Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2

Article abstract of DOI:10.1016/j.ejmech.2012.07.001

As part of an effort to identify novel selective modulators of sirtuins, we synthesized and tested several isosteres and constrained analogues of nicotinamide. Biological data suggest that compound 2 is selective for Sirt3 over Sirt1 and Sirt2.

Full text of DOI:10.1016/j.ejmech.2012.07.001

European Journal of Medicinal Chemistry 55 (2012) 58e66
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of a sirtuin 3 inhibitor that displays selectivity over sirtuin 1 and 2
Ubaldina Galli, Ornella Mesenzani, Camilla Coppo, Giovanni Sorba, Pier Luigi Canonico,
*
*
Gian Cesare Tron , Armando A. Genazzani
Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale “A. Avogadro”, Largo Donegani 2, 28100 Novara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of an effort to identify novel selective modulators of sirtuins, we synthesized and tested several
isosteres and constrained analogues of nicotinamide. Biological data suggest that compound 2 is selective
for Sirt3 over Sirt1 and Sirt2.
Received 3 April 2012
Received in revised form
26 June 2012
Accepted 1 July 2012
Available online 15 July 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Sirtuins
Histone deacetylase
Isostere
Rigid analogues
N-Unsubstituted-1,2,3-triazolyl pyridine
1. Introduction
modulators of Sirt1 and Sirt2. On the other hand, the character-
ization of the pharmacology of other sirtuin members is partly
Acetylation and deacetylation are crucial post-translational
mechanisms to alter the activity of proteins. For example, it is
now well established that this mechanism plays a crucial role in
regulating gene expression programs via the acetylation and
deacetylation of histones [1]. Although the enzymes deputed to
deacetylation can recognize many substrates, they are historically
referred to as histone deacetylases (HDACs), and are considered as
good drug targets in a number of contexts, including cancer therapy
[2]. Sirtuins are set apart from other HDACs as they use NAD to
remove the acetyl group from lysine. Seven mammalian members
of the sirtuin family have been described to date, with different
cellular localizations and different substrate specificity [3,4]. The
fact that their activity is dependent on NAD has led to the
hypothesis that these proteins may act as energy sensors in cells,
and indeed they have been shown to participate in a number of
functions, ranging from gene expression to longevity and metab-
olism [3,5]. In this respect, the possibility to use modulators of
sirtuin activity to combat a number of diseases, including the
metabolic syndrome, diabetes and cancer, has been postulated and
drugs are currently being developed in this direction [6,7]. In
particular, the main focus of the industry is at present on
neglected. For example, for Sirt3, which is predominantly or
exclusively mitochondrial, and has been demonstrated to modulate
the acetylation of a number of mitochondrial proteins [4], no
specific activators or inhibitors are known and most information
gathered on this enzyme comes from cellular and molecular
approaches [6,7].
A common feature of most NAD-consuming enzymes is their
sensitivity to nicotinamide inhibition, and sirtuins are no exception
[7]. Indeed, it has also been postulated that nicotinamide could
function as an endogenous negative modulator of sirtuin activity,
although the concentrations required are particularly high. With
this in mind, we set out to screen nicotinamide analogues to find
molecules which are able to discriminate between the different
sirtuins. We therefore decided to follow two classical medicinal
chemistry strategies. In the first one, we replaced the amide bond of
nicotinamide (1) with suitable isosteres, while the second strategy
relied on the synthesis of constrained analogues of nicotinamide.
Although a number of sirtuin modulators have been described to
date [8], it is surprising that this classical medicinal chemistry
strategy has not been fully exploited for nicotinamide analogues,
while it has been employed with success for acetyl-lysine
analogues [8]. In total, 10 compounds (2e11) were synthesized
(Fig. 1). While none of these compounds is novel in the literature, it
is, to our knowledge, the first time these have been investigated
with respect to sirtuin activity.
* Corresponding authors. Tel.: þ390321375827; fax: þ390321375821.
E-mail
addresses:
tron@pharm.unipmn.it
(G.C.
