Study of 1,4-dihydropyridine structural scaffold: Discovery of novel sirtuin activators and inhibitors

Article abstract of DOI:10.1021/jm9008289

NAD⁺-dependent sirtuin deacetylases have emerged as potential therapeutic targets for treatment of human illnesses such as cancer, metabolic, cardiovascular, and neurodegenerative diseases. The benefits of sirtuin modulation by small molecules

Full text of DOI:10.1021/jm9008289

5496 J. Med. Chem. 2009, 52, 5496–5504
DOI: 10.1021/jm9008289
Study of 1,4-Dihydropyridine Structural Scaffold: Discovery of Novel Sirtuin Activators and Inhibitors
Antonello Mai,*,† Sergio Valente,† Sarah Meade,‡ Vincenzo Carafa,§ Maria Tardugno,† Angela Nebbioso,§ Andrea Galmozzi,
Nico Mitro, ,^ Emma De Fabiani, Lucia Altucci,§ and Aleksey Kazantsev‡
†Istituto Pasteur;Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Universitaꢀ degli Studi di Roma “La
Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, ‡Harvard Medical School, Massachusetts General Hospital, CNY114 16th Street, Charlestown,
Massachusetts 02129, §Dipartimento di Patologia Generale, Seconda Universitaꢀdegli Studi di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy,
Dipartimento di Scienze Farmacologiche, Universitaꢀ degli Studi di Milano, Via Balzaretti 9, 20133 Milano, Italy, and ^Giovanni Armenise
Harvard Foundation Laboratory, Boston, Massachusetts
Received May 27, 2009
NADþ-dependent sirtuin deacetylases have emerged as potential therapeutic targets for treatment of
human illnesses such as cancer, metabolic, cardiovascular, and neurodegenerative diseases. The benefits
of sirtuin modulation by small molecules have been demonstrated for these diseases. In contrast to the
discovery of inhibitors of SIRT1, -2, and -3, only activators for SIRT1 are known. Here, we rationalized
the potential of the previously unexplored dihydropyridine scaffold in developing sirtuin ligands, thus
we prepared a series of 1,4-dihydropyridine-based derivatives 1-3. Assessment of their SIRT1-3
deacetylase activities revealed the importance of the substituent at the N1 position of the dihydropyr-
idine structure on sirtuin activity. Placement of cyclopropyl, phenyl, or phenylethyl groups at N1
conferred nonselective SIRT1 and SIRT2 inhibition activity, while a benzyl group at N1 conferred
potent SIRT1, -2, and -3 activation. Senescence assays performed on hMSC and mitochondrial function
studies conducted with murine C2C12 myoblasts confirmed the compounds’ novel and unique SIRT-
activating properties.
Introduction
SIRT1, the sirtuin member with the highest sequence similar-
ity (48%) with yeast Sir2, has recently been found to be
involved in a variety of diseases such as cancer and metabolic
disorders as well as cardiovascular and neurodegenerative
diseases, mainly through deacetylation of nonhistone sub-
strates such as the tumor suppressor protein p53, the histone
acetyltransferase (HAT) enzyme p300, members of the fork-
head box class O (FOXO) family, peroxisome proliferator-
activated receptor γ (PPARγ) coactivator 1R (PGC-1R), and
nuclear factor-κB (NF-κB).11-13
SIRT1, through its negative regulation of p53 and con-
comitant positive regulation of the oncogene B-cell lym-
phoma 6 (BCL6) protein, and through induction of the
FOXO-1-dependent vascular endothelial growth factor-C
(VEGF-C), may repress apoptosis and enhance tumor
growth, angiogenesis, and metastasis.14-16 In addition,
SIRT1 has been found to be upregulated in human lung
cancer, prostate cancer, colon carcinoma, and leukemia,
highlighting the potential use of SIRT inhibitors (SIRTi) as
anticancer agents.17,18
Indeed, inhibitors such as sirtinol have been reported to
induce senescence-like growth arrest in human breast cancer
MCF-7 cells and lung cancer H1299 cells, cambinol induced
apoptosis in BCL6-expressing Burkitt lymphoma cells,20 and
salermide, recently reported by us, was well tolerated by mice
at concentrations up to 100 μM and prompted tumor-specific,
p53-independent apoptosis in a wide range of human cancer
cell lines.21 Furthermore, tenovins22 were identified via a yeast
genetic screen for p53 activators and decreased tumor growth
in vivo as single agents at low micromolar concentrations
(Chart 1).
