Benzodeazaoxaflavins as sirtuin inhibitors with antiproliferative properties in cancer stem cells

Article abstract of DOI:10.1021/jm301115r

Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence.

Full text of DOI:10.1021/jm301115r

Brief Article
pubs.acs.org/jmc
Benzodeazaoxaflavins as Sirtuin Inhibitors with Antiproliferative
Properties in Cancer Stem Cells
Dante Rotili,† Domenico Tarantino,† Vincenzo Carafa,‡ Chantal Paolini,§ Jorg Schemies,∥ Manfred Jung,∥
̈
Giorgia Botta,† Salvatore Di Maro,⊥ Ettore Novellino,⊥ Christian Steinkuhler,§ Ruggero De Maria,#
̈
Paola Gallinari,§ Lucia Altucci,‡,▽ and Antonello Mai*,†
†Istituto Pasteur−Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
̀
di Roma,
Piazzale Aldo Moro 5, 00185 Roma, Italy
‡Dipartimento di Patologia Generale, Seconda Universita
̀
degli Studi di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy
§Exiris s.r.l., via Castelfidardo 8, Rome, Italy
∥Institut fur Pharmazeutische Wissenschaften, Albert-Ludwigs-Universitat Freiburg, Albertstrasse 25, 79104 Freiburg, Germany
̈
⊥Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli,
Italy
#Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy
▽CNR-IGB, Institute of Genetics and Biophysics, via P. Castellino, 80100 Napoli, Italy
S
* Supporting Information
ABSTRACT: Inhibition of sirtuins has recently been proposed as a promising anticancer strategy. Some of the new
benzodeazaoxaflavins (2a, 2b, and 2d) here reported as SIRT1/2 inhibitors were endowed with pro-apoptotic properties in
human U937 leukemia cells and, most importantly, together with the prototype MC2141 (1) displayed antiproliferative effects in
cancer stem cells from patients with colorectal carcinoma and glioblastoma multiforme, known to be highly tumorigenic, resistant
to conventional cancer chemotherapy, and responsible, at least in part, for cancer relapse or recurrence.
proposed for the treatment of some age-related diseases and
specific metabolic disorders.1−3 SIRTs (SIRT1/3/7) have been
found to be upregulated in many tumor types (such as
leukemia, lung, and prostate cancer) and are able to inactivate
at transcriptional and posttranslational level some tumor
suppressor proteins such as p53 and p73 (SIRT1). In addition,
sirtuins have been shown to activate the oncoprotein BCL6
(SIRT1) to exert antiapoptotic and antidifferentiation activities
(SIRT1) through deacetylation of specific transcription factors
(E2F1, FOXO3a, etc.) and to regulate DNA repair (SIRT1/6),
cell cycle progression, and chromosomal stability (SIRT1/2).
Thus, sirtuin inhibitors (SIRTi) have been proposed as
potential anticancer agents.5 However, the role of SIRTs in
tumor progression is controversial, as these enzymes are
downregulated in some cancer types and have potential tumor
suppressor effects (SIRT1−3).2,6 Because heterogeneous SIRT
levels have been observed in subsets of tumors, it is conceivable
that SIRTs display tumor suppressor or oncogenic functions
depending on genetics and stage of the tumor and on the
varying cellular environments and signals.2,6 To date, only a few
SIRT1/2 inhibitors have been validated in cancer. Sirtinol was
reported to induce senescence-like growth arrest in human
breast cancer MCF-7 cells and lung cancer H1299 cells,7
cambinol induced apoptosis in BCL6-expressing Burkitt
lymphoma cells and mouse xenografts,8 salermide, recently
INTRODUCTION
■
Class III histone deacetylases (HDACs), also indicated as
sirtuins (SIRTs), are a family of seven enzymes in humans
(SIRT1−7) which have NAD+ as cosubstrate for their catalytic
activity, show no homology with other classes of HDACs, and
are sensitive to specific modulators.1 Sirtuins catalyze the
removal of the acetyl group from the lysine residues of their
substrates, coupling this event with the hydrolysis of NAD+ to
generate nicotinamide, deacetylated protein, and O-acetyl-
