Novel acridinedione derivatives: Design, synthesis, SIRT1 enzyme and tumor cell growth inhibition studies

Article abstract of DOI:10.1016/j.bmcl.2012.03.030

A new scaffold N-(9-(ortho/meta/para-(benzyloxy)phenyl)-3,3,6,6- tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-octahydroacridin-10(9H)-yl) isonicotinamide (H1-3) was discovered as a hSIRT1 inhibitor through virtual screening of in-house database. Based on these h

Full text of DOI:10.1016/j.bmcl.2012.03.030

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3256–3260
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel acridinedione derivatives: Design, synthesis, SIRT1 enzyme and tumor
cell growth inhibition studies
Mallika Alvala a, Shubhmita Bhatnagar a, Alvala Ravi b, Variam Ullas Jeankumar a,
Thimmappa H Manjashetty a, Perumal Yogeeswari a, Dharmarajan Sriram a,
⇑
a Drug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology and Science, Pilani-Hyderabad Campus, Hyderabad 500 078, India
b G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad 500 028, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new scaffold N-(9-(ortho/meta/para-(benzyloxy)phenyl)-3,3,6,6-tetramethyl-1,8-dioxo-1,2,3,4,5,6,7,8-
octahydroacridin-10(9H)-yl) isonicotinamide (H1-3) was discovered as a hSIRT1 inhibitor through virtual
screening of in-house database. Based on these hits, a library of compounds were designed, synthesized
and tested for in vitro hSIRT1 activity. The most potent compound 4d in the series showed a significant
inhibition of SIRT1 activity. Further antitumor studies of compound 4d, showed a dose dependent
Received 9 January 2012
Revised 27 February 2012
Accepted 7 March 2012
Available online 13 March 2012
increase in acetylation of p53K382 and decrease in SIRT1 with an IC50 of 0.25 lM in MDA-MB231 breast
Keywords:
cancer cell lines. Individual 3D-QSAR analysis using Schrödinger showed distribution of hydrophobic and
non polar positive co-efficient at ortho position essential for bioactivity based on 4d.
Ó 2012 Elsevier Ltd. All rights reserved.
Antiproliferative activity
Acridinedione derivatives
hSIRT1
MDA-MB231
SIRT1 also known as NAD-dependent deacetylase sirtuin-1, the
closest mammalian homolog of yeast Sir2, belongs to the family of
the class III histone deacetylase. The complicated interaction loops
established between human sirtuins and various critical cellular
pathways suggest them to be possible drug targets.1–5 Several lines
of evidence have indicated that SIRT1 deacetylates not only the ly-
sine residues of histones, preferentially H3 and H46 but also non
histone proteins. It has been found to be up-regulated in many tu-
mor types and are able to deacetylate various substrates including
p53,7–9 BCL6,10 HIV Tat,11 PGC-1a,12,13 AceCS1,14 Ku70, Smad7, and
NF-kB15 and Rb16 which are associated with various disease condi-
tions such as cancer and HIV.17 Therefore, SIRT1 inhibitors are of
interest as potential therapeutic agents.4 Several inhibitors of
SIRT1 have been reported,18 including nicotinamide,18 EX-527 ana-
logues,19 splitomicin analogues20–22 sirtinol analogues,23 cambi-
nol,10 2-anilinobenzamides,24 aristoforin25and various other
compounds reported26–29 (Fig. 1). The first synthetic inhibitor that
was discovered is sirtinol.30 In the present study we describe the
synthesis and structural characterization of a series of acridinedi-
one derivatives as well as their hSIRT1 inhibitory activity sup-
ported with in silico, biochemical and in vitro cell-proliferation
assay.
using PRIME homology modeling program (Schrödinger L.L.C.,
USA) as described previously31 and the fitness of the model was
checked by PROCHECK program.32 This model has 83.7% residues
in most favored regions and ProSA-Web Z score of ꢀ6.38 similar
to the template structure scores. Binding site pocket was identified
by using SITEMAP (Schrödinger L.L.C., USA). With an aim of identi-
fying novel hSIRT1 inhibitors, virtual screening of in-house data-
base of 2500 molecules was carried out by using GLIDE
(Schrödinger L.L.C., USA), GOLD and AUTODOCK 4.0 software’s
against catalytic core of hSIRT1. Based on docking score, binding
energy and fitness values compounds H1, H2, and H3 were selected
for (Fig. 2) in vitro enzymatic assay at 50 lM concentration along
with reference compound suramin. Binding energy, docking score,
fitness and percentage inhibition values are presented in Table 1.
Further to explore the structure–activity relationship of hit
compounds (Fig. 2) as SIRT1 inhibitors, different derivatives were
designed by computational and medicinal chemistry approaches.
Docking studies for the designed compounds were performed by
using three different softwares (Schrödinger, GOLD, and Autodock
4.0). Docking score, fitness values and binding affinity values of all
the designed molecules along with reference compound suramin
are shown in Table 2. The designed compounds having better
scores than the initial hit compounds (H1-3), were derivatized by
using a one pot synthetic protocol previously reported by our
group.33
Since the crystal structure of human SIRT1 is not available, the
three dimensional model of catalytic domain of the hSIRT1
(Q96EB6; 244–498 AA) was developed by comparative modeling
The target molecules (4a–r) were synthesized via a microwave
assisted one-pot three component reaction (Scheme 1) of either
ortho, meta or para substituted benzyloxy benzaldehydes (1a–c),
⇑
Corresponding author. Tel.: +91 40 66303506; fax: +91 40 66303998.
E-mail address: drdsriram@yahoo.com (D. Sriram).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.030