Acetylation/deacetylation of the ε-amino group of lysine
residues in histone tails is a well-known mechanism of regula-
tion of gene expression and transcription.1 Among the histone
deacetylase (HDACa) enzyme families, the class III HDACs,
also named sirtuins from their founding member in yeast,
silent information regulator 2 (Sir2), share peculiar features
that make them very different from the “classical” class I/II/
IV HDACs.2,3 Indeed, the human sirtuins (SIRT1-SIRT7)
do not have any sequence similarity with the other HDACs,
they act through a NADþ-dependent mechanism of catalysis
(instead of the Zn2þ-dependent deacetylation used by
HDACs), and are not sensitive to the common HDAC
inhibitors.4-6 In regard to their roles in physiology and
pathology, sirtuins are multifaceted enzymes that regulate a
variety of cellular functions, from controlling gene expression
and genome maintenance to longevity and metabolism.7-10
*To whom correspondence should be addressed. Phone: þ3906-4991-
3392. Fax:þ3906-491491. E-mail: antonello.mai@uniroma1.it.
a Abbreviations: BCL6, B-cell lymphoma 6; CD11c-PE, phycoerythr-
ine-conjugated CD11c; DHP, 1,4-dihydropyridine; eNOS, endothelial
NO synthase; FOXO, forkhead box class O; HAT, histone acetyltrans-
ferase; HDAC, histone deacetylase; HDL, high-density lipoprotein;
hMSC, human mesenchimal stem cells; HUVEC, human umbilical vein
endothelial cells; LXR, liver X receptor; mTFA, mitochondrial tran-
scription factor A; NF-κB, nuclear factor-κB; NRF, nuclear respiratory
factor; PBS, phosphate buffered saline; PGC-1R, peroxisome prolifera-
tor-activated receptor γ coactivator 1R; PI, propidium iodide; PML,
promyelocytic leukemia; PPARγ, peroxisome proliferator-activated
receptor γ; RSV, resveratrol; SAHA, suberoylanilide hydroxamic acid;
SA-β-gal, senescence-associated β-galactosidase; Sir2, silent informa-
tion regulator 2; STACs, sirtuin activating compounds; VEGF-C,
vascular endothelial growth factor-C; β-gal, β-galactosidase.
r
pubs.acs.org/jmc
Published on Web 08/10/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5497
Chart 1. Structures of Known SIRT Inhibitors
Figure 1. Novel 1,4-dihydropyridine (DHP) compounds and
related derivatives.
Chart 2. Structures of Known SIRT Activators
Scheme 1a
Conversely, SIRT1 may have some beneficial effects
in carcinogenesis, through its deacetylation activity on
Ku70 and resultant Ku70-associated increase of DNA repair
activity.23 Additionally, deacetylation of β-catenin results in
inactivation of transcription and halts cell proliferation.24
SIRT1 displays a protective role in cardiovascular diseases,
likely through its activation of the liver X receptor (LXR)
gene, resulting in beneficial effects in HDL synthesis, choles-
terol transport, and atherosclerosis.25 In addition, through
eNOSdeacetylation, SIRT1 improves endothelialcell survival
and functions.26 A neuroprotective role has been evocated for
SIRT1, associated with its downregulation of pro-apoptotic
factors and activation of PGC-1R,12,27,28 as PGC-1R mediates
the regulatory role of SIRT1 in metabolic homeostasis, consu-
mption of oxygen in muscle fibers, mitochondrial function,
and mitochondrial biogenesis.29 Last but not least, an increase
in Sir2 levels extended lifespan in yeast, Caenorhabditis ele-
gans, and Drosophila, while a deletion or mutation in Sir2 gave
an opposite effect.2,3,9 In humans, SIRT1 activity can over-
come p53/PML-induced senescence30 and negatively regu-
lates mSIN3A/HDAC1 transcriptional repression activity,
inhibiting cell growth arrest and cellular senescence.31 Resver-
atrol (RSV, Chart 2), the first reported SIRT1 activator, has
been shown to extend lifespan in yeast, C. elegans, Drosophila,
and rodents.29,32 With respect to the number of SIRTi de-
scribed in the literature, only a few SIRT activators have been
disclosed up to now. Aside from RSV and other polyphenolic
compounds (sirtuin activating compounds, STACs),33 two
series of small molecule SIRT activators, imidazo[1,2-
b]thiazoles (such as SRT1720, Sirtris Pharmaceuticals)34 and
pyrrolo[3,2-b]quinoxalines (such as PQX, S*Bio Pte Ltd.)35
(Chart 2), have been recently described as oral antidiabetic
and lipolytic/anti-inflammatory agents, respectively.
