Discovery of salermide-related sirtuin inhibitors: Binding mode studies and antiproliferative effects in cancer cells including cancer stem cells

Article abstract of DOI:10.1021/jm3011614

Chemical changes performed on 1a (sirtinol) led to a series of SIRT1/2 inhibitors, in some cases more potent than 1a mainly against SIRT1. Tested in human leukemia U937 cells, the benzamide and anilide derivatives 1b, 1c, 2b, and 2c as well as the 4-(2-phenylpropyl)thioanalogue 4c showed huge apoptosis induction, while some sulfinyl and sulfonyl derivatives (5b, 5c, and 6a-c) were highly efficient in granulocytic differentiation. When assayed in human leukemia MOLT4 as well as in human breast MDA-MB-231 and colon RKO cancer cell lines, the anilide 2b (salermide) and the phenylpropylthio analogue 4b emerged as the most potent antiproliferative agents. Tested on colorectal carcinoma and glioblastoma multiforme cancer stem cells (CSCs) from patients, 2b was particularly potent against colorectal carcinoma CSCs, while 4b, 6a, and the SIRT2-selective inhibitor AGK-2 showed the highest effect against glioblastoma multiforme CSCs. Such compounds will be further explored for their broad-spectrum anticancer properties.

Full text of DOI:10.1021/jm3011614

Article
pubs.acs.org/jmc
Discovery of Salermide-Related Sirtuin Inhibitors: Binding Mode
Studies and Antiproliferative Effects in Cancer Cells Including Cancer
Stem Cells
Dante Rotili,† Domenico Tarantino,† Angela Nebbioso,‡ Chantal Paolini,§ Covadonga Huidobro,∥
Ester Lara,⊥ Paolo Mellini,†,# Alessia Lenoci,† Riccardo Pezzi,† Giorgia Botta,† Maija Lahtela-Kakkonen,#
Antti Poso,# Christian Steinkuhler,§ Paola Gallinari,§ Ruggero De Maria,▽ Mario Fraga,∥,⊥
̈
Manel Esteller,○,◆,¶ Lucia Altucci,‡,+ and Antonello Mai*,†
†Istituto PasteurFondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Universita
̀
degli Studi di Roma
“La Sapienza”, P.le A. 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
∥Cancer Epigenetics Laboratory, Instituto Universitario de Oncología del Principado de Asturias (IUOPA), HUCA, Universidad de
Oviedo, Oviedo, Spain
⊥Department of Immunology and Oncology, National Centre for Biotechnology (CNB-CSIC), Cantoblanco, Madrid, Spain
#School of Pharmacy (Pharmaceutical Chemistry), University of Eastern Finland, Kuopio Campus, P.O. Box 1627, 70211 Kuopio,
Finland
▽Regina Elena National Cancer Institute, Via Elio Chianesi 53, 00144 Rome, Italy
○Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908 L’Hospitalet,
Barcelona, Catalonia, Spain
◆Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain
¶Institucio
́
Catalana de Recerca i Estudis Avanca̧ ts (ICREA), Barcelona, Catalonia, Spain
+CNR-IGB, Institute of Genetics and Biophysics, via P. Castellino, 80100, Napoli, Italy
S
* Supporting Information
ABSTRACT: Chemical changes performed on 1a (sirtinol) led to a series of
SIRT1/2 inhibitors, in some cases more potent than 1a mainly against
SIRT1. Tested in human leukemia U937 cells, the benzamide and anilide
derivatives 1b, 1c, 2b, and 2c as well as the 4-(2-phenylpropyl)thioanalogue
4c showed huge apoptosis induction, while some sulfinyl and sulfonyl
derivatives (5b, 5c, and 6a−c) were highly efficient in granulocytic
differentiation. When assayed in human leukemia MOLT4 as well as in
human breast MDA-MB-231 and colon RKO cancer cell lines, the anilide 2b
(salermide) and the phenylpropylthio analogue 4b emerged as the most
potent antiproliferative agents. Tested on colorectal carcinoma and
glioblastoma multiforme cancer stem cells (CSCs) from patients, 2b was
particularly potent against colorectal carcinoma CSCs, while 4b, 6a, and the
SIRT2-selective inhibitor AGK-2 showed the highest effect against
glioblastoma multiforme CSCs. Such compounds will be further explored for their broad-spectrum anticancer properties.
Among deacetylases, sirtuins (class III HDACs, SIRT1−7)
use NAD+ as cosubstrate for their unique deacetylation
chemistry, show no homology with the other classes of
HDACs, and are sensitive to specific inhibitors.6,7 Besides
histones, sirtuins deacetylate and/or ADP-ribosylate many
other proteins (transcription factors, enzymes, nuclear
INTRODUCTION
■
Reversible protein acetylation is an important posttranslational
modification that regulates the function of histones and
nonhistone proteins and, by this way, the gene expression at
epigenetic level.1−3 Acetylation of lysine residues, carried out by
histone/protein acetyltransferases (HATs), is counteracted by
the activity of histone/protein deacetylases (HDACs), which
remove acetyl groups from the same targets.4,5
Received: August 7, 2012
Published: November 29, 2012
© 2012 American Chemical Society
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Figure 1. Known SIRT1/2 inhibitors sharing the 2-hydroxy-1-naphthyl pharmacophore moiety.