ADP-ribose.1 Mono-ADP-ribosyl transferase (mART) activity
has been found to be the only enzymatic activity of SIRT4,
whereas SIRT1−3 and SIRT6 display both deacetylase (strong)
and mART (weak) activities.1,2 In addition to histones, SIRTs
can deacetylate and/or ADP-ribosylate with different substrate
specificities many other important proteins, such as tran-
scription factors, enzymes, nuclear receptors, and other
regulatory and structural proteins, so modulating their
activity.1−3 Very recently, SIRT5 has been found to be able
to catalyze lysine demalonylation and lysine desuccinylation
both in vitro and in vivo.4
These data can explain the multiple biological functions of
sirtuins that range from repression of gene expression to
regulation of circadian rhythm, DNA repair, cellular differ-
entiation, and/or apoptotic processes, from control of energetic
metabolism to aging.1−3 Moreover, because these enzymes in
humans regulate cell survival under stress conditions as well as
neuro/cardio protection, stimulate insulin secretion and
lipolysis, and inhibit adipogenesis, sirtuin activators have been
Received: July 31, 2012
Published: August 30, 2012
© 2012 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
reported by us, showed tumor-specific apoptosis in a wide
range of human cancer cell lines,9 and tenovins decreased
tumor growth in vivo as single agents at low micromolar
concentrations (Chart 1).10 Very recently, tenovin 6 in
The effects of the novel compounds on cell cycle, apoptosis,
and granulocytic differentiation in U937 cells have also been
explored. Selected benzodeazaoxaflavins (1, 2a, 2b, and 2d)
have been finally tested on four cancer stem cell (CSC) lines
for their effects on cell proliferation/survival. CSCs are a cancer
cell subset highly tumorigenic, highly resistant to conventional
cancer therapies, and supposed to play a crucial role in cancer
relapse or recurrence.21,22
Chart 1. Structures of Known SIRT Inhibitors (SIRTi)
CHEMISTRY
■
The synthetic routes followed for the preparation of 2a−g and
3a−d are depicted in Scheme 1. The 10-substituted-9H-
Scheme 1
combination with the BCR-ABL tyrosine kinase inhibitor
imatinib was shown to effectively inhibit leukemia stem cells in
chronic myelogenous leukemia (CML).11 Differently, the
SIRT1-selective inhibitor EX-52712 (Chart 1) efficiently
increased p53 acetylation in cells without effects on cell
viability, or p53-controlled gene expression.13
Pursuing our efforts for identification of novel small molecule
sirtuin modulators,9,14−19 we recently identified the 10-phenyl-
9H-benzo[5,6]chromeno[2,3-d]pyrimidine-9,11(10H)-dione
MC2141 (1) as the prototype of a series of benzodeazaoxa-
flavins (BDF4s) as SIRTi endowed with significant inhibitory
potency and high antiproliferative activity against Raji, DLD1,
and HeLa cells.17 Here we report the preparation and the
biological evaluation of novel simplified analogues (2a−g, 3a−
d) of 1 aimed to improve its solubility and drug-like properties,
to increase its pharmacodynamic properties, and to acquire
structure−activity relationship (SAR) information (Figure 1).
benzo[5,6]chromeno[2,3-d]pyrimidine-9,11(10H)-dione deriv-
atives 2a−g were prepared by condensation of the appropriate
N-alkylbarbituric acids, synthesized by standard methods,17
with 2-hydroxy-1-naphthaldehyde in dry ethanol at reflux
temperature for 1 h. The 3-substituted-2H-chromeno[2,3-
d]pyrimidine-2,4(3H)-dione compounds 3a−d were obtained
by condensation of the corresponding barbituric acids with o-
chlorobenzaldehyde in ethanol dry in the presence of a catalytic
amount of pyridine followed by the intramolecular dehydroha-
logenation of the crude mixture of E and Z 5-(2′-
chlorobenzylidene)barbituric acid intermediates by heating
them in an oven at 240−260 °C without solvent for 0.5 h.