Tron),
genazzani@
pharm.unipmn.it (A.A. Genazzani).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.001

U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
59
Me
O
O
NH2
NH
NH
N
NH
N
N
N
N
N
N
6
7
O
rigid
analogues
O
NH2
O
isosteres
N
N
2
3
NH2
N
O
O
S
O
N
1
N
Me
8
9
NH2
NH2
NH
NH
O
N
N
O
4
5
N
N
10
11
Fig. 1. Synthesized isosteres and rigid analogues of nicotinamide.
2. Result and discussion
The methyl group can be considered as a steric blocker that reduces
the conformational freedom of nicotinamide.
2.1. Chemistry
To prepare compound 7, quinolinic acid was heated at reflux
with Ac2O and acetamide to give quinolimide [12]. Afterwards,
quinolimide was treated at ꢀ20 ꢁC with NaBH4. The two
regioisomers a and b were formed with a 3:1 ratio [13,14]. After
purification by crystallization in EtOH, the regioisomer a was
reduced to 7 refluxing it with zinc in acetic acid [15]. The same
reduction was carried out on the commercially available 3,4-
pyridindicarboxyimide to give the rigid analogue 10, although in
very low yield. The synthesis of the rigid analogue 8 commenced
with a Sonogashira coupling between the methyl ester of the 2-
chloronicotinic acid and trimethylsilylacetylene to afford the pro-
tected ethynyl derivative which was subsequently deprotected in
the presence of TBAF. The triple bond was then partially hydroge-
nated under Lindlar reduction conditions and the corresponding 2-
vinyl nicotinic acid was cyclized in the presence of an excess of
ammonium chloride and acetic acid to give the desired lactam [16].
Following the same strategy, compound 11 was prepared. In brief,
Sonogashira coupling between the ethyl ester of 4-chloronicotinic
acid and trimethylsilylacetylene, followed by deprotection gave
the 4-ethynyl derivative, which was then reduced to olefin. The
alkene was then cyclizated affording in 20% yield the desired lac-
tam. Finally, the two methyl derivatives of nicotinamide (6 and 9)
were easily synthesized by converting the corresponding carbox-
ylic acids to amides using ammonium chloride as ammonia source
under EDCI, HOBt condition (Scheme 2).
At first, we replaced the amide bond of nicotinamide with four
different isosteres which, in principle, should still be able to give rise
to hydrogen bond interactions, either as donors or acceptors. In
particular, we synthesized the N-unsubstituted and C-unsubstituted
1,2,3-triazoles [9], (2 and 3) the sulphonamide derivative (4) and the
vinylogous nicotinamide (5). Compound 2 was prepared in 64% yield
by reacting 3-ethinylpyridine with trimethylsilylazide using copper
(I) iodide as catalyst [10]. The reaction conditions allowed the direct
cleavage of the trimethylsilyl group. The same conditions were
employed for the synthesis of the regioisomer 3, starting from the 3-
azidopyridine prepared by the diazotationeazidation reaction of 3-
aminopyridine. The silyl protected triazole was obtained with
a fair 51% yield [11]. Finally, the silyl triazole was deprotected with
TBAF yielding 3 in quantitative yield. The pyridine sulphonamide 4,
was prepared starting from 3-pyridine sulphonic acid, which was
converted to sulphonyl chloride with PCl5 and then reacted with
ammonia carbonate. Finally, the vinylogous of nicotinamide 5, was
obtained by coupling the trans 3-pyridyl acrylic acid with ammo-
nium chloride using the classical EDCI/HOBt protocol (Scheme 1).
The generation of some constrained analogues of nicotinamide
can freeze different conformations of the amide. Therefore, this
strategy might be helpful either to identify compounds with
different selectivity for the different isoforms of sirtuins or to
increase the potency thanks to the decrease of the entropic penalty.
We have therefore synthesized four lactams (7, 10 and 8, 11) (five
and six-membered rings respectively) through an intramolecular
cyclization at the 2 and 4 position of the pyridine ring and two
methyl derivatives (6 and 9) at the 2 and 4 position of nicotinamide.