a (a) CH3COOH, 80 ꢀC. (b) 5 N KOH, EtOH, 80 ꢀC. (c) (1) Et3N, Bop
reagent, dry DMF, rt, 30 min, N2 atmosphere; (2) 33% aq NH3, dry
DMF, rt, 30 min, N2 atmosphere.
To pursue our researches on sirtuin modulating com-
pounds,21,36 we started with the information that nicotina-
mide inhibited SIRT at the micromolar level,37 whereas
isonicotinamide has been reported as a SIRT activator38
(Figure 1). In addition, Suzuki et al. screened a focused
library containing hydroxy-, alkoxy-, and anilino-benza-
mides, phthalimide, quinazolones, amino- and alkoxy-ni-
cotinamides, and 3-carboxamidopyridinium ions, identi-
fying a series of 2-anilinobenzamides as SIRT1 inhibitors39
(Figure 1). Thus, we decided to explore the 1,4-dihydro-
pyridine scaffold for the design of new compounds, poten-
tially active as sirtuin modulators. Using a three-com-
ponent (aliphatic/aromatic amine, ethyl propiolate, and
benzaldehyde), one-pot reaction, we prepared some 3,5-
dicarbethoxy-, 3,5-dicarboxy-, and 3,5-dicarboxamido-4-
phenyl-1,4-dihydropyridines 1a-d, 2a-d, and 3a-d, re-
spectively, and we tested them against human SIRT1,
SIRT2, and SIRT3.
Chemistry
The 3,5-dicarbethoxy-4-phenyl-1,4-dihydropyridines 1a-d
were prepared by cyclocondensation between benzaldehyde,
ethyl propiolate and the opportune amine, heated at 80 ꢀC
in glacial acetic acid. Furthermore, compounds 1a-d under-
went alkaline hydrolysis at 80 ꢀC in ethanol to furnish the

5498 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Table 1. Chemical and Physical Data of Compounds 1-3
Mai et al.
Figure 2. SIRT1, -2, and -3 modulating activities of compounds 1-3 tested at 50 μM.
corresponding 3,5-dicarboxy derivatives 2a-d. Finally, these
last compounds reacted with triethylamine, benzotriazole-1-
yloxytris(dimethylamino)phosphonium hexafluoropho-
sphate (Bop reagent), and 33% aqueous ammonia under N2
atmosphere to give the 3,5-diamides 3a-d (Scheme 1). Among
the synthesized derivatives, compounds 1b, 1c, and their
corresponding acids 2b and 2c are known in the literature
(1b and 2b,40 1c,41 2c42). Chemical and physical data for
compounds 1-3 are reported in Table 1.
Results and Discussion
SIRT1, -2, and -3 Modulator Activity: Enzyme and Func-
tional Assays. All the prepared compounds were tested against
human SIRT1, SIRT2, and SIRT3 at 50 μM, using the Fluor

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5499
Figure 3. Dose-dependent SIRT1, -2, and -3 activation by the N-benzyl DHPs 1c, 2c, and 3c.
HDAC inhibitor SAHA45 (suberoylanilide hydroxamic acid,
3 h, 5 μM) to amplify the acetylation of R-tubulin.43,44 After
the wash out at 3 h of SAHA incubation, the effects of 1c, 2c,
and 3c, used at 50 μM, in comparison with vehicle or SAHA
treatment (5 μM) on tubulin acetylation levels after 5 h of
treatment, have been assessed. As depicted in Figure 4, the
tested N-benzyl-DHP compounds induced hypoacetylation
on R-tubulin in U937 cells. In SAHA-untreated U937 cells,
the SIRT activators showed no effects on tubulin acetylation
(data not shown).