Figure 2. Strategy for the design of novel (2-hydroxy-1-naphthylmethylene)aminophenyl derivatives related to 1a.
receptors, and other regulatory and structural proteins) and
play a critical role in regulation of a variety of cell processes,
from apoptosis to control of energetic metabolism and
aging.8−12
SIRT1 and −2 has been suggested as the best therapeutic
option.31
Among the SIRT1/2 inhibitors described so far, only
nicotinamide,32 sirtinol (1a),33 cambinol,34 JGB1741,35 and
the benzodeazaoxaflavin (BDF4) MC2141,36 the last four all
sharing the same 2-hydroxy-1-naphthyl pharmacophore moiety
(Figure 1), and the structurally different tenovins37 were found
active as antiproliferative agents, alone or in combination with
other chemotherapeutics, in a variety of cancer cells.38−40
Compound 1a (2-[(2-hydroxy-1-naphthylmethylene)-
amino]-N-(1-phenylethyl)benzamide) is a very attractive
molecule because its structure can be dissected into three
fragments (A, 2-hydroxy-1-naphthylmethylene; B, 2-iminoben-
zamide; C, N-1-phenethylamino) that can individually be
manipulated to obtain novel analogues with potential improved
activity (Figure 2). The cited JGB1741,35 with the replacement
of the anthranilic moiety of 1a with the 2-amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylic acid (Figure 1),
offers an example of a successful fragment B modification.
In 2005, we reported some 1a analogues with changes at the
B or C fragments.41 Among them, only compounds bearing the
N-(1-phenylethyl)amino side chain shifted from ortho- to meta-
(1b) or para- (1c) position of the benzene ring (Figure 2)
showed improved activity against SIRT1 and SIRT2.41 In yeast
phenotypic screening, 1b and 1c were as potent as 1a.41 No
data on their effects on human cells were reported.
In cancer, the role of sirtuins is actually highly debated. In
particular, SIRT1 has been reported as either tumor promoter
or tumor suppressor protein.13,14 In fact, it was found
overexpressed in some malignancies (melanoma, prostate,
liver, breast, colorectal, and ovarian epithelial cancer)15−19
and reduced in others (glioblastoma, bladder, colon, ovary, and
prostate cancer).20−24 SIRT1 is also able to inactivate at
transcriptional and posttranslational level some tumor
suppressor proteins (p53, p73, etc.) and to activate the
oncoprotein BCL6, to exert antiapoptotic and antidifferentia-
tion activities through deacetylation of specific transcription
factors (E2F1, FOXO3a, Ku70, etc.), and to regulate DNA
repair, cell cycle progression, and chromosomal stability.25 On
the other hand, in APC−/+ mice transgenic expression of SIRT1
reduced polyp formation, a potential precursor to colorectal
cancer26 (however, APC−/+/SIRT null mice did not show
increased number of polyps),27 and SIRT1+/−/p53+/− mice
were reported to develop tumors in multiple organs.24
Altogether, these and other findings suggest that SIRT1 could
play different roles in tumorigenesis according to the specific
tissue and/or context of tumor.13,14,25 SIRT2 was previously
considered less involved in cancer development than SIRT1
due to its predominantly cytosolic localization and its ability to
deacetylate α-tubulin in addition to histones. Nevertheless,
recent reports highlighted the role of SIRT2 in the patho-
genesis and development of cancer,28−30 and targeting both
Pursuing our researches on sirtuin modulators,36,42−46 we
designed a number of 1a-related derivatives by replacing the
benzoyl amide linkage of the prototype with other bioisosteric
groups such as retroamide, sulfonamide, thiomethyl, sulfinyl-
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a
Scheme 1
a
Reagents and conditions: (a) glacial acetic acid, dry ethanol/benzene 3/1, reflux.
a
Scheme 2
a
Reagents and conditions: (a) triethylamine, dry dichloromethane, room temperature; (b) stannous chloride dihydrate, 35% HCl, ethanol, room
temperature; (c) potassium carbonate, dry DMF, room temp; (d) 30% hydrogen peroxide (w/w), sodium tungstate dihydrate, glacial acetic acid/
water, 65 °C.
methyl, and sulfonylmethyl and bearing the 1-phenylethyl side
chain with ortho, meta, or para regiochemistry (Figure 2). The
compounds 2−6 were tested in vitro against human
recombinant (hr) SIRT1 and SIRT2, and in human myeloid
leukemia U937 cells, together with 1a−c, to evaluate their
effects on cell cycle, apoptosis induction, and differentiation.
From this preliminary screening, salermide (2b)46 and some
analogues were chosen for further experiments.46 Among the
tested cancer cell lines, the leukemia MOLT4 cell line was the
most sensitive to the antiproliferative action of 2b and related
derivatives.
Cancer stem cells (CSCs) have been described in many solid
and hematologic malignancies as a subset of highly tumorigenic
cells with distinct properties such as self-renewal, activation of
pluripotency genes, and multidrug resistance. CSCs were
shown to resist to conventional cancer therapies, and are
reputed to play a major role in cancer relapse.47,48 Thus, 2b and
selected analogues were tested against two colorectal carcinoma
(CRO and 1.1) and two glioblastoma (30P and 30PT) CSCs
from patients to determine their potential antiproliferative
effects.