2.2. Biological results
To determine the activity of the compounds synthesized, we
proceeded with an enzymatic assay, testing all compounds at
Scheme 1. Synthesis of compounds 2e5. Reagents, conditions and yields: (a) TMS-azide, CuI cat., DMF/MeOH, 100 ꢁC, 65%; (b) NaNO2, urea, NaN3, H2SO4/H2O, 0 ꢁC to r.t., 95%; (c)
TMS-acetylene, CuI cat., DMF/MeOH, 100 ꢁC, 51%; (d) TBAF, THF, r.t., 99%; (e) PCl5, 130 ꢁC, 66%; (f) (NH4)2CO3, 100 ꢁC, 17%; (g) NH4Cl, HOBt, EDCI, N-methylmorpholine, DMF, r.t., 85%.

60
U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
O
O
O
OH
NH
O
a,b
c
OH
OH
NH
OH
+
NH
N
N
N
N
O
b
a
7
O
NH
NH
d
O
O
N
N
10
O
O
O
e, f
OMe
g, h
OMe
OEt
NH
N
Cl
O
N
N
8
Cl
O
NH
OEt
i, j
k, l
O
N
N
N
11
O
CH3
N
O
O
CH3
N
O
m
OH
n
NH2
OH
CH3
NH2
HCl
N
N
CH3
6
9
Scheme 2. Synthesis of compounds 6e11. Reagents, conditions and yields: (a) Ac2O, AcNH2, reflux, 46%; (b) NaBH4, CHCl3/CH3OH, ꢀ20 ꢁC, 85%; (c) Zn/CH3COOH, reflux, 38%; (d) Zn/
CH3COOH, reflux, 17%; (e) TMS-acetylene, CuI, Pd/C, PPh3, DIPEA, 80 ꢁC, 50%; (f) TBAF, THF r.t., 56%; (g) Pd Lindlar, H2, EtOAc r.t., 84%; (h) NH4Cl, CH3COOH, H2O, reflux, 25%; (i) TMS-
acetylene, CuI, DIPEA, Pd(PPh3)4, 80 ꢁC, 51%; (j) TBAF, THF, r.t., 70%; (k) Pd Lindlar, H2, EtOAc, r.t., 88%; (l) NH4Cl, CH3COOH, H2O, reflux, 20%; (m) NH4Cl, HOBt, EDCI, N-methyl-
morpholine, DMF, r.t., 79%; (n) NH4Cl, HOBt, EDCI, N-methylmorpholine, DMF, r.t., 69%.
a concentration of 1 mM. For Sirt1 and Sirt2, we used a commer-
cially available kit while for Sirt3 we used an in-house protocol
which employs Escherichia coli extracts over-expressing Sirt3 and
Z-MAL as the substrate. As shown in Table 1, none of the
compounds tested inhibited significantly the activity of Sirt1 or
Sirt2. The well known non-specific sirtuin inhibitor suramin, as
expected, was able to inhibit the activity of both enzymes. To our
surprise, compounds 2 and 6 inhibited Sirt3 activity to a similar
extent compared to nicotinamide.
more potent than nicotinamide (38 ꢂ 5
m
M and 23 ꢂ 3
mM respec-
tively) at inhibiting Sirt3. Compound 2 was as efficacious as nicotin-
amide at inhibiting the enzyme, while also high concentrations of 6
were unable to give maximal inhibition. The reason for the incom-
plete inhibition by high concentrations of 6 was not investigated
further. Nonetheless, the above data show that compounds 2 and 6
selectively inhibit Sirt3 over Sirt1 and Sirt2 and are more potent than
nicotinamide. Given that compound 2 was able to give full inhibition
we then decided to focus on the characterization of this compound.
We first proceeded to evaluate the possible cytotoxicity of this
compound through the NIH-NCI Developmental therapeutics
program (https://dtps7.ncifcrf.gov/). In brief, this program tests
cytotoxicity of selected compounds at a fixed concentration of
Given this result, we next proceeded to perform a concen-
trationeresponse curve of nicotinamide, and compounds 2 and 6
(Fig. 2). Nicotinamide inhibited Sirt3 activity with an IC50 of approx.
