Amberlite IR-120H catalyzed MCR: Design, synthesis and crystal structure analysis of 1,8-dioxodecahydroacridines as potential inhibitors of sirtuins

Article abstract of DOI:10.1016/j.bmcl.2013.01.026

A rapid, inexpensive and high yielding method has been developed for the synthesis of 1,8-dioxodecahydroacridines using Amberlite IR-120H as a reusable catalyst under open air. These compounds were designed as potential inhibitors of sirtuins and prepared

Full text of DOI:10.1016/j.bmcl.2013.01.026

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1828–1833
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Amberlite IR-120H catalyzed MCR: Design, synthesis and crystal
structure analysis of 1,8-dioxodecahydroacridines as potential
inhibitors of sirtuins
Ali Nakhi a, P. T. V. A. Srinivas b, Md. Shafiqur Rahman a,c, Ravada Kishore d, G. P. K. Seerapu a,
K. Lalith Kumar a, Devyani Haldar a, M. V. Basaveswara Rao e, , Manojit Pal a,
⇑
⇑
a Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli, Hyderabad 500 046, India
b Department of Chemistry, K.L. University, Vaddeswaram, Guntur 522 502, Andhra Pradesh, India
c Chemical Synthesis & Process Technologies, Department of Chemistry, University of Delhi, New Delhi 110 007, India
d School of Chemistry, University of Hyderabad, Gachibowli, Hyderabad 500 046, India
e Department of Chemistry, Krishna University, Machilipatnam 521 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A rapid, inexpensive and high yielding method has been developed for the synthesis of 1,8-dioxodecahy-
droacridines using Amberlite IR-120H as a reusable catalyst under open air. These compounds were
designed as potential inhibitors of sirtuins and prepared via the MCR of 5,5-dimethyl-1,3-cyclohexanedi-
one, (hetero)aryl aldehydes and (hetero)aromatic amines under mild conditions. Further structure elab-
oration of a representative compound was performed via Pd catalyzed C–C bond forming reactions. The
crystal structure analysis and H-bonding patterns along with in vitro inhibitory activity against yeast Sir2
of the same compound is presented. Docking studies indicated that the compound interacts well with the
yeast Sir2.
Received 9 November 2012
Revised 24 December 2012
Accepted 7 January 2013
Available online 19 January 2013
Keywords:
Amberlite IR-120H
MCR
Ó 2013 Elsevier Ltd. All rights reserved.
Acridine
Sirtuin
Cancer
The identification of suitable drug targets is one of the major
challenges in cancer drug discovery. In the recent years, majority
of the efforts have been devoted by focusing on signaling mole-
cules mostly kinases as drug targets. However, with the emergence
of kinase inhibitor resistant cancers, there is a growing need to
identify newer targets and develop new drugs. Recently, inhibition
of sirtuins has been described as a new approach for the discovery
of novel anticancer drugs. The sirtuins (class III NAD-dependent
deacetylases that catalyze NAD+ dependent removal of acetyl
group to generate deacetylated proteins, nicotinamide, and
O-acetyl-ADP-ribose) function in diverse biological processes such
as transcriptional silencing, regulation of apoptosis by deacetyla-
tion of p53, fatty acid metabolism, cell cycle regulation, and aging.1
The mammalian sirtuin family consists of seven members for
example SIRT1–7 and among the seven human sirtuins, SIRT1
has been studied well which has several substrates such as p53,
Ku70, NF-B, forkhead proteins etc.2 Since sirtuins are up-regulated
in many cancers hence they are considered as important targets for
cancer therapeutics. Indeed, inhibition of sirtuins allows re-
expression of silenced tumor suppressor genes, leading to reduced
growth of cancer cells. Several small molecule inhibitors of sirtu-
ins, such as nicotinamide, sirtinol, splitomicin, cambinol, tenovins,
and the indole derivative EX5273 have been shown to induce cell
death in cancer cells. However, no sirtuin inhibitors except
EX527 (which is presently undergoing Phase 1a clinical trial for
the treatment of Huntington’s disease) have progressed into clini-
cal trials as anticancer agents. Recently we have reported the
synthesis of 1,8-dioxo-octahydroxanthenes (A, Fig. 1) as potential
anticancer agents.4 In continuation of that work and our interest
in the identification of inhibitors of sirtuins5 we now report the
(Het)
Ar
Ar
O
O
O
O
N
O
(Het)
Ar
B
A
⇑
Corresponding authors. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail addresses: palmanojit@yahoo.co.uk, manojitpal@rediffmail.com (M. Pal).
Figure 1. Novel 1,8-dioxodecahydroacridine derivatives (B) derived from A.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.026