CHEMISTRY
■
The (2-hydroxy-1-naphthylmethylene)amino-N-phenylderiva-
tives 2−6 were obtained by final condensation between 2-
hydroxy-1-naphthaldehyde and the appropriate aniline deriva-
tives 7−11 in acidic medium (Scheme 1).The N-(2-, 3-, and 4-
aminophenyl)-2-phenylpropanamides 7a−c were prepared
according to literature by reaction of (R/S)-2-phenylpropionic
acid with the proper phenylendiamine in the presence of BOP
reagent or similar.46,49−51 Commercially available 2-, 3-, and 4-
nitrobenzenesulfonyl chlorides treated with (R/S)-1-phenyl-
ethylamine under standard conditions led to the 2-, 3-, and 4-
nitro-N-(1-phenylethyl)benzenesulfonamides 12a−c, which
were in turn reduced to the corresponding 2-, 3-, and 4-
amino-N-(1-phenylethyl)benzenesulfonamides 8a−c using
stannous chloride dihydrate in acidic medium. (See Scheme
2A for the synthesis of the unknown 3-nitro-N-(1-phenylethyl)-
benzenesulfonamide 12b and of the 2- and 3-amino analogues
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8a,b. The 2- and 4-nitro derivatives as well as the 4-amino-N-
(1-phenylethyl)-benzenesulfonamide 8c are known.)43,52 The
2-, 3-, and 4-(2-phenylpropylthio)anilines 9a−c were prepared
by alkylation under basic conditions of the proper amino-
thiophenols with 1-bromo-2-phenylpropane.43 Further oxida-
tion of 9a−c with sodium tungstate dihydrate afforded a
mixture of the corresponding 2-, 3-, and 4-(2-
phenylpropylsulfinyl)anilines 10a−c and 2-, 3-, 4-(2-
phenylpropylsulfonyl)anilines 11a−c, which were separated
by column chromatography, recrystallized, and used in the
subsequent condensation with 2-hydroxy-1-naphthaldehyde
(Scheme 2B).
Chemical and physical data of compounds 2−6 are listed in
Table S1 in Supporting Information. Chemical and physical
data of the intermediate compounds 8a, 8b, 9a−c, 10a−c,
11a−c, and 12b are listed in Table S2 in Supporting
Information.
series, the highest SIRT2 inhibiting activity was shown by the
meta (4b) and the para (4c) analogues, 4b being the most
potent (IC50 = 19.2 μM) among all the tested derivatives.
Similar potency against SIRT2 was also observed with the 2-(2-
phenylpropyl)sulfonyl derivative 6a (IC50 = 24.2 μM).
Docking Studies. The structural variability of the
compounds with ortho, meta, and para substituents offers the
possibility of different interaction modalities in the binding site
that could result in some cases in a differential activity on
SIRT1 and SIRT2. To study the effect of the substitution of the
(2-hydroxy-1-naphthylmethylene)amino-N-phenyl derivatives
2−6 on the binding in the NAD+ pocket, 2−6 were docked
into SIRT1 and SIRT2. Our docking studies showed that there
were little differences in docking poses for most of the
compounds. In contrast, in the case of compound 1a, the β-
naphthol ring is completely out of SIRT1 binding site (Figure
4A), while for SIRT2 (Figure 4B) the 1a β-naphthol ring is
placed in the B-pocket (the ribose moiety binding pocket of
NAD+, in blue in Figure 4) and the phenyl ring is oriented
toward the C-pocket (a nicotinamide binding site, in yellow in
Figure 4). Compound 1a forms two hydrogen bonds with
Thr262 and Ser98 of SIRT2, increasing the affinity of binding
compared to SIRT1. Differently from 1a, 4b, the most efficient
inhibitor reported in this study, has the β-naphthol ring placed
in the B-pocket in both SIRT1 (Figure 4C) and SIRT2 (Figure
4D).
RESULTS AND DISCUSSION
■
SIRT1 and SIRT2 Assays. The (2-hydroxy-1-
naphthylmethylene)amino-N-phenyl derivatives 2−6 were
tested in vitro against SIRT1 and SIRT2 using a fluorogenic
substrate and NAD+.44 The IC50 (inhibitory concentration 50,
compound dose required to inhibit the enzyme activity of 50%)
values of 2−6 are graphed in Figure 3. IC50 values of 1a, 1b,
and 1c against SIRT1 and SIRT2 were added for comparison.
Nevertheless, in SIRT1, 4b does not have a perfect fit in the
binding pose because there are different exposed areas. In
SIRT2, 4b is binding from the A to C pocket and shows a
correct fit in NAD+ binding site. This might be due to openness
of C-pocket in SIRT2 and to favorable interactions in the C-
pocket between Phe96 and His187 established by 4b in SIRT2
(Figure 4D).
The conclusion from docking studies is that the most potent
compounds show a common binding mode for SIRT2: the β-
naphthol ring has enough space to fit in B-pocket and the rest
of the scaffold can enter into the C-pocket. On the contrary, in
the case of SIRT1, there is less space in B-pocket, the scaffold
has no optimal fit in it, and the C-pocket is practically empty, so
that only suboptimal complementary binding is achieved in
SIRT1 (see Supporting Information).
Cell Cycle Effect, Apoptosis Induction, And Gran-
ulocytic Differentiation on Human Leukemia U937 Cells.
The (2-hydroxy-1-naphthylmethylene)amino-N-phenyl deriva-
tives 1−6 were tested at 5, 25, and 50 μM in human leukemia
U937 cells in order to determine their effects on cell cycle,
apoptosis, and granulocytic differentiation. At 5 μM, no effect
was detected, and at 50 μM, many compounds showed massive
apoptosis (see Figures S11−S12 in Supporting Information).