M. Alvala et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3256–3260
3257
Figure 1. Known sirt1 inhibitors.
The results of virtual screening were validated by invitro screen-
ing of the best hits (H1-3) against hSIRT1 activity. Among them H1
showed 72.63% inhibition at 50
icantly active. Based on these result we further generated a series
of compounds and tested against hSIRT1 activity at 50 M using
lM and H2 and H3 were not signif-
l
the Fluor-de-Lys peptide substrate from Cayman chemicals
(USA). Compounds showing more than 50% inhibition were se-
lected for IC50 calculation. Compound 4a and 4d were found to
be the most potent inhibitors compared to reference compound
suramin with an IC50 of 11 0.02 lM, and 10 0.08 lM, respec-
tively. Others like 4b, 4c, 4f, 4i, 4l, and 4n were found to be better
inhibitors than suramin and the remaining compounds showed no
inhibition. The percentage inhibition and IC50 values are presented
in Table 2. The higher inhibitory activity of compounds 4a and 4d
might be attributed to the presence of benzyloxy phenyl substitu-
tion at ortho position and the presence of phenyl and 2,4 dinitro-
phenyl groups. Binding pose of most active compound 4d
(Fig. 3A and B) clearly demonstrated the orientation of ortho ben-
zyloxy group in the hydrophobic groove of the protein (red-hydro-
phobic; blue-hydrophilic).
Figure 2. In-house database hit compound.
It was observed that substituent at C9 (–R position) of aminoac-
ridine dione dictates the bioactivity, wherein introduction of bulky
substituents like benzoyl or 4-nitrobenzoyl or 2,4-dichlorobenzoyl
or 2,6-dimethoxynicotionoyl or shift of benzyloxy substituent (at
R’ position)on phenyl ring of aminoacridine dione to meta position
showed either reduced or no inhibition (4b, 4e, 4h, 4k, 4n, 4q).
However shift of benzyloxyphenyl substitution to para position re-
sulted in moderate inhibition of enzyme in analogues 4c, 4f, 4i, and
4r.
Over expression of SIRT1 is observed in many cancers and inhi-
bition of SIRT1 by small molecules demonstrated inhibition of can-
cer cell proliferation.35–37 To investigate the effectiveness of these
SIRT1 inhibitors as anticancer agents, compounds 4a, 4b, 4c, 4d, 4f,
4i and 4r were evaluated for their ability to inhibit growth of tu-
5,5-dimethylcyclohexane-1,3-dione (2), and corresponding phenyl
hydrazine’s or aryl/heteroaryl acid hydrazides (3a–f) in the ratio
1:2:1 in dry DMF media with catalytic amount of HCl. The use of
microwave condition’s in the present protocol not only improved
the yield, but also reduced the reaction time drastically to 5–
8 min compared to conventional heating which took about 3–8 h.
Both analytical and spectral data (1H NMR, 13C NMR, and mass
spectra) of all the synthesized compounds were in full agreement
with the proposed structures.34
To explore the scope and limitations of these conditions a series
of the six simplified derivatives, (4a–r) (Table 2) were prepared by
introduction of either unsubstituted phenyl/benzoyl derivatives or
with various electron withdrawing/donating groups like 2,4-dini-
trophenyl or 4-nitrobenzoyl or 2,4-dichlorobenzoyl or 2,6-dimeth-
oxynicotionoyl group at C9 (R position) and also with either ortho,
meta or para benzyloxy substituent on C9 phenyl ring of aminoac-
ridine dione [3,4,6,7-tetrahydro-3,3,6,6-tetramethyl-10-amino-9-
phenyl acridine—1,8(2H,5H,9H,10H)—dione]. As apparent from
the Table 2, the compounds were found to react well to give the
corresponding amino acridinedione in excellent yields (64–84%).
mor cells at 10 lM compared with suramin as standard SIRT1
inhibitor and doxorubicin as a standard anticancer drug. Human
chronic myeloid leukemia (K562) cells and metastatic breast can-
cer (MDA-MB231) cells were used as tumor cells and HEK 293 as
control cells. Percentage inhibition of 4a, 4b, 4c, 4d, 4f, 4i and 4r
at 10 lM can be found in Figure 4. As shown in Table 2, all com-
pounds showed better growth inhibition except 4a in MDA-
MB231 cells compared to K562 cells. There is no significant growth

