed
encouraging inhibition of sir2 protein (a yeast homologue of
URA3
URA3
+
Sir2
Integration of URA3 gene
in the telomeric region
Scheme 2. Cell based sir2 mediated reporter silencing assay in yeast. (I) Growth in
presence of 5-fluoroorotic acid (5-FOA): sir2 mediated silencing of URA3 gene
permits growth in FOA; (II) no growth in presence of FOA: inhibition of sir2 results
in expression of URA3 gene and cell death in presence of FOA.
mammalian SIRT1) when tested using yeast based assay.
A
representative compound 6g showed dose dependent inhibition

4204
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
Figure 4. The binding mode of (S)- and (R)-isomers of compound 6g and their 2D interaction plot.
(IC50 = 78.05 lM) of yeast sir2 and good interactions in silico (dock
Acknowledgments
score À6.7) when docked into this protein. Overall, the pyrano[4,3-
b]pyran-5(4H)-one framework presented here could be an attrac-
tive template for the identification of novel sir2 inhibitors and
the corresponding synthetic strategy described could be useful
for generating diversity based library of small molecules related
to this scaffold.
The authors thank Professor Javed Iqbal for his encourage-
ment. D.H. and M.P. thank DBT, New Delhi, India for financial
support (Grant No. BT/PR13997/Med/30/310/2010). A.N. thanks
CSIR—New Delhi, India for awarding
Fellowship.
a
Senior Research

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4195–4205
4205
Figure 5. Binding mode of 6g docked into the catalytic domain of Yeast sir2 (PDB ID: 2HJH).
Table 8
Factors contributing docking score of (S)- and (R)-isomers of compound 6g with sir2 protein
Molecule
Dock scorea
Steric
Protein desolvation
Ligand desolvation H-bond
Clash
Ligand desolvation
Hydrogen bond
Splitomicin
6g (S-isomer)
6g (R-isomer)
À8.5
À6.7
À6.0
À14.4
À14.4
À17.9
6.5
3.9
4.8
À0.5
À0.01
À0.6
0.1
0.3
0.6
0.5
1.3
2.1
À0.7
0
À0.6
a
FRED Chemgauss4 score.
E.; Baker, L.; Thompson, A.; Mathers, J.; Holland, S. J.; Stark, M. J. R.; Pass, G.;
Woods, J.; Lane, D. P.; Westwood, N. J. Cancer Cell 2008, 13, 454; (e) Harborne, J.
B. Prog. Clin. Biol. Res. 1988, 280, 17.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
05.014.
8. Khan, A. T.; Lal, M.; Ali, S.; Khan, M. Md. Tetrahedron Lett. 2011, 52, 5327.
9. Pal, M. Synlett 2009, 2896.
10. For a review on the use of iodine as a reagent, see: (a) Mphahlele, M. J.
Molecules 2009, 14, 5308; For leading references on iodocyclization, see: (b)
Mancuso, R.; Mehta, S.; Gabriele, B.; Salerno, G.; Jenks, W. S.; Larock, R. C. J. Org.
Chem. 2010, 75, 897; (c) Verma, A. K.; Aggarwal, T.; Rustagi, V.; Larock, R. C.
Chem. Commun. 2010, 46, 4064; (d) Mehta, S.; Larock, R. C. J. Org. Chem. 2010,
75, 1652; (e) Cho, C.-H.; Neuenswander, B.; Larock, R. C. J. Comb. Chem. 2010,
12, 278; (f) Barange, D. K.; Batchu, V. R.; Gorja, D.; Pattabiraman, V. R.; Tatini, L.
K.; Babu, J. M.; Pal, M. Tetrahedron 2007, 63, 1775.
11. Crystallographic data (excluding structure factors) for 6d have been deposited
with the Cambridge Crystallographic Data Center as supplementary
publication number CCDC No: 923597.
12. Layek, M.; Kumar, Y. S.; Islam, A.; Karavarapu, R.; Sengupta, A.; Halder, D.;
Mukkanti, K.; Pal, M. Med. Chem. Commun. 2011, 2, 478.
References and notes
1. Pal, S.; Chatare, V.; Pal, M. Curr. Org. Chem. 2011, 15, 782.
2. Oikawa, T.; Sasaki, M.; Inose, M.; Shimamura, M.; Kuboki, H.; Hirano, S.;
Kumagai, H.; Ishizuka, M.; Takeuchi, T. Anticancer Res. 1997, 17, 1881.
3. Mulakayala, N.; VNS Murthy, P.; Rambabu, D.; Aeluri, M.; Adepu, R.; Krishna, G.
R.; Reddy, C. M.; Prasad, K. R. S.; Chaitanya, M.; Kumar, C. S.; Rao, M. V. B.; Pal,
M. Bioorg. Med. Chem. Lett. 2012, 22, 2186.
4. Nakhi, A.; Raju, A.; Rambabu, D.; Kishore, R.; Vanaja, G. R.; Arunasree, M. K.; Pal,
M. Bioorg. Med. Chem. Lett. 2012, 22, 4418.
5. Sauve, A. A.; Wolberger, C.; Schramm, V. L.; Boeke, J. D. Annu. Rev. Biochem.
2006, 75, 435.
6. Stunkel, W.; Campbell, R. M. J. Biomol. Screen. 2011, 16, 1153.
7. (a) Milne, J. C.; Denu, J. M. Curr. Opin. Chem. Biol. 2008, 12, 11; (b) Balcerczyk, A.;
Pirola, L. Biofactors 2010, 36, 383; (c) Heltweg, B.; Gatbonton, T.; Schuler, A. D.;
Posakony, J.; Li, H.; Goehle, S.; Kollipara, R.; DePinho, R. A.; Gu, Y.; Simon, J. A.;
Bedalov, A. Cancer Res. 2006, 66, 4368; (d) Lain, S.; Hollick, J. J.; Campbell, J.;
Staples, O. D.; Higgins, M.; Aoubala, M.; McCarthy, A.; Appleyard, V.; Murray, K.
13. Splitomicin has been reported to inhibit yeast sir2 with an IC50 value of 60 lM
in vitro, see: (a) Bedalov, A.; Gatbonton, T.; Irvine, W. P.; Gottschling, D. E.;
Simon, J. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 15113; (b) Grozinger, C. M.;
Chao, E. D.; Blackwell, H. E.; Moazed, D.; Schreiber, S. L. J. Biol. Chem. 2001, 276,
38837.
14. Hall, B. E.; Buchberger, J. R.; Gerber, S. A.; Ambrosio, A. L. B.; Gygi, S. P.; Filman,
D.; Moazed, D.; Ellenberger, T. doi: http://www.rcsb.org/pdb/explore/
explore.do?structureId=2hjh.