Figure 5 shows cellular data for 1−6 tested on U937 cells at 25
μM for 40 h. EX-527,53 a well-known potent and selective
SIRT1 inhibitor typically devoid of effects on cell growth and
viability,54 and AGK-2,55 a SIRT2-selective inhibitor useful in
Parkinson’s disease models, were used as reference drugs both
at 25 μM.
Figure 3. IC50 values of 1−6 against SIRT1 and SIRT2.
Against SIRT1, compounds 2−6 showed quite similar
inhibiting activity, with IC50 values ranging from 40.3 to 67.3
μM regardless of the nature or regiochemistry of the side chain.
The only exceptions were 3a and 6c, which displayed lower
potency, whereas the 4-phenylpropionamide derivative 2c and
the (2-phenylpropyl)thio derivative 4b were the most efficient
inhibitors, with IC50 values of 40.9 and 40.3 μM, respectively. In
comparison with the reference compounds, the majority of 2−6
derivatives displayed inhibition potencies higher than 1a and
similar to 1b and 1c.
Under the tested conditions, among the 2 series the 2-(2-
phenylpropionamide) derivative 2a displayed a block at the S
phase of cell cycle, whereas the corresponding 3- and 4-isomers
2b and 2c gave an evident arrest at the G1 phase, similar to that
detected with 1b and 1c. Cells treated with the 3-N-(1-
phenylethyl)sulphonamide compound 3b showed a clear arrest
at the S phase, whereas with the 4-(2-phenylpropylsulfonyl)
analogue 6c, a block of cell cycle at the G1 phase was observed
In general terms, in anti-SIRT2 assay the tested compounds
2−6 exhibited similar or higher inhibiting activities than against
SIRT1, albeit with some exceptions (2a and 3b). Among
compounds substituted with 2-phenylpropionamide function,
the meta analogue 2b (salermide) displayed the highest activity
(IC50 = 25.0 μM), while in the 3 series the 4-N-(1-
phenylethyl)benzenesulphonamide 3c was the most efficient
(IC50 = 22.6 μM). In the (2-phenylpropyl)thio-substituted
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Figure 4. Docking poses for 1a in SIRT1 (A) and SIRT2 (B), and docking poses for the most potent derivative 4b in SIRT1 (C) and SIRT2 (D).
The B-pocket (the ribose moiety binding pocket of NAD+) of the enzymes is depicted in blue, and the C-pocket (a nicotinamide binding site) of the
enzymes is depicted in yellow.
(Figure 5A). EX-527 and AGK-2 did not show any change in
the U937 cell cycle at the tested conditions. Also combination
attempts of EX-527 and AGK-2 failed to give cell cycle
alteration and/or apoptosis (see Figure S13 in Supporting
Information). The evidence that different phases of cell cycle
appear blocked by different compounds that share the same
targets (SIRT) might both be related to the different effects on
SIRT1 versus SIRT2 or be related to the different inhibitory
action in living cells of the compounds. Clearly, we can not
exclude that also additional targets might be involved.
Most of the tested compounds exhibited a high pre-G1 peak
(an index of apoptosis induction) at the FACS analysis. The
reference compound 1a caused an apoptosis induction of 47%
and its regioisomers 1b and 1c, as well as the corresponding
retroamide analogues 2b and 2c, led to a percentage of
apoptosis near (or over) 70%. In this assay, the 4-(2-
phenylpropyl)thio derivative 4c showed massive apoptosis so
that we fixed the cutoff at 90% (Figure 5B). Contrarily, EX-527
and AGK-2, alone or in combination (Figure S13 in Supporting
Information), were ineffective in inducing apoptosis.
Granulocytic differentiation was evaluated by counting the
number of cells expressing the superficial antigen CD11c and
subtracting from this value the number of dead [propidium
iodide (PI) positive] cells. Under the tested conditions, mainly
the (2-phenylpropyl)sulfinyl (5b, 5c) and the (2-
phenylpropyl)sulfonyl (6a−c) derivatives displayed high
percentage of CD11c positive/PI negative cells, with values
ranging from 57.3 to 85.6% (Figure 5C). Also, in this test, no
effects were detected using EX-527 and AGK-2.
Antiproliferative Effect on Several Cancer Cell Lines.
All the described compounds 2−6, including 1b and 1c for
comparison, were tested first at 25 μM on human leukemia
MOLT4 cells for 24, 48, and 72 h to determine their
antiproliferative effects (MTT method) (Figure S14 in
Supporting Information). In addition, selected compounds
bearing the (2-hydroxy-1-naphthylmethylene)amino-N-phenyl
moiety linked to N-(1-phenylethyl)benzamide (1b), 2-phenyl-
propionamide (2a and 2b), N-(1-phenylethyl)sulphonamide
(3b), (2-phenylpropyl)thio (4a and 4b), and (2-phenylpropyl)-
sulfonyl (6a and 6b) groups were tested first at 25 μM on
human breast cancer MDA-MB-231 and human colon cancer
RKO cells (Figure S15 in Supporting Information). At 25 μM,
in MOLT4 cells 1c, 2b, 4a, 4b, 5a, 5b, and 6a were able to
arrest to some extent the cell growth. At the same
concentration, only 2b (salermide), among the tested
derivatives, showed some antiproliferative effects on MDA-
MB-231 and RKO cells and was therefore chosen for further
studies.46
Selected compounds 2b, 4a, 4b, and 6b were then tested at
50 μM in MOLT4, MDA-MB-231, and RKO cells for 24, 48,
and 72 h, and their antiproliferative effect was determined
(Figure 6). In these assays, 2b and 4b emerged as the most
efficient compounds to hamper the growth of the three tested
cancer cell lines. Finally, some compounds (1c, 2b, 2c, 3a, 3c,
4c, 5a, 5b, 5c, and 6c) were tested at 100 μM in MOLT4 cells,
and at this concentration 1c, 2b, 2c, 5a, and 5c displayed high,
time-dependent growth arrest (Figure S16 in Supporting
Information).