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1828–1833
1829
O
R1
N
O
O
Amberlite IR-120H
1
+
R2NH2
+
R CHO
EtOH, 60 oC, 2-4 h
O
R2
3
4
2
1
Scheme 1. Amberlite IR-120H catalyzed synthesis of 1,8-dioxodecahydroacridines.
Table 1
Effect of catalysts on the MCR of 1, 2a and 3aa
OH
O
O
CH3
O
OH
+
Catalyst
+
N
EtOH, 60oC
O
CHO
NH2
4a
2a
3a
1
CH3
Entry
Catalyst (amount)
Time (h)
Yieldb (%)
1
2
3
4
5
6
7
8
9
Con. HCl (1–2 drops)
p-TSA (30 mg)
Amberlyst-15 (50 mg)
Amberlite IR-120H (50 mg)
Amberlite IR-120H (50 mg)
No catalyst
Amberlite IR-120H (50 mg)
Amberlite IR-120H (50 mg)
Amberlite IR-120H (50 mg)
5
5
4
2
1
5
4
4
2
88
62
80
95c (91, 87)
72
0
58d
67e
62f
a
All the reactions were carried out by using 1 (2 mmol), 2a (1 mmol), 3a (1 mmol) and a catalyst in EtOH (4 mL) at 60 °C under open air.
Isolated yields.
Catalyst was reused for additional two runs and figures within parentheses indicate the corresponding yield for each run.
DMF was used in place of EtOH.
MeCN was used in place of EtOH. The product isolated was found to be 9-(3-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione (4aa)
b
c
d
e
instead of 4a in this case.
f
Dichloromethane was used in place of EtOH.
synthesis and sirtuin inhibiting properties of analogous 1,
8-dioxodecahydroacridine derivatives (B, Fig. 1) the design and
selection of which was further supported by the docking studies
(see later for a discussion).
Initially, we carried out the reaction of diketone 1, 3-hydroxy-
benzaldehyde (2a), and p-toluidine (3a) in EtOH at 60 °C in the
presence of con. HCl and p-TSA when the desired product 4a was
isolated in 88% and 62% yield, respectively (entry 1 and 2, Table
1). However, to identify an environmental friendly condition the
same reaction was performed in the presence of known catalyst
Amberlyst-15 when the reaction was completed within 4 h afford-
ing 4a in 80% yield (entry 3, Table 1). The use of another resin based
catalyst for example Amberlite IR-120H was found to be more
effective as the reaction was completed within 2 h affording 4a in
95% yield (entry 4, Table 1). A further decrease in reaction time de-
creased the product yield (entry 5, Table 1) whereas the reaction
did not proceed in the absence of catalyst indicating the key role
played by the catalyst in the present MCR. To assess the recyclabil-
ity of Amberlite IR-120H the catalyst was recovered by filtration
(followed by washing with EtOAc and drying) and reused when
4a was isolated without significant loss of its yield (entry 4, Table
1). While all these reactions were performed in EtOH the use of
other solvents for example DMF (entry 7, Table 1), MeCN (entry 8,
Table 1), dichloromethane (entry 9, Table 1) etc. was also explored
and found to be less effective. In case of MeCN a different product
that is 9-(3-hydroxyphenyl)-3,3,6,6-tetramethyl-3,4,5,6,7,9-hexa-
hydro-1H-xanthene-1,8(2H)-dione (4aa) was isolated instead of
4a whereas a mixture of both was formed when water was used
as a solvent.
The synthesis of 1,8-dioxodecahydroacridines is generally car-
ried out by using a multi-component reaction (MCR) of dimedone,
aldehydes, and different anilines or ammonium acetate in the pres-
ence of a range of catalysts for example p-dodecylbenezenesulfonic
acid (DBSA),6 [B(C6F5)3],7 proline,8 carbon-based solid acid (CBSA),9
NH4Cl,10 Brønsted acidic imidazolium salts11 and ceric ammonium
nitrate (CAN).12 Synthesis of these compounds by the classical
Hantzsch’s procedure13 or reaction of aldoximes with dimedone,
under microwave irradiation have also been reported.14 The use
of a resin based catalyst for example Amberlyst-15 was also found
to be effective.15 While, many of these methods are quite effective
and can be performed under environmental friendly conditions we
were in search for a resin based inexpensive, faster, operationally
simple and high yielding method for the synthesis of our target
compound B. We have found that the rapid synthesis of B (or 4,
Scheme 1) can be carried out in high yields by using Amberlite
IR-120H as an inexpensive and recyclable catalyst16 in the MCR
of 5,5-dimethylcyclohexane-1,3-dione (1), aldehyde (2), and ani-
line (3) (Scheme 1). To the best of our knowledge this is the first
example of synthesis of compound B (or 4) catalyzed by Amberlite
IR-120H. The preliminary results of this study are presented.