The reference compounds 1 and cambinol have been prepared
as previously reported by us.17,18 EX-527 has been synthesized
according to literature,12 while AGK-2 was purchased from
Sigma-Aldrich.
Figure 1. Novel MC2141 (1) analogues described in the present
study.
RESULTS AND DISCUSSION
■
The IC50 (inhibitory concentration 50, compound dose
required to inhibit the enzyme activity of 50%) values for the
novel compounds 2a−g and 3a−d against human recombinant
(hr) SIRT1 and SIRT2 were determined using a fluorescent
biochemical assay. As shown in Table 1, the majority of the
newly synthesized compounds displayed SIRT1/2 inhibition
with IC50 values in the low micromolar range. In particular, the
introduction of a methyl or allyl substituent at the N-10 of the
tetracyclic scaffold led to compounds (2a,d) more potent than
the parent compound 1 against both the enzymes, while an
ethyl or cyclohexyl group at the same position (2b,f) caused a
decrease of the inhibitory activities of the derivatives, mainly
against SIRT2. In the series of the tricyclic deazaoxaflavins 3a−
d, all the compounds retained inhibitory potency against SIRT1
Because the previously reported N-unsubstituted analogue of
1 had shown similar inhibitory potency against SIRT1 as 1,
suggesting that the 10-phenyl substitution is not crucial for
SIRT inhibition,17 we introduced different (aryl)alkyl sub-
stituents at the N-10 position (2a−g), and we simplified the
tetracyclic scaffold of the BDF4 prototype 1 by removal of the
condensed benzene ring and by preparing a small series (3a−d)
of different 3-substituted-2H-chromeno[2,3-d]pyrimidine-
2,4(3H)-diones (also indicated as 5-deaza-10-oxaflavins)
(Figure 1). All the synthesized compounds (2a−g and 3a−d)
were tested for their ability to inhibit the human recombinant
SIRT1 and SIRT2 enzymes together with 1, cambinol, AGK-2
(a SIRT2-selective inhibitor),20 and EX-527 as reference drugs.
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Journal of Medicinal Chemistry
Brief Article
Table 1. Inhibitory Activity of 1−3 against Human
a
Recombinant (hr) SIRT1 and SIRT2
IC50, μM
compd
R
hrSIRT1
9.8 0.3
hrSIRT2
1
Ph
Me
Et
12.3 0.5
11.2 0.3
30.0 1.2
12.5 0.5
10.8 0.3
16.9 0.8
58.5 2.9
15.9 0.6
24.6 1.0
22.5 0.9
29.9 1.5
30.8 1.5
42.1 2.1
5.5 0.2
2a
7.0 0.2
2b
20.2 0.8
9.4 0.3
2c
n-Pr
2d
Allyl
n-Bu
c-Hex
CH2−Ph
Me
6.6 0.1
2e
10.4 0.4
21.1 1.0
12.0 0.4
10.5 0.3
11.8 0.3
12.5 0.5
14.1 0.6
38% @ 150 μM
38.0 1.5
0.16 0.008
2f
2g
3a
3b
Et
3c
n-Pr
3d
n-Bu
cambinol
AGK-2
EX-527
48.5 1.9
a
Values are the means of two determinations.
comparable to the parent compound 1 with a slight decrease of
activity along with the increase of the size of the alkyl
substituent (from Me to n-Bu), and, with the only exception of
3b, they were about 2-fold weaker than the corresponding
tetracyclic counterparts against both SIRT1 and SIRT2. In the
previous study, the lead 1 showed 8.4 and 191.2 μM inhibiting
activities against SIRT1 and SIRT2, respectively.17 Differently,
under the present assay conditions, 1 resulted in activity against
SIRT2 with IC50 values in the low μM range, too. Such SIRT2
inhibiting activity by 1 was also confirmed using a fluorescent
acetylated lysine derivative, Z-MAL, that is widely employed for
SIRT inhibition assays,23 (see Supporting Information (SI),
Table S3).