Antiproliferative Activity on Cancer Stem Cells (CSCs):
Effect of Selected 2−6 Derivatives on Two Colorectal
Carcinoma (CRC) CSC Lines and Two Glioblastoma
Multiforme (GBM) CSC Lines. Taking into account the
apoptotic, cytodifferentiating, and/or antiproliferative activities
displayed in previous assays, we selected compounds 2b, 4b, 5a,
5b, 6a, and 6c to detect their capability to inhibit viability of
CSCs, a cancer cell subset highly tumorigenic, resistant to
conventional cancer therapies, and believed to play a major role
in cancer relapse.48 The SIRT1-selective inhibitor EX-527 and
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Figure 6. Percentage of antiproliferative activity of 2b, 4a, 4b, and 6b
tested at 50 μM in leukemia MOLT4, breast MDA-MB-231, and colon
RKO cancer cells (MTT method).
Figure 5. Cell cycle analysis (A), apoptosis induction (B), and
granulocytic differentiation (C) obtained treating human leukemia
U937 cells with 1−6 at 25 μM for 40 h.
Table 1. Cell Viability Inhibition by 2b, 4b, 5a, and 6a in
CRC and GBM CSCs
a
its SIRT2-selective counterpart AGK-2 were added to the assay.
Two colorectal carcinoma (CRC, CRO, and 1.1) and two
glioblastoma multiforme (GBM, 30P, and 30PT) CSCs 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 1. The
corresponding dose−response curves are reported in Support-
ing Information Figure S17.
Against the tested CSCs, EX-527 failed to produce significant
growth inhibition up to 50 μM, whereas AGK-2 displayed good
antiproliferative activity against GBM CSCs in which it was the
most potent among the tested derivatives. Compound 2b
(salermide), under the tested conditions, was highly efficient in
reducing CRC CSC viability, bearing single-digit μM activity
[CC50 = 6.7 (CRO) and 9.7 (1.1) μM], while it was much less
potent in GBM CSCs (CC50 = 41.0 (30P) and 36.5 (30PT)
μM).
CC50, μM
CRC CSCs
GBM CSCs
compd
CRO
1.1
30P
30PT
2b
4b
5a
6.7 1.2
14.5 1.2
33%
9.7 3.1
20.0 2.8
46%
inhibition
23.4 0.6
19.5 0.7
34.0 2.2
no inhibition
41.0 5.2
15.5 1.9
42%
36.5 2.5
15.6 3.6
39%
b
b
b
b
inhibition
inhibition
35.0 1.9
17.4 1.7
30.2 1.8
10%
inhibition
ND
c
5b
ND
6a
15.7 1.0
ND
15.3 3.1
ND
6c
b
EX-527
20%
inhibition
20%
inhibition
b
b
b
inhibition
AGK-2
50%
inhibition
50%
inhibition
12.5 0.5
9.6 1.0
b
b
a
b
c
Values are means of three experiments. At 50 μM. ND, not
detected.
With respect to 2b, compounds 4b and 6a exhibited lower
potency on CRC CSCs, with CC50 values ranging from 14.5 to
20 μM, and higher potency on GBM CSCs (CC50 = 15.3 to
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Figure 7. Effects of EX-527 plus AGK-2 combination in CRC 1.1 and GBM 30 P CSCs.
17.4 μM), in this last case being only slightly less efficient than
AGK-2. Remaining compounds 5b and 6c, chosen because of
their high cytodifferentiation effect in U937 cells, displayed low
antiproliferative activities against both the tested CSC lines.
Combination assays of EX-527 and AGK-2 were carried out
to ascertain the potential benefit derived from dual SIRT1/2
inhibition. The combined cytotoxic effects of EX-527 and
AGK-2 on CRC 1.1 and GBM 30P CSC clones are shown in
Figure 7. While for the CRC 1.1 clone the experimental
combination data conformed to the Bliss’s additive model, the
two compounds resulted to act synergistically on the GBM 30P
clone.
GBM 30P CSC clones the two compounds acted synergisti-
cally.
Because the most efficient compounds in cells showed
activity up to single-digit micromolar concentration, while
against SIRTs they displayed inhibition in the 20−50 μM range,
the involvement of off-target side effects can not be ruled out.56
In conclusion, compounds 2b, 4b, and 6a emerged as highly
potent small molecules for anticancer treatment, also against
CSCs, that are normally refractory to conventional anticancer
therapy and are believed to play a role in cancer relapse.
Further studies should be addressed to improve pharmacoki-
netic and drug-like properties of such compounds.
EXPERIMENTAL SECTION
CONCLUSION
■
■
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. H NMR spectra were
Chemical manipulations (replacing the benzamide with an
anilide, sulphonamide, thiomethyl, sulfinylmethyl, or sulfonyl-
methyl moiety and/or shifting the side chain from ortho to meta
or para position of the benzene ring) were performed on the 1a
scaffold to obtain (2-hydroxy-1-naphthylmethylene)amino-N-
phenyl derivatives 2−6, many of which with improved activity
against both SIRT1 and SIRT2. The higher inhibitory activity
of 4b, the most potent compound in this series, with respect to
the prototype 1a has been rationalized through docking studies.