1830
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1828–1833
Table 2
Synthesis of 1,8-dioxodecahydroacridines using Amberlite IR-120H (Scheme 1)a
Entry
Aldehyde;2; R1
=
Aniline;3; R2
=
Time (h)
Product (4)
Yieldb (%)
1
2
3
4
5
6
7
8
2a; 3-OHC6H4
2a
2a
2a
3a; 4-Me C6H4
3b; 4-BrC6H4
3c; 4-IC6H4
3d; 2-IC6H4
3e; 4-FC6H4
3c
3f; 4-ClC6H4
3h; C6H5
3i; 4-MeCOC6H4
3j; 4-CNC6H4
3k; 4-CF3C6H4
3b
3
3
4
3
2
4
2
3
4
3
4
3
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
91
89
94
90
95
86c
88
85
92
85
88
90c
87
2a
2b; 3-NO2C6H4
2b
2c; 2-OHC6H4
2a
2a
2a
9
10
11
12
13
4j
4k
4l
2d; 2-Naphthyl
2e; 2-Thienyl
3c
4m
CO2Et
S
14
2e
4
4n
87c
15
16
2f; 3-FC6H4
2g; C6H5
3e
3a
3
3
4o
4p
88
73
a
All the reactions were carried out by using 5,5-dimethyl-1,3-cyclohexanedione 1 (2 mmol), aldehyde 2 (1 mmol), amine 3 (1 mmol) and Amberlite IR-120H (50 mg) in
EtOH (4 mL) at 60 °C under open air.
b
Isolated yields.
After purification by column chromatography.
c
All the compounds (4a–4p) synthesized were characterized by
spectral data (1H and 13C NMR, IR, MS and HPLC) and the molecular
structure of a representative compound 4c was confirmed unam-
biguously by single crystal X-ray diffraction studies (Fig. 2).18 Fur-
ther, crystal structure analysis was carried out to understand the
packing and/or hydrogen bonding patterns in crystal of this mole-
cule and results are summarized in the following section.
The crystal structure analysis of 4c indicated supramolecular
interactions between carbonyl oxygen (C@O) and methylene
hydrogen (CH2) that were within the range of 2.459–2.503 Å. Since
only two supramolecular interactions (O4Á Á ÁH22B = 2.502 Å and
O4Á Á ÁH8A = 2.460 Å) were present between hydrogen and oxygen,
hence it gave very short network as shown in Figure 3. These
supramolecular interactions were within the range of van der
Waals radii.19
Having prepared the iodo derivative 4c we then focused on fur-
ther structure elaboration of this compound by using various Pd-
catalyzed C–C bond forming reactions such as Sonogashira, Suzuki,
and Heck coupling (Scheme 2).
Some of the synthesized compounds were tested for sirtuin
inhibitory potential in vitro by using a yeast cell based reporter
silencing assay as a model system for primary screening. Com-
pounds were tested at the concentration of 50 lM for their ability
to inhibit yeast sirtuin family NAD-dependent histone deacetylase
(HDAC) Sir2 protein (a yeast homologue of mammalian SIRT1).
Splitomicin,20a a known inhibitor of sirtuin, was used as a reference
compound in this assay. Compounds (4) were tested for their abil-
ity to inhibit Sir2 protein by estimating inhibition of growth of
yeast strain containing URA3 gene at telomeric locus, in presence
of 5-fluoroorotic acid (5-FOA).20b In this assay a yeast strain (TEL::-
Figure 2. Thermal ellipsoidal plot compound 4c (30% probability, hydrogen atoms
and another asymmetric molecule has been omitted for clarity).
Encouraged by the utility of Amberlite IR-120H as a catalyst in
the present MCR in EtOH various functionalized 1,
8-dioxodecahydroacridines (4) were prepared by using structurally
diverse aromatic aldehydes (2) and amines (3) bearing various
electron donating and electron withdrawing groups (Table 2). Both
aryl and heteroaryl aldehydes as well as amines were found to be
effective in this reaction. While all the reactants used in the pres-
ent MCR are commercially available the amine that is ethyl
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 3d
(entry 14, Table 2) used were prepared according to the reported
method.17
URA3 strain (MATa ura3-52 lys2-801 ade2-101 trpD63 his3D200
leu3 200 leu2- 1 TEL adh4::URA) was used in which, a reporter
D
D
gene URA3 was inserted in the silenced telomeric region where it
is silenced by yeast Sir2 protein (Scheme 3). Inhibition of Sir2 pro-
tein by an inhibitor would allow the URA3 gene to be expressed
thereby resulting in death of the yeast cell in presence of 5-FOA
through the formation of toxic 5-fluorouracil. Among all the com-
pounds tested (see ESI) the compound 4c showed ꢀ40% inhibition
of Sir2. The other compounds that showed inhibition of Sir2