3258
M. Alvala et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3256–3260
Table 1
Docking scores, % inhibition of hsirt1 and cell growth inhibition at 50
l
M concentration of hit compounds
R0
Autodock score (Kcal/mol)
Gold Fitness
GLIDE Score
% inhibition of hSIRT1 at 50
lM
MDA-MB231 (IC50
lM)
K562 (IC50 lM)
H1
H2
H3
Suramin
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
ꢀ6.79
ꢀ7.46
ꢀ7.68
ꢀ4.67
52.50
57.84
55.80
45.7
ꢀ4.92
ꢀ3.41
ꢀ3.76
ꢀ3.33
72.63343
53.67
No inhibition @50
82.28
5
10
10
>50
18.49
>50
>50
31.19
lM
Table 2
Docking scores, physical constants and in vitro inhibition studies. (— indicates not determined)
No
R
R0
Yield (%) % inhibition of
Glide Docking Gold fitness Auto dock SIRT1_IC50
Score
(
l
M) MDA MB231 IC50
hSIRT1 at 50
l
M
(lM)
4a
4b
4c
4d
4e
4f
Phenyl
Phenyl
Phenyl
2,4-Dinitrophenyl
2,4-Dinitrophenyl
2,4-Dinitrophenyl
Benzoyl
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
2-Benzyloxy
3-Benzyloxy
4-Benzyloxy
84
75
64
85
65
75
68
75
80
77
73
65
71
70
69
69
79
75
—
82.1
51.4
55.2
83.2
33.2
54.8
39.7
21.5
52.0
39.0
33.6
35.6
ꢀ19.7
4.7
ꢀ3.53
ꢀ6.09
ꢀ3.36
ꢀ4.28
—
45.1
51.1
48.0
57.6
—
55.4
—
—
51.9
—
—
—
—
—
—
—
—
42.8
45.7
—
ꢀ8.85
ꢀ8.95
8.54
ꢀ10.84
—
11.08 0.02
40.06 0.72
43.14 0.1
10.13 0.08
50
0.99
1.38
0.25
—
6.63
—
—
3.16
—
—
—
—
—
ꢀ5.19
ꢀ8.52
43.29 0.49
—
—
4g
—
—
4h Benzoyl
—
—
4i
4j
4k
4l
Benzoyl
ꢀ5.90
ꢀ7.84
45.97 0.71
4-Nitrobenzoyl
4-Nitrobenzoyl
4-Nitrobenzoyl
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
ꢀ3.08
ꢀ3.33
—
—
—
—
—
—
—
—
ꢀ9.23
ꢀ4.67
—
4m 2,4-Dichlorobenzoyl
4n 2,4-Dichlorobenzoyl
4o
4p
4q
4r
—
—
—
—
2,4-Dichlorobenzoyl
3.6
2,6-Dimethoxynicotionoyl 2-Benzyloxy
2,6-Dimethoxynicotionoyl 3-Benzyloxy
2,6-Dimethoxynicotionoyl 4-Benzyloxy
Suramin
Doxorubicin
35.2
35.6
55.1
53.9
—
25.018 0.02
2.8
—
0.31
—
—
—
10
R'
O
CHO
1a-c
O
O
i)
+
2
RNHNH2
3a-f
R'
+
O
N
NH-R
2
4a-r
Scheme 1. Synthesis of compounds. Reagents and conditions: (i) HCl, Dry DMF, MWI, 5–8 min.
Figure 3. (A) Docking pose of most active compound 4d. (B) Ligand interaction diagram showing important amino acids with in 5A0. Dotted line represents hydrogen bond to
the side chain. Blue-charged positive, green-Hydrophobic group, Yellow-Solvent exposure, Red-acidic group, purple-basic, gray-other groups.