When tested in U937 human leukemia cells (25 μM, 40 h), the
4-(2-phenylpropyl)thio derivative 4c displayed massive apop-
tosis, and the meta- and para-benzamide and -anilide derivatives
1b,c and 2b,c showed cell cycle arrest at the G1 phase joined to
high apoptosis. Differently, the sulfinyl and sulfonyl analogues
5b,c and 6a−c were the most efficient in inducing granulocytic
differentiation in U937 cells at the same conditions. Selected
2−6 compounds were screened for assessing their antiprolifer-
ative activity against human leukemia MOLT4 cell lines, human
breast MDA-MB-231, and colon RKO cancer cell lines at 25,
50, and (in some cases) 100 μM for 24, 48, and 72 h. From the
overall results, 2b (salermide) and 4b emerged as two of the
most potent derivatives able to block cell growth in the tested
cancer cell lines. Thus, 2b and 4b as well as the sulfinyl and
sulfonyl derivatives 5a,b and 6a,c were tested against two
colorectal carcinoma (CRO and 1.1) and two glioblastoma
multiforme (30P and 30PT) CSCs, in comparison with the
SIRT1-selective inhibitor EX-527 and the SIRT2-selective
inhibitor AGK-2. Against the tested CSCs, 2b showed the
highest antiproliferative activity on colorectal carcinoma CSCs,
with single-digit CC50 values, while 4b and 6a were 2-fold less
potent than 2b. Against glioblastoma multiforme CSCs, 4b, 6a,
and AGK-2 displayed the highest potency, thus suggesting a
positive, additional role of SIRT2 in the viability and
proliferation of CSCs. Combination of EX-527 and AGK-2 in
CRC 1.1 gave an additive antiproliferative effect, whereas in
1
recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical
shifts are reported 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. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Concen-
tration of solutions after reactions and extractions involved the use of a
rotary evaporator operating at reduced pressure of ca. 20 Torr.
Organic solutions were dried over anhydrous sodium sulfate.
Elemental analysis has been used to determine purity of the described
compounds, that is >95%. Analytical results are within 0.40% of the
theoretical values. All chemicals were purchased from Aldrich Chimica,
Milan (Italy) or from Lancaster Synthesis GmbH, Milan (Italy) and
were of the highest purity.
General Procedure for the Preparation of Derivatives (2−6).
Example: N-(4-[(2-Hydroxynaphthalen-1-yl)methyleneamino]-
phenyl)-2-phenylpropanamide (2c). A mixture of 2-hydroxy-1-
naphthaldehyde (0.36 g, 2.1 mmol) and N-(4-aminophenyl)-2-
phenylpropanamide51 (0.50 g, 2.1 mmol) in absolute ethanol/benzene
(3/1) (30 mL) was heated at reflux for 4 h in the presence of a
catalytic amount of glacial acetic acid. After cooling at room
temperature, from the mixture reaction a yellow solid separated,
which was collected by filtration, washed with chloroform, and purified
1
by crystallization from toluene/ethanol. H NMR (CDCl3) δ 1.63 (d,
3H, CHCH3), 3.76 (m, 1H, CHCH3), 6.63 (m, 2H, aromatic rings),
7.07−8.01 (m, 15H, aromatic rings), 9.30 (s, 1H, CHN), 15.58 (s,
1H, OH). Anal. C, H, N.
(R/S)-3-Nitro-N-(1-phenylethyl)benzenesulfonamide (12b).
3-Nitrobenzenesulfonyl chloride (1 equiv, 1 g, 4.51 mmol) was
dissolved in 20 mL of dichloromethane and then was added dropwise
to a mixture of (R/S)-1-phenylethylamine (1.1 equiv, 0.64 mL, 4.96
mmol) and triethylamine (1.2 equiv, 0.73 mL, 5.41 mmol) in
dichloromethane (50 mL) while cooling with water bath. The resulting
mixture was stirred at room temperature for 2 h and then was diluted
with water (20 mL), and the aqueous solution was extracted with ethyl
acetate (3 × 50 mL). The organic layers were collected, washed with
0.2 N hydrochloric acid (3 × 50 mL), 0.2 N sodium hydroxide (3 × 50
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Article
mL), and brine (3 × 50 mL), dried (sodium sulfate), and evaporated
under reduced pressure. The solid residue was then purified by
crystallization from toluene to furnish the desired derivative 12b. H
NMR (CDCl3) δ 1.50 (d, 3H, CHCH3), 4.63 (q, 1H, CHCH3), 5.35
(d, 1H, NH exchangeable with D2O), 7.01−7.10 (m, 5H, phenyl ring),
7.51 (m, 1H, nitrophenyl ring), 7.93 (m, 1H, nitrophenyl ring), 8.23
(m, 1H, nitrophenyl ring), 8.33 (m, 1H, nitrophenyl ring).Anal. C, H,
N, S.
NM_012237), full length, MW = 43 kDa, expressed in E. coli.