Figure 4. Effects of SIRT activators 1c, 2c, and 3c (50 μM) on
R-tubulin acetylation in SAHA-pretreated U937 cells.
de Lys fluorescent biochemical assay (BioMol) (Figure 2.
Absolute values of SIRT modulation activities by 1-3 have
been reported as Supporting Information, Figure S1). The
new 1,4-dihydropyridine (DHP) derivatives 1-3 showed a
wide range of SIRT modulating activities, depending on their
substituent at the N1 position and/or at the 3,5 position of the
DHP ring. In particular, compounds carrying a cyclopropyl, a
phenyl, or a phenylethylgroup at N1 inhibited SIRT1 and, toa
lesser extent, SIRT2, while they were inactive against SIRT3.
The highest SIRT1 inhibition activities were seen with the N1-
phenyl analogues bearing a carboxy or a carboxamido group
at the 3,5 positions (2b and 3b, 86.6% and 89.1% of SIRT1
inhibition at 50 μM), followed by the 1-phenethyl- and 1-
phenyl-3,5-dicarbethoxy derivatives 1d and 1b (76.6% and
63.6% of inhibition, respectively). Against SIRT2, the diester
compounds 1a, 1b, and 1d showed the highest inhibition
activity, with comparable potency (1a: 53.7%; 1b: 51.3%;
1d: 49.6% of SIRT2 inhibiting activity at 50 μM).
Surprisingly, the DHPs bearing a benzyl group at the N1
position, regardless of their substitution at the 3,5 positions
(either a carbethoxy (1c), carboxy (2c), or carboxamido (3c)
moiety), did not inhibit the tested sirtuins but behaved as
potent, dose-dependent SIRT1, -2, and -3 activators
(Figure 3). In the SIRT1 assay, 1c and 2c were the most
potent derivatives, with a EC150 (effective concentration able
to increase the enzyme activity of 150%) around 1 μM, while
the 3,5-dicarboxamide 3c was less effective (EC150=36 μM).
Against SIRT2, the dicarboxy derivative 2c displayed the
highest activating potency (EC150=15 μM), followed by the
dicarbethoxy compound 1c (EC150=25 μM), while 3c was
less efficient. Also in the SIRT3 assay, although being less
effective than in SIRT1 and -2 assays, 1c and 2c exhibited
higher activation activity than 3c (EC150 values: 1c and 2c,
around 50 μM; 3c, >50 μM).
Biological Cellular Activities. All the epigenetic modula-
tors prepared in our lab are routinely tested, initially, in the
human U937 leukemia cell line, to study their effects on cell
cycle, apoptosis induction, and granulocytic differentia-
tion.46 As such, compounds 1-3 were tested in U937 cells
at 50 μM for 30 h and displayed marginal effects in these
experimental conditions (Supporting Information, Figure
S2). In particular, the three SIRT activators 1c, 2c, and 3c
arrested the cell cycle at the G1/S phase, with an effect that
could be related to p53 deacetylation as well as to effects on
multiple targets within the cell, while a slight if at all
apoptotic induction (3-4%) was observed with the diester
inhibitors 1a, 1b, and 1d. Granulocytic differentiation was
evaluated by the increase of expression of CD11c positive,
propidium iodide (PI) negative cells: in this assay, only the
diethyl 1-phenethyl-4-phenyl-1,4-dihydropyridine-3,5-di-
carboxylate 1d gave a significant (12.3%) increase, with
an effect that seems not to be related to its SIRT1 and -2
inhibition activity. When tested in the MCF-7 breast
cancer cell lines, these compounds did not show significant
effects, thus suggesting to exert marginal antitumor activ-
ities both in hematological and solid cancer models (data
not shown).