The novel SIRTi 2a−g and 3a−d were tested at 50 μM for
30 h in the human U937 leukemia cell line to detect their
effects on cell cycle progression and apoptosis induction
(Figure 2). Cambinol, EX-527, and 1 (all at 50 μM) were
added as reference drugs. After the treatment, most of
compounds 2 and 3 (in particular 2f and 3a−d) elicited cell-
cycle arrest at the G1 phase (Figure 2A). The FACS analysis for
apoptosis indicated that all the novel derivatives 2 and 3 were
able to induce programmed cell death more efficiently than EX-
527 and cambinol (CMB), with compounds 2a, 2b, and 2d
being the most effective (18.8%, 23.5%, and 19.6% of apoptosis,
respectively, Figure 2B) and even stronger than 1.
Figure 2. Cell cycle effect (A) and apoptosis induction (B) of 2a−g
and 3a−d (50 μM, 30 h) in U937 cells. Values are the means of three
experiments.
the assay in order to achieve information about the effect of
isoform-selective SIRTi in CSCs. Two colorectal carcinoma
(CRC) CSCs (CRO and 1.1 cells) and two glioblastoma
multiforme (GBM) CSCs (30P and 30PT cells) isolated from
patients were chosen for this assay. After 72 h of treatment, the
CC50 (cytotoxic concentration able to kill 50% of the cells)
values were determined and are reported in Table 2. The
relative dose−response curves are reported in SI (Figure S2).
Against the tested CSCs, the SIRT1-selective inhibitor EX-
527 failed to produce significant growth inhibition at 50 μM,
whereas the SIRT2-selective inhibitor AGK-2 displayed good
antiproliferative activity only against GBM CSCs. In contrast,
compounds 1 and 2d under the same conditions were highly
Table 2. Cell Viability Inhibition by 1, 2a, 2b, and 2d in CRC
a
and GBM CSCs
CC50, μM
CRC CSCs
GBM CSCs
compd
CRO
1.1
30P
30PT
In addition, granulocytic differentiation was evaluated for 2
and 3 (50 μM, 30 h) in U937 cells by the increase of % CD11c
positive/propidium iodide (PI) negative cells. At the tested
conditions, a significant induction of granulocytic differ-
entiation was not detected (see SI).
Because of their pro-apoptotic properties, we selected 2a, 2b,
and 2d in addition to the lead 1 for evaluating their potential in
the inhibition of cell viability of CSCs. We also added SIRT1-
selective (EX-527) and SIRT2-selective (AGK-2) inhibitors to
1
7.0 0.6
75.4 11.9
23.6 2.6
9.6 0.5
5.5 0.7
33.5 10.7
14.9 2.1
8.5 0.4
4.9 0.2
34.2 4.6
15 1.7
6.6 1.2
3.9 0.7
33.4 2.8
15.6 6.0
5.0 3.8
2a
2b
2d
b
b,
c
b
b
EX-527
AGK-2
20%
NI
10%
20%
b
b
50%
50%
12.5 0.5
9.6 1.0
a
b
c
Values are the means of three experiments. Inhibition at 50 μM. NI,
no inhibition.
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Journal of Medicinal Chemistry
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polycyclic system), 7.73 (m, 1H, C8-H polycyclic system), 7.92 (m,
1H, C9-H polycyclic system), 8.12 (m, 1H, C6-H polycyclic system),
9.01 (s, 1H, C5-H polycyclic system). 13C NMR (DMSO) δ 11.8, 38.5,
112.4, 114.2, 116.3, 121.2, 128.7, 128.9, 129.3, 154.3, 158.5, 160.0,
160.4 ppm. Anal. (C13H10N2O3) C, H, N. MS, m/z: 243 [M + H]. The
chemical and physical data of the other compounds of general formula
3 are reported in SI.
SIRT1/2 Inhibition Assays. See SI.