Compounds were tested using a modified Fluor de Lys fluorescence-
based assay kit (AK-555, AK-556 Biomol). The assay procedure has
two steps: in the first part the SIRT1/2 substrate (50 μg), an
acetylated Lys side chain comprising amino acids 379−382 (Arg-His-
Lys-Lys(Ac)) (for SIRT1 assay), or 317−320 (Gln-Pro-Lys-Lys(Ac))
(for SIRT2 assay) of human p53 conjugated with aminomethylcou-
marin was deacetylated during incubation at 37 °C for 1 h by SIRT1 or
SIRT2 in the presence of NAD+ (500 μg) and the tested compounds
(reaction buffer: 50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2; add fresh 1 mg/mL BSA, 1% DMSO). The second
stage was initiated by the addition of the Developer II, including 2 mM
nicotinamide (NAM), a sirtuin inhibitor that stops the SIRT1/2
activity, and the fluorescent signal is produced. The fluorescence was
measured on a fluorometric reader (Inphinite 200 TECAN) with
excitation set at 360 nm and emission detection set at 460 nm.
Counter assay was performed without enzyme: 1 μM standard
(fluorogenic unacetylated peptide) + compounds added as above and
then developed and fluorescence measured as above. The fluorescence
from compounds was subtracted from all signals if there was.
Experiments on the SIRT1 and -2 inhibition have been performed in
triplicate. IC50 data were analyzed using GraphPad Prism Software.
Molecular Modeling. No crystal structure is available for SIRT1,
and for SIRT2 there is only apo structure available. Therefore, we were
forced to build also homology model for SIRT2 in bioactive
conformation. The template used for the homology model of SIRT1
and SIRT2 was the crystal structure of Sir2 homologue from
Archeaoglobus fulgidus (Sir2-Af1) with NAD+ (PDB code: 1ici). The
homology models of SIRT1 and SIRT2 were built using FUGUE and
ORCHESTRA in SYBYL 1.3 (Alignments are presented in Supporting
Information).48 The side chains were optimized by geometry
optimization with Amber99 using Molecular Operating Environment
(MOE) 2010.10.49 The ligand structures were built with MOE
software and minimized using MMFF94 force field until a rmsd
gradient of 0.05 kcal mol−1 Å−1 was reached. The docking simulations
were performed in the NAD+ binding site of the homology models
SIRT1 and -2 using MOE. Default values were used for all docking
settings. The best-ranked poses of each docked ligand was included in
the analysis. The docking results were visually inspected.
Cell Cycle and Apoptosis Analysis on Human Leukemia
U937 Cells. Cells were collected and suspended in 500 μL of
hypotonic buffer [0.1% Triton X-100, 0.1% sodium citrate, 50 μg/mL
propidium iodide (PI)]. Then the cells were incubated in the dark for
30 min. Samples were acquired on a FACS-Calibur flow cytometer
using the Cell Quest software (Becton Dickinson). Analyses were
performed with standard procedures using ModFit LT version3
software (Verity). All experiments were performed in triplicate.
Granulocytic Differentiation on U937 Cells. Cells were
suspended in 10 μL of phycoerythrine-conjugated CD11c (CD11c-
PE). Control samples were incubated with 10 μL of PE conjugated
mouse IgG1. The incubations were performed at 4 °C in the dark for
30 min, and after, cells were collected, washed in phosphate buffered
saline (PBS), and suspended in 500 μL of PBS containing propidium
iodide (PI) (0.25 μg/mL). Samples were analyzed by FACS with Cell
Quest technology (Becton Dickinson). PI positive cells have been
excluded from the analysis.
1
General Procedure for the Preparation of (R/S)-2- and -3-
Amino-N-(1-phenylethyl)benzenesulfonamides (8a and 8b).
E x a m p l e : ( R / S ) - 3 - A m i n o - N - ( 1 - p h e n y l e t h y l ) -
benzenesulfonamide (8b). A solution of 12b (1 equiv, 0.99 g,
3.23 mmol) in ethanol (9 mL) was slowly added to a solution of
stannous chloride dihydrate (3.5 equiv, 2.55 g, 11.31 mmol) and 37%
hydrochloric acid (3 mL) while cooling with ice bath. After 30 min, the
reaction was allowed to reach room temperature and then stirred for
further 2 h. After the completion of the reaction, the solvent was
evaporated under reduced pressure and the residue treated with 2N
potassium hydroxide (20 mL) and ethyl acetate (3 × 20 mL). Then,
the organic phase was dried (sodium sulfate) and evaporated to afford
the desired compound 8b. 1H NMR (CDCl3) δ 1.42 (d, 3H, CHCH3),
3.66 (s, 2H, NH2 exchangeable with D2O), 4.46 (q, 1H, CHCH3), 5.15
(s, 1H, NH exchangeable with D2O), 6.78 (m, 1H, benzenesulfona-
mide ring), 7.05−7.24 (m, 8H, benzenesulfonamide ring and phenyl
ring). Anal. C, H, N, S.
General Procedure for the Preparation of (R/S)-2-, -3-, and
-4-(2-Phenylpropylthio)anilines (9a−c). Example: (R/S)-4-(2-
Phenylpropylthio)aniline (9c). (R/S)-1-Bromo-2-phenylpropane
(1.2 equiv, 1.45 mL, 9.58 mmol) was added to a suspension of 4-
aminothiophenol (1 equiv, 0.84 mL, 1.0 g, 7.98 mmol) and anhydrous
potassium carbonate (2 equiv, 2.2 g, 15.97 mmol) in dry N,N-
dimethylformamide (5 mL), and the mixture was stirred at room
temperature for 2 h. Afterward, the reaction mixture was quenched
with water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The
organic phase was then washed with brine (2 × 30 mL) and dried over
sodium sulfate. After evaporation in vacuum, the crude was purified by
column chromatography on silica gel eluting with ethyl acetate/
chloroform (1/20) to furnish the desired compound 9c as a pure oil.