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1828–1833
1831
Figure 3. The supramolecular interactions between hydrogen and oxygen in compound 4c.
(CH2)3CH3
10% Pd/C, PPh3,
Ar
CuI, Et3N
EtOH, 60oC, 3h
OH
O
(CH2)3CH3
5a
O
(64%)
PhB(OH)2
Pd(OAc)2, K2CO3
Ar =
Ar
N
ArI
Ph
5b (68%)
DMF, 80oC, 4h
4c
CO2Et
Pd(OAc)2, K2CO3,
DMF, 80oC, 3h
Ar
CO2Et
5c (52%)
Scheme 2. Structure elaboration of 4c via Pd catalyzed C–C bond forming reactions.
Sir2 (I)
Sir2
Integration of URA3 gene
in the telomeric region
Scheme 3. 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.
includes 4a (30%), 4b (32%), 4e (33%) and 4f (35%) indicating a po-
lar group preferably an OH at the m-position of the 9-phenyl ring
was beneficial for Sir2 inhibition. Similarly, a substituent prefera-
bly a halo group at the p-position of the 10-phenyl ring seemed
to be favorable for inhibitory effect.
NH of Asp 43 and (ii) hydroxyl group of 4c with OH of COOH in
Asp 43 (Fig. 4). The presence of N-aryl group facilitated 4c to adopt
the favorable conformation suitable for binding with Sir2 protein.
The glide score obtained for 4c including other parameters are
listed in Table 3.
To understand the binding mode of the compound 4c with yeast
Sir2 protein docking21 was carried out using the crystal structure of
yeast Sir2 (PDB ID: 1Q1A). The lowest energy conformation was se-
lected and the ligand interactions (H-bonding and hydrophobic
interaction) with the active sites of yeast Sir2 were determined.
Two H-bond interactions were observed between Asp 43 and the
molecule 4c involving (i) one of the carbonyl group of 4c with
In conclusion, a rapid, inexpensive and high yielding method has
been developed for the synthesis of 1,8-dioxodecahydroacridines
using Amberlite IR-120H as a recyclable catalyst under open air.
To the best of our knowledge this is the first example of synthesis
of these compounds catalyzed by Amberlite IR-120H. These acri-
dine derivatives were designed as potential inhibitors of sirtuins
(that are considered as emerging targets in cancer drug discovery)

1832
A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1828–1833
Figure 4. Binding mode and interactions orientation of 4c with Yeast Sir2 (PDB ID: 1Q1A).
Table 3
The crystal structure analysis and H-bonding patterns along with
Glide score and contributing parameters
in vitro inhibitory activity against yeast Sir2 of the same compound
is presented. Docking studies indicated that the compound inter-
acts well with yeast Sir2. Overall, the Amberlite IR-120H catalyzed
MCR described here could be useful in constructing library of small
molecules based on 1,8-dioxodecahydroacridine framework lead-
ing to the identification of novel inhibitors of sirtuins.
Molecule
GScore
LipophilicEvdW
HBond
Electro
PhobicPenal
0.34
4c
À3.0
À2.35
À0.48
À0.52
LipophilicEvdW: Chemscore lipophilic pair term and fraction of the total protein–
ligand vdw energy.
HBond: Rewards for hydrogen bonding interaction between ligand and protein.
Electro: Electrostatic reward.
PhobicPenal: Penalty for solvent-exposed ligand groups.
Acknowledgments
The authors thank Professor Javed Iqbal for his encouragement.
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 a Senior Research Fellowship.
and prepared via the MCR of 5,5-dimethyl-1,3-cyclohexanedione,
(hetero)aryl aldehydes and (hetero)aromatic amines under mild
conditions. Further structure elaboration of a representative com-
pound was performed via Sonogashira, Suzuki and Heck reactions.

A. Nakhi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1828–1833
1833
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.026.
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