Inhibition of SIRT1 by sirtinol or splitomicin (another
well-known SIRTi identified through a phenotypic screening
in yeast) induced senescence in the H1299,19 HUVEC,47 and
MCF-719 cell lines. In addition, SIRT1 has been reported to
gradually decrease in aging cells,48 whereas SIRT1 activity
was able to overcome p53/PML-induced senescence.30 Thus,
agents able to improve SIRT1 activity could be useful against
senescence, and indeed RSV has been shown to increase
lifespan in many species including rodents.32 Normal pri-
mary cells proliferate in culture for a limited number of
doublings prior to undergoing an irreversible growth-ar-
rested stage called replicative senescence, acquiring a senes-
cent phenotype. The most used biomarker for senescent and
aging cells is senescence-associated β-galactosidase (SA-β-
gal), which identifies the senescent cells after cytoplasmatic
coloration in blue color.49 From these data, we tested our
SIRT activators 1c, 2c, and 3c (all used at 50 μM) in primary
human mesenchimal stem cells (hMSC), to determine their
potential protection against senescence. RSV (50 μM) has
The new SIRT activators 1c, 2c, and 3c were tested in
functional assay to determine their effect on acetylation level
of R-tubulin, a known substrate for SIRT2. Western blot
analysis was performed on the human leukemia U937 cell
line. Because U937 cells typically show weak basal acetyla-
tion levels for R-tubulin,43,44 the expected hypoacetylation
effect from the SIRT activators may be difficult to appreci-
ate. Thus, we pretreated the cells with the well-known

5500 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Mai et al.
Figure 5. β-Galactosidase assay in hMSC. (A) Percent of β-gal positive hMSC treated with RSV, 1c, 2c, or 3c at 50 μM for 48 h. (B) Optic
microscope images (10ꢀ) of treated hMSC.
Figure 6. Mitochondrial function in C2C12 myoblasts. (A) Fluorescence intensity of C2C12 myoblasts treated for 16 h with DMSO (0), or
increasing concentrations of 1c, or 50 μM RSV, and stained with Mitotracker Green. The data represent the values obtained after subtraction
of the mean fluorescence background (unstained cells). (B) Scheme of the constructs carrying the wild type and mutant mTFA promoters.
(C) Transcriptional activity of wild type (white bars) and mutant (black bars) mTFA promoter in C2C12 myoblasts treated for 16 h with
increasing concentrations of 1c (left panel) and RSV (right panel). * p < 0.05, ** p < 0.01 versus DMSO (0) treated cells (Student’s t-test).
been used as a reference drug. After 48 h of treatment, the
number of β-galactosidase positive cells, used as a marker of
senescence, have been counted (Figure 5A). Optic micro-
scopy images (10ꢀ) of the cell cultures have been also
obtained (Figure 5B). In these conditions, considering the
amount of senescent positive cells in the control population
as 100%, 1c, 2c, and 3c gave 60%, 70%, and 65% positive
cells, respectively, in comparison with 50% positive cells by
RSV.
Because RSV was reported to improve mitochondrial
function in skeletal muscle, largely due to PGC-1R activa-
tion,29 we tested the ability of our SIRT activator 1c to
modulate mitochondrial function in murine C2C12 myo-
blasts. As shown in Figure 6A, treatment with compound 1c

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5501
for 16 h led to a significant increase of fluorescence intensity
due to the mitochondrial specific probe Mitotracker Green,
indicating increased mitochondrial density. The effect was
similar to that observed with 50 μM RSV. Mitochondrial
transcription factor A (mTFA) is crucial for mitochondrial
DNA replication/transcription, and it was described that its
expression is controlled by transcription factors belonging to
the nuclear respiratory factor (NRF) family and by PGC-
1R.50 Therefore, we investigated whether SIRT activators
were able to increase the transcriptional activity of the
mTFA promoter bearing the binding site for NRF1
(Figure 6B), whose activity is PGC-1R-dependent. We found
that both compound 1c (Figure 6C, left panel) and RSV
(Figure 6C, right panel) stimulated the promoter activity in a
dose-dependent manner. The effect of both the SIRT acti-
vators is PGC-1R-dependent as the effect was not seen upon
deletion of the NRF1 site.