Cell Lines. See SI.
Cell Cycle and Cell death Analysis on U937 cells. See SI.
efficient in reducing the viability of the four CSC lines tested,
bearing CC50 values in the single-digit μM range. These effects
seem to be quite sensitive to the substitution on the N-10
position of the tetracyclic system because the N-Me analogue
2a showed a drop of antiproliferative activity of about 6−10-
fold (depending on the specific cell line) in comparison with
the lead 1, and its N-Et counterpart 2b exhibited a 3-fold lower
potency in all the four CSC lines tested. Maybe a quite large,
unsaturated substituent at N-10 seems to improve the cell
penetration of the corresponding derivatives 1 and 2d.
Cell Viability Inhibition in Cancer Stem Cells (CSCs): Cell
culture. CSC cultures were established and propagated under
spherogenic conditions as described.24,25 Cells were grown for 20−
30 days in CSC serum-free medium containing epidermal growth
factor (EGF) and basic fibroblast growth factor (bFGF) and
maintained at a concentration of 50000−200000 cells/mL. Medium
was replaced every three−five days with fresh medium complemented
with 25% conditioned medium, depending on the growth rate and
viability of each cell line. Cell viability assay. Spheroid cultures were
enzymatically dissociated in Accumax Reagent (Sigma-Aldrich), cell
density was determined, and single cell suspensions were dispensed
into 96-well black microplates with clear, flat bottoms. Initial densities
of 2000−3000 viable cells were chosen to be within the linear range of
the assay at the chosen time point for all CSC lines. After plating, cells
were incubated at 37 °C in 5% CO2 for 24 h and then were treated
with 1:3 serial dilutions of each compound (eight concentration points
over a 0.023−50 μM concentration range) or with 0.1% DMSO as
negative control. Three replicates for each experimental point were
included. Cells were incubated at 37 °C in 5% CO2 for 72 h, and then
cell viability was determined using the CellTiter-Glo luminescent cell
viability assay (Promega), essentially by following the manufacturer’s
recommendations. Compound cytotoxicity was evaluated in compar-
ison to cells treated with DMSO. The CC50 values were determined by
4P logistic fitting of the experimental data with Kaleida Graph software
based on the residual luminescence in the presence of increasing
concentrations of the inhibitors.
In conclusion, we have identified a series of low micromolar
SIRT1/2 inhibitors endowed with pro-apoptotic properties in
U937 human leukemia cells and, most importantly, with single-
digit micromolar antiproliferative activity in CSCs from patients
(coloncarcinoma and glioblastoma multiforme CSCs), which
represent one of the most attractive targets of the modern
anticancer therapy. Further studies are presently ongoing to
improve the pharmacological profile of such novel compounds.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. H NMR spectra were
■
1
recorded at 400 MHz on a Bruker AC 400 spectrometer; reporting
chemical shifts in δ (ppm) units relative to the internal reference
tetramethylsilane (Me4Si). All compounds were routinely checked by
1
TLC and H NMR. TLC was performed on aluminum-backed silica
gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots
visualized by UV light. Yields of all reactions refer to the purified
products. All chemicals were purchased from Aldrich Chimica, Milan
(Italy) and were of the highest purity. Mass spectra were recorded on a
API-TOF Mariner by Perspective Biosystem (Stratford, Texas, USA),
samples were injected by an Harvard pump using a flow rate of 5−10
μL/min, infused in the Electrospray system. Elemental analyses were
obtained by a PE 2400 (Perkin- Elmer) analyzer and have been used to
determine purity of the described compounds, that is >95%. Analytical
results are within 0.40% of the theoretical values (see SI).