M. Alvala et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3256–3260
3259
Figure 4. (A) Graph showing % inhibition on K562, MDA-MB231 cells. (B) Graph showing % growth on HEK 293 cells.
Table 3
The QSAR results table shows the statistics of the fit for the training set and test set
Activity(lM)
Predicted activity
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4.955
4.398
4.365
4.994
4.301
4.364
4.301
4.301
4.338
4.301
4.301
4.301
4.301
4.355
4.301
4.301
4.301
4.602
4.78347
4.5601
4.54989
4.73862
4.3524
4.313
4.3354
4.289
4.22116
4.35061
4.36279
4.26662
4.28422
4.30994
4.2141
4.56794
4.42566
4.46849
Figure 5. Western blot analysis of MDA-MB231 cells treated with compound 4d at
M, 0.1, 0.25 and 0.5 M concentrations. (A) A representative Western blot
0
l
l
showing the dose dependent decrease of expression levels of SIRT1. (B) Inhibition of
SIRT1 by compound 4d resulted in increased Acp53levels dose dependently. (C) b-
actin was used as control.
Figure 6. (A) QSAR visualization of most active compound 4d. Cubes at that position shows positive threshold effect. Red-Hydrogen bond donor; Cyan-Hydrophobic groups;
Green-Negative ionic; Yellow-Positive ionic; Pink-electron withdrawing groups. (B) QSAR of most active compound 4d in which benzyloxy is present at ortho, meta and para
positions (4d, 4e, 4f).
inhibition on HEK 293 cells indicating the specificity of these com-
pounds towards cancer cells. Further, IC50 values were calculated
on MDA-MB231 cells (Table 2). The compound 4d emerged as
the most promising inhibitor in MDA-MB231 cells with an IC50 of
250 nM. These results suggested that acridinedione derivatives
bearing the benzyloxy substitution at ortho position are potential
antitumor agents.
To investigate further the distinct effects of the compound 4d as
SIRT inhibitor, on cell fate, we performed Western blot analysis on
MDA-MB231 cells treated with compound 4d at different concentra-

3260
M. Alvala et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3256–3260
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Interestingly compound 4d has shown dose dependent decrease in
SIRT1 (Fig. 5A) and increase in acetylation of p53 at lysine 382
(Fig. 5B). These results are in accordance with the previous results.35
3D-QSAR model was developed by phase module of Schröding-
er. All dataset compounds were divided into a training set of 80%
and a representative test set of 20%. Individual QSAR model of
phase was used for construction of the simplest hypotheses
that best correlates the experimental and predicted activities
(Table 3). Statistically best QSAR model was generated with a
R2 = 0.6283, F = 20.3, SD = 0.1506, RMSE = 0.08 and Q2 = 0.6278.
For the highly active compound 4d, the QSAR feature is
shown in Figure 6A. Accumulation of highest positive coefficient
(1.1 ꢁ 10ꢀ3)threshold of cyan colored hydrophobic or non polar
cubes at the ortho position of 4d infers the importance of
substitution with hydrophobic moieties at ortho position rather
than para and meta positions as shown in Figure 6B.
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NMR (300 MHz, CDCl3): d = 0.92 (s, 6H, 2⁄CH3), 0.99 (s, 6H, 2 ⁄CH3) 1.92 (d,
J = 9.1 Hz, 2H), 2.07 (d, J = 9.3 Hz, 2H,), 2.41 (dd,, JGem = 8.9 Hz, 4H, (2⁄CH2)H2,
H7), 4.84 (s, 1H, H9), 5.2 (s, 2H,–O– CH2) 6.7–6.96 (m, 4H, ArH of phenyl at C9),
7.2–7.4 (m, 5H, ArH of benzyl), 8.4-9.3(m, 3H, ArH of benzoyl), 8.2 (s, 1H, NH);
13C NMR (75 MHz, CDCl3): d = 195.6, 163.8, 158.5, 153.5, 147.6, 143.2, 141.2,
134.2, 129.8, 128.3, 127.7, 127.0, 126.5, 121.1, 120, 116.4, 112.8, 111, 73.16,
52.3, 38.7, 32, 30.3, 27.1; EI-MS m/z 665.3 (M+H)+;Anal calcd. for C37H36N4O8:
C, 66.86; H, 5.46; N, 8.43. Found: C, 66.91; H, 5.41; N, 8.47.
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Acknowledgments
The authors are thankful to the Department of Biotechnology
(BT/PR13326/BRB/10/719/2009), Government of India, for their
financial assistance. We thank Dr. Srikanth Viswanadha and
Sumalatha from Incozen Therapeutics (P) Ltd for providing cell cul-
ture facilities.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.030.
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