solid product was filtered and washed with Et2O (3ꢀ 30 mL) to
give pure 1a, which was recrystallized by cyclohexane. 1H NMR
(400 MHz, CDCl3) δ 0.83-0.90 (m, 4 H, cyclopropane protons),
1.14-1.17 (t, 6 H, COOCH2CH3), 2.94-2.99 (m, 1 H, cyclo-
propane proton), 3.97-4.11 (q, 4 H, COOCH2CH3), 4.83 (s, 1
H, PhCH), 7.08-7.23 (m, 5 H, benzene protons), 7.31 (s, 2 H,
dihydropyridine protons) ppm. 13C NMR (400 MHz, CDCl3) δ
6.10 (2C), 14.20 (2C), 38.50, 44.50, 61.70 (2C), 108.0 (2C),
125.80, 128.70 (2C), 129.10 (2C), 142.20, 146.10 (2C), 167.20
(2C) ppm. MS (EI): m/z: 341 (M)þ.
General Procedure for the Synthesis of 1-Aryl-(or Arylalkyl, or
Cycloalkyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylic Aci-
ds (2a-d). Example: 1-Phenethyl-4-phenyl-1,4-dihydropyridine-
3,5-dicarboxylic acid (2d). A mixture of 1d (1.23 mmol, 0.5 g) and
5 N KOH (6.15 mmol, 0.34 g, 1.23 mL) in ethanol (10 mL) was
stirred at 80 ꢀC overnight. Afterward, the solvent was evapo-
rated, the residue was eluted with water (30 mL), and the
resulting solution acidified with 2 N HCl. The obtained pre-
cipitate was filtered, washed with water (3ꢀ 30 mL) and dried to
afford pure compound 2d, which was recrystallized by acetoni-
1
trile/methanol. H NMR (400 MHz, DMSO-d6) δ 2.95-2.99
Conclusion
(t, 2 H, PhCH2CH2), 3.61-3.65 (t, 2 H, PhCH2CH2), 4.82 (s, 1
H, PhCH), 7.07-7.34 (m, 12 H, benzene protons and dihydro-
pyridine protons), 12.00 (bs, 2 H, COOH) ppm. 13C NMR
(400 MHz, DMSO-d6) δ 42.00, 43.80, 57.70, 107.30 (2C),
125.70, 125.90, 127.70 (4C), 128.60 (4C), 139.40, 144.40,
148.90 (2C), 171.30 (2C) ppm. MS (EI); m/z: 349 (M)þ.
In conclusion, we have reported the first investigation of
DHP-based compounds on the sirtuin modulation activity.
The insertion of either a cyclopropyl, phenyl, or phenethyl
group at the N1 position of the DHP ring led to SIRT1 and, to
a lesser extent, SIRT2 inhibition. Conversely, the insertion of
a benzyl group at the same position furnished three new
potent SIRT1, -2, and -3 activators, compounds 1c, 2c, and
3c, lacking the polyphenolic structure of RSV, a pleiotropic
drug reported as the first SIRT1 activator. Similarly to RSV,
in a senescence assay performed with hMSC, 1c, 2c, and 3c
were able to reduce the number of senescent cells up to 40%.
Furthermore, when tested in murine C2C12 myoblasts, 1c
showed a dose-dependent increase of mitochondrial function,
with a mechanism involving PGC-1R. Further studies are
ongoing to improve the SIRT activation activity of our
N-benzyl-DHPs and to optimize the inhibition profile of the
N-cyclopropyl and N-phenyl DHPs.
General Procedure for the Synthesis of 1-Aryl-(or Arylalkyl, or
Cycloalkyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxamides
(3a-d). Example: 1-Phenyl-4-phenyl-1,4-dihydropyridine-3,5-di-
carboxamide (3b). Triethylamine (10.38 mmol, 1.44 mL) and
BOP reagent (3.12 mmol, 1.38 g) were added under nitrogen
atmosphere to a solution of compound 2b (0.47 mmol, 0.150 g)
in dry DMF (5 mL), and the resulting mixture was stirred for
30 min at room temperature. Afterward, 33% aqueous ammo-
nia (4.7 mmol, 0.27 mL) was added, and the resulting mixture
was stirred for further 30 min. The reaction was quenched with
water (30 mL), the filtered precipitate was washed with water
(3ꢀ 30 mL) and Et2O (3ꢀ 30 mL) to provide 3b, which was
recrystallized by acetonitrile/methanol. 1H NMR (DMSO-d6) δ
5.07 (s, 1 H, PhCH), 6.73 (bs, 4 H, CONH2), 7.02-7.53 (m, 12 H,
benzene protons and dihydropyridine protons) ppm. 13C NMR
(400 MHz, DMSO-d6) δ 45.10, 110.20 (2C), 116.30 (2C), 118.80,
125.80, 128.70 (2C), 129.60 (2C), 138.10 (2C), 141.30, 142.20,
171.0 (2C) ppm. MS (EI): m/z: 319 (M)þ.