ASSOCIATED CONTENT
* Supporting Information
■
S
General Procedure for the Synthesis of 10-Substituted-9H-
benzo[5,6]chromeno[2,3-d]pyrimidine-9,11(10H)-diones (2a−
g). Example: 10-Propyl-9H-benzo[5,6]chromeno[2,3-d]-
pyrimidine-9,11(10H)-dione (2c). A mixture of N-propylbarbituric
acid (0.87 g, 5.1 mmol) and 2-hydroxy-1-naphthaldehyde (0.97 g, 5.6
mmol) in dry ethanol (22 mL) was heated at reflux temperature for 1
h. The hot suspension was then filtered off, and the resulting yellow
solid purified by recrystallization from a mixture acetic acid/acetic
Chemical and physical data for compounds 2−3. SIRT1/2
inhibition assays. Cell culture, cell cycle, and cell death
induction analysis on U937 cells. Granulocytic differentiation
investigation on U937 cells. Dose−response curves on CSCs.
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
anhydride (9:1); mp >260 °C; yield 85%. H NMR (DMSO) δ 0.87
AUTHOR INFORMATION
Corresponding Author
*Phone: +3906-4991-3392. Fax: +3906-49693268. E-mail:
antonello.mai@uniroma1.it.
(t, 3H, CH3), 1.58 (m, 2H, CH2CH3), 3.83 (t, 2H, NCH2CH2CH3),
7.72−7.83 (m, 3H, C1−6-H polycyclic system), 8.14 (m, 1H, C1−6-H
polycyclic system), 8.49 (m, 1H, C1−6-H polycyclic system), 8.83 (m,
1H, C1−6-H polycyclic system), 9.57 (s, 1H, C12-H polycyclic system).
13C NMR (DMSO) δ 10.8, 19.9, 46.16, 115.5, 116.3, 117.7, 122.3,
■
Notes
123.6, 126.8, 128.5, 128.7, 128.8, 130.1, 130.3, 150.5, 158.5, 160.0,
160.4 ppm. Anal. (C18H14N2O3) C, H, N. MS, m/z: 307 [M + H]. The
chemical and physical data of the other compounds of general formula
2 are reported in SI.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
General Procedure for the Synthesis of 3-Substituted-2H-
chromeno[2,3-d]pyrimidine-2,4(3H)-diones (3a−d). Example:
3-Ethyl-2H-chromeno[2,3-d]pyrimidine-2,4(3H)-dione (3b). A
mixture of N-ethylbarbituric acid (0.55 g, 3.5 mmol), o-chlorobenzal-
dehyde (0.54 g, 3.9 mmol), and a catalytic amount of pyridine (5
drops) in dry ethanol (14 mL) was heated at reflux temperature for 2
h. The mixture was then evaporated under reduced pressure, the
residue triturated from diethyl ether, and, after filtration, the crude
solid over the filter, without further purification, was heated in an oven
at 260 °C for 0.5 h. After cooling at room temperature, the final solid
was purified by recrystallization from a mixture acetic acid/acetic
anhydride (9:1) to provide the desired product 3b as a yellow solid;
mp 236−238 °C (with decomposition); yield 67%. 1H NMR (DMSO)
δ 1.15 (t, 3H, CH3), 3.88 (t, 2H, NCH2CH3), 7.57 (m, 1H, C7-H
This work was supported by AIRC (L.A.), PON01_01227
(L.A.), PRIN 2009PX2T2E (A.M. and L.A.), FIRB
RBFR10ZJQT (A.M.), and FP7 Project BLUEPRINT/
282510 (A.M. and L.A.). M.J. was supported by the Deutsche
Forschungsgemeinschaft (Ju295/8-1).
ABBREVIATIONS USED
■
HDAC, histone deacetylase; SIRT, sirtuin; FoxO, forkhead box
class O; NAD+, nicotinamide adenine dinucleotide; mART,
mono-ADP-ribosyl transferase; NF-kB, nuclear factor-kappaB;
BCL-6, B-cell lymphoma 6; BDF4s, benzodeazaoxaflavins;
CMB, cambinol; FACS, fluorescence-activated cell sorting;
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Fraga, M. F.; Altucci, L.; Mai, A. Identification of tri- and tetracyclic
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CSCs, cancer stem cells; CRC, colorectal carcinoma; GBM,
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