377 ꢂ 52
mM. Compounds 2 and 6 were both approximately 10-fold
10 mM. Cell growth was not significantly affected in 45 out of the 60
Table 1
tumoral cell lines (Table 2). This was estimated as cell growth in the
range of 80e120% of control. On the contrary, cell growth was
significantly affected (less than 50% of control) in 5 cell lines: CCRF-
CEM, COLO 205, OVCAR-4, OVCAR-8, and SK-OV-3.
Synthesized compounds and biological activity. Values represent activity compared
to control. Values are mean þ S.E.M. of 3e4 replicates. Similar results were obtained
at a concentration of 100
m
M. NA ¼ not assessed.
Structures
Sirt1 activity
[1 mM]
Sirt2 activity
[1 mM]
Sirt3 activity
[1 mM]
Given that the concentration used (10
mM) is below the IC50
determined for enzyme inhibition, we reasoned that this effect on
viability/cell growth may or may not be attributable to Sirt3 inhibi-
tion. Asouraimwasto determine the cellulareffectofSirt3 inhibition,
we then searched also for a cell line in which high concentrations
could be used without a significant impact on viability. The two
extremes of the cell lines analysed were HeLa (in which the IC50 for
Control
Suramin
Nicotinamide
2
3
4
5
6
7
8
9
10
11
100 ꢂ 5
18 ꢂ 1
NA
100 ꢂ 2
20 ꢂ 1
100 ꢂ 7
NA
71 ꢂ 3
20 ꢂ 2
16 ꢂ 2
77 ꢂ 3
110 ꢂ 6
110 ꢂ 25
36 ꢂ 1
110 ꢂ 9
118 ꢂ 25
89 ꢂ 3
113 ꢂ 17
84 ꢂ 9
88 ꢂ 6
97 ꢂ 14
108 ꢂ 3
99 ꢂ 2
105 ꢂ 5
88 ꢂ 9
86 ꢂ 4
101 ꢂ 2
117 ꢂ 4
125 ꢂ 4
92 ꢂ 2
100 ꢂ 5
115 ꢂ 3
109 ꢂ 1
110 ꢂ 1
111 ꢂ 3
115 ꢂ 3
107 ꢂ 2
145 ꢂ 2
136 ꢂ 2
cell death was 4.7 ꢂ 0.02
m
M) and SK-MEL-28 (in which 69 ꢂ 3% of
cells survived a 48 h treatment with a concentration of 100
mM).
Sirt3 is an understudied protein. It is thought that this protein is
localized in mitochondria, although there are reports that it may
also be elsewhere in the cell [16]. Mice deficient of Sirt3 has been
shown to be viable and to be metabolically normal under basal and

U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
61
Furthermore, it has been shown, via a proteomic approach, that
global acetylation of mitochondrial proteins is altered in Sirt3
deficient animals [17]. With this in mind, we prepared mitochon-
drial extracts of HeLa cells and probed them with an antibody that
recognizes acetylated-lysine. As shown in Fig. 3, acetylation of
mitochondrial proteins increased upon addition of 1 mM of
compound 2 for 1e6 h to cells prior to extraction. Our compound is
therefore most likely capable of inhibiting Sirt3 also in a cellular
context.
It is thought that Sirt3 is activated upon cellular stress and can
thereby regulate ATP production [4,18]. Indeed, it has been shown
that the absence of Sirt3 in mice results in a marked alteration of
ATP levels in vivo [18]. To probe whether our compound could
replicate these effects, we decided to incubate cells for 24 h with
compound 2 and to measure overall ATP levels with a commercial
kit based on luciferase. For these experiments, we opted to use SK-
MEL-28 cells as these cells did not display marked sensitivity in
terms of toxicity after 24 h and used compound 2 at a concentration
of 100 mM. When the incubation took place in regular medium,
containing growth factors and glucose, no significant changes on
ATP levels were observed. To unmask a possible effect of compound
2, therefore, we decided to incubate cells in a balanced saline
solution in the absence of glucose and growth factors (i.e. foetal calf
serum). As shown in Fig. 4, in this setting incubation for 24 h with
compound 2 reduced significantly the levels of ATP compared to
control. Similar results were obtained with nicotinamide.