SIRT Assay. Modulation of sirtuin activity by compounds
was assessed using the Fluor de Lys fluorescent biochemical
assay available through BioMol International, LP in 96-well
format. In the first part of a two-step reaction, an acetylated
lysine side chain present on the substrate is deacetylated during
incubation at 37 ꢀC for an hour with active enzyme (SIRT1,
SIRT2, or SIRT3), compounds 1-3, and NADþ in white,
96-well polystyrene luminescence plates (Perkin-Elmer-
6005680). The latter half of the reaction produces a fluorophore
upon treatment with a developing reagent. The reaction is read
by a Perkin-Elmer Victor2V 1420 Multilabel Counter plate
reader with filters that excite at 355 nm and detect emitted light
at 460 nm with a read time of 0.1s per well.
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. Infrared (IR)
spectra (KBr) were recorded on a Perkin-Elmer Spectrum One
1
instrument. H NMR and 13C NMR spectra were 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 TLC and 1H NMR. TLC was performed on
aluminum-backed silica gel plates (Merck DC, Alufolien Kie-
selgel 60 F254) with spots visualized by UV light. All solvents
were reagent grade and, when necessary, were purified and dried
by standard methods. Concentration of solutions after reactions
and extractions involved the use of a rotary evaporator operat-
ing at reduced pressure of ca. 20 Torr. Organic solutions were
dried over anhydrous sodium sulfate. Analytical results are
within (0.40% of the theoretical values. All chemicals were
purchased from Aldrich Chimica, Milan (Italy), or from Lan-
caster Synthesis GmbH, Milan (Italy), and were of the highest
purity.
General Procedure for the Synthesis of Diethyl 1-Aryl-(or
Arylalkyl, or Cycloalkyl)-4-phenyl-1,4-dihydropyridine-3,5-di-
carboxylates (1a-d). Example: Diethyl 1-cyclopropyl-4-phenyl-
1,4-dihydropyridine-3,5-dicarboxylate (1a). Ethyl propiolate
(18.84 mmol, 1.9 mL), benzaldehyde (9.42 mmol, 0.96 mL),
and cyclopropylamine (9.42 mmol, 0.65 mL) in glacial acetic
acid (0.5 mL) were heated at 80 ꢀC for 30 min. After cooling, the
mixture was poured into water (20 mL) and stirred for 1 h. The
Sirtuin 1 enzyme (BioMol-SE-239) is a 747 amino acid protein
with a molecular weight of 82 kDa, while Sirtuin 2 enzyme
(BioMol-SE-251) is a 389 amino acid protein with a molecular
weight of 43 kDa; both were purified from human cDNA
expressed in Escherichia coli and stored in 25 mM Tris, pH
7.5, 100 mM NaCl, 5 mM DTT, and 10% glycerol. Sirtuin 3
enzyme (BioMol-SE-270) is inactive prior to removal of its N-
terminus, thus this assay only utilizes the catalytically active
fragment spanning amino acids 102-299 with a molecular
weight of 32.7 kDa. Like sirtuins 1 and 2, the fragment was
purified from cDNA expressed in E. coli and stored in 25 mM
Tris, pH 7.5, 100 mM NaCl, 5 mM DTT, and 10% glycerol.

5502 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Mai et al.