1H NMR (CDCl3) δ 1.39 (d, 3H, CHCH3), 2.93 (m, 2H,
SCH2CHCH3), 3.09 (m, 1H, CH2CHCH3), 6.60−7.33 (m, 9H,
aromatic rings). Anal. C, H, N, S.
General Procedure for the Preparation of (R/S)-2-, -3-, and
-4-(2-Phenylpropylsulfinyl)anilines (10a−c) and (R/S)-2-, -3-,
and -4-(2-Phenylpropylsulfonyl)anilines (11a−c). Example: (R/
S)-4-(2-Phenylpropylsulfinyl)aniline (10c) and (R/S)-4-(2-
Phenylpropylsulfonyl)aniline (11c). A mixture of sodium tungstate
dehydrate (0.001 g, 0.003 mmol), 1 drop of acetic acid, and water (1
mL) was placed in a flask and heated at 65 °C. (R/S)-4-(2-
Phenylpropylthio)aniline (9c) (0.50 g, 2.05 mmol) was added,
followed by dropwise addition of 30% w/w hydrogen peroxide (0.36
mL), and the mixture was stirred at 65 °C for 1.5 h. After cooling, 5
mL of water and 5 mL of ethyl acetate were added. The layers were
separated, and the aqueous phase was extracted with ethyl acetate (3 ×
5 mL). The organic phase was washed with brine and dried over
sodium sulfate. The solvent was removed under reduced pressure to
furnish a mixture of compounds (0.60 g), which was separated by silica
gel column chromatography using ethyl acetate/n-hexane (1/1) as
eluent. Pure compounds 10c [Rf = 0.2 (ethyl acetate); yield 57%] and
Antiproliferative Activity on Human Leukemia MOLT4,
Human Breast Cancer MDA-MB-231, and Human Colon Cancer
RKO Cell Lines. Cell lines were obtained from the American Type
Culture Collection (VA, USA). Cell viability was determined using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as
described earlier.46
1
11c [Rf 0.6 (ethyl acetate); yield 31%] were obtained. 10c: H NMR
(CDCl3) δ 1.42−1.50 (d, 3H, CHCH3), 2.84−3.16 (m, 2H,
SCH2CHCH3), 3.29 (m, 1H, CH2CHCH3), 3.99 (s, 2H, NH2), 6.75
(m, 2H, aromatic rings), 7.21−7.46 (m, 7H, aromatic rings). Anal. C,
1
H, N, S. 11c: H NMR (CDCl3) δ 1.43 (d, 3H, CHCH3), 3.32 (m,
Cell Viability Inhibition in Cancer Stem Cells (CSCs). CSC
cultures were established and propagated under spherogenic
conditions as described.57,58 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
3H, CH2CHCH3 and SCH2CHCH3), 4.18 (s, 2H, NH2) 6.63 (m, 2H,
aromatic rings), 7.08−7.26 (m, 5H, aromatic rings), 7.57 (m, 2H,
aromatic rings). Anal. C, H, N, S.
SIRT1/2 Inhibition Assay. The SIRT activity assay was performed
using: for SIRT1, human Sirtuin 1 (GenBank accession no.
NM_012238), amino acid 193−741 with N-terminal GST tag, MW
= 87.2 kDa, expressed in Escherichia coli expression system. For SIRT2:
human Sirtuin
2 (hSir2SIRT2) (GenBank accession no.
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each cell line. For the 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 bottom. 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 KaleidaGraph software
based on the residual luminescence in the presence of increasing
concentrations of the inhibitors.
For the EX-527/AGK-2 combination experiments, cells were
treated in triplicate with 1:2 serial dilutions of each compound or
with the combination of the two compounds (6.25−50 μM
concentration range for CRC CSC 1.1 clone and 3.125−50 μM
concentration range for GBM CSC 30P clone). Fractional response
values were calculated relative to samples treated with DMSO, and
dose−response curves for compounds EX-527 and AGK-2 adminis-
tered individually or together were obtained by sigmoidal dose−
response fitting of the experimental data using GraphPad Prism
software. Bliss’s independence (additivity) curves were derived by
fitting the experimental data obtained with each individual compound
into the Bliss’s equation: Y = FA + FB − (FA × FB), where Y is the
fractional response of the combination of the two drugs A and B
assuming Bliss’s independence.
HOXA10, homeobox A10; MEF2, myocyte enhancer factor-2;
MOE, molecular operating environment; MTT, (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NF-
kB, nuclear factor-kappa B; PGC1-α, peroxisome proliferator-
activated receptor-γ coactivator 1-α; PI, propidium iodide
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ASSOCIATED CONTENT
* Supporting Information
■
S
Chemical and physical data for compounds 2−6 and for the
intermediate compounds 8a, 8b, 9a−c, 10a−c, 11a−c, and
12b. Modeling data and docking poses. Cell cycle and apoptosis
induction at 5 and 50 μM in U937 cells. Antiproliferative
activity in MOLT4 (at 25 and 100 μM), MDA-MB-231 (25
μM), and RKO (25 μM) cancer cells. Dose−response curves of
the tested SIRT inhibitors in CSCs. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +3906-4991-3392. Fax: +3906-491491. E-mail:
antonello.mai@uniroma1.it.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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ABBREVIATIONS USED
■
BCL-6, B-cell lymphoma 6; BDF4s, benzodeazaoxaflavins;
bFGF, basic fibroblast growth factor; BOP reagent, benzo-
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HeLa, Henrietta Lacks; HepG2, hepatocellular carcinoma G2;
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