While there is variability in activity among individual lots, each
experiment normalizes SIRT1 activity to 1U/reaction well and
SIRT2 and SIRT3 activity to 5U/reaction well (where U = 1
pmol/min at 37 ꢀC, 250 μM substrate, and 500 μM NADþ). All
reagents are diluted on ice in the following reaction buffer:
50 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM
MgCl2, and 1 mg/mL BSA. Thus for each reaction well, the
requisite amount of enzyme (as detailed above) is added to
500 μM NADþ (BioMol-KI-282), 250 μM fluorgenic deacety-
lase substrate (BioMol-KI-177), and the compound of interest
at a given concentration in a total reaction volume of 50 μL.
After an hour incubation at 37 ꢀC, the reaction is stopped upon
addition of 1ꢀ Developer (BioMol-KI-176) for a final reaction
volume of 100 μL. The reaction is incubated at 37 ꢀC for an
additional 15 min and then read on the plate reader. Experi-
mental replicates were done in triplicate with appropriate con-
trols. Positive controls contained only enzyme, substrate,
NADþ, and DMSO while background controls contained sub-
strate, NADþ, and DMSO only. Autofluorescent controls were
also included for testing of activators and included substrate,
NADþ, and the compound at the same concentration as its
experimental counterpart. Background signal was subtracted
from positive control well signals, and autofluorescent back-
ground was subtracted from experimental well signals at all
tested compound doses.
well) and treated overnight with the compounds under investi-
gation. Mitochondria were stained with the Mito-Tracker
Green FM dye (200 nM) from Molecular Probes (Invitrogen,
Milan, Italy). Fluorescence intensities were measured using the
EnVision multilabel reader platform (PerkinElmer, Italia Spa,
Monza, Italy). The data shown represent the values obtained
after subtraction of the mean fluorescence background mea-
sured in wells with unstained cells.
Transcriptional Activity of mTFA-Promoter in Murine
C2C12 Myoblasts. Murine C2C12 myoblasts (CRL-1772,
ATCC, Teddington, UK) were grown in Dulbecco’s Modified
Eagle’s Medium supplemented with 10% heat inactivated fetal
calf serum. Cells were retrotransfected with the plasmids bearing
either the mTFA-promoter or the mutated form lacking the
NRF1 binding site,50 fused to the luciferase gene. Lipofectamine
(Invitrogen, Milan, Italy) was used as transfecting reagent
(2.5 μL/μg DNA). Transfected cells were then plated in 96-well
clusters (3000 cells/well) and treated overnight with the com-
pounds under investigation. The luciferase activity was mea-
sured using the EnVision multilabel reader platform
(PerkinElmer Italia Spa, Monza, Italy). The constructs used in
the transfection assays were first validated overexpressing PGC-
1R or SIRT1. In both conditions the wild type promoter was
stimulated by 2-3 fold, whereas the mutant promoter lacking
the NRF1 binding site did not respond.
Determination of r-Tubulin Specific Acetylation. For R-tubu-
lin acetylation studies, 50 μg of total protein extracts (U937
cells) were separated on a 10% polyacrylamide gels and
blotted.51 Western blots were shown for acetylated R-tubulin
(Sigma, dilution 1:500) and total ERKs (Santa Cruz) were used
to normalize for equal loading.
Acknowledgment. This work was supported by grants
from AIRC, RETI FIRB, European Union (APOSYS, AT-
LAS), Fondazione Luigi Califano ONLUS, Fondazione
Roma, and RJG and Carmen’s Foundations.
Cell Cycle Analysis on U937 Cells. First, 2.5ꢀ 105 cells were
collected and resuspended in 500 μL of a hypotonic buffer (0.1%
Triton X-100, 0.1% sodium citrate, 50 μg/mL propidium iodide
(PI), RNase A). Then 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) and analyzed
with standard procedures using the Cell Quest software (Becton
Dickinson) and the ModFit LT version 3 Software (Verity) as
previously reported.51 All experiments were completed in tripli-
cate.
Supporting Information Available: Elemental analyses and 1H
NMR/13C NMR/mass spectra of compounds 1-3. Absolute
values of modulation of SIRT1, -2, and -3 activities by 1-3.
Effects of 1-3 (50 μM, 30 h) on cell cycle, apoptosis induction,
and granulocytic differentiation in human U937 leukemia cells.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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