Last, it has been shown that Sirt3 null mice display increased
levels of superoxide levels and difficulty in responding to increased
reactive oxygen species [21]. To investigate whether our compound
was capable of influencing cellular superoxide levels, we employed
an identical protocol compared to the one described above and
measured the oxidation the dihydroethidium (DHE) by fluores-
cence analysis. Surprisingly, compound 2 induced a significant
increase of superoxide both in basal conditions and when cells
were maintained in Locke’s solution (a balanced saline solution).
Fig. 2. Concentration-response curves of nicotinamide, 2 and 6. Sirt3 activity was
evaluated as described in the text. Values are mean þ S.E.M. of 8 replicates from 2
separate experiments.
mild-stress conditions [17]. Nonetheless, it has been reported that
Sirt3 may regulate the acetylation status of a number of crucial
mitochondrial proteins, such as complex I, acetyl-CoA dehydroge-
nase, glutamate dehydrogenase and cyclophilin
D [17e20].
Table 2
Effect of compound 2 on cell proliferation/viability in different cell lines. Experi-
ments were performed by the NIH/NCI therapeutics program as described in the
text. Values represent cell viability compared to the respective control.
Cell line
Growth percent
Cell line
Growth percent
(compound 2,
(compound 2,
10
m
M)
10 mM)
Leukaemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Breast cancer
MCF7
3. Conclusion
24.80
92.89
75.16
101.41
66.94
97.35
102.49
99.04
72.54
In conclusion, the present manuscript describes, to our knowl-
edge, the first inhibitor of Sirt3 over Sirt1 and Sirt2. The cellular
effects elicited by this compound (a decrease of ATP levels and an
increase of superoxide production) are compatible with reports on
Sirt3 null animals. Therefore, 2 might represent a useful tool to
investigate the role of Sirt3 in cells in acute settings. Yet a number
of limitations must be acknowledged. The fact that 2 is a simple
bioisosteric analogue of nicotinamide suggests that it might also
recognize other molecular targets and indeed this is the case.
Compound 2 has recently been shown to be an inhibitor of
methionine aminopeptidase 2 [22] at concentrations lower than
those reported here for Sirt3 and to be an inhibitor of indoleamine
2,3-dioxygenase at concentrations above those reported here [23].
Similarly, it is likely that these compounds will also inhibit NADþ-
dependent enzymes, including dehydrogenases. As a consequence,
compound 2 might have a number of off-targets that would require
examination and should be examined in the future.
55.99
109.32
105.77
102.72
90.30
101.28
76.43
90.88
80.75
80.62
90.69
113.25
89.52
47.38
100.38
49.76
71.31
48.03
89.61
104.40
114.66
101.45
23.77
77.34
82.36
69.18
39.86
115.89
105.83
103.65
105.42
110.59
95.13
98.58
Notwithstanding, we are confident that this compound will be
a novel starting point for further chemical modifications for the
identification of more potent and more selective Sirt inhibitors.
102.16
103.25
75.98
112.55
106.82
106.01
102.34
92.97
116.78
103.09
118.05
97.69
102.41
103.82
4. Experimental protocols
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
U251
Prostate cancer
PC-3
110.94
4.1. Chemistry
88.47
96.29
Commercially available reagents and solvents were used
without further purification and were purchased from
DU-145

62
U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
Brockmann III (Fluka Type 507) using the indicated eluants. Thin
layer chromatography (TLC) was carried out on 5 ꢃ 20 cm plates
with a layer thickness of 0.25 mm (Merck Silica gel 60 F254). When
necessary they were developed with KMnO4 or Dragendorff
reagent. Purity of tested compounds was established by elemental
analysis. Elemental analysis (C, H, N) of the target compounds are
within ꢂ0.4% of the calculated values, confirming ꢄ95% purity.
4.1.1. General methods
4.1.1.1. 3-(1H-1,2,3-Triazol-4-yl)pyridine (2). To a solution of 3-
ethynylpyridine (0.20 g, 1.94 mmol, 1 eq) in DMF/MeOH 9:1
(4.0 mL), CuI (0.02 g, 0.097 mmol, 0.05 eq) and trimethylsilylazide
(0.38 mL, 2.91 mmol,1.5 eq) were added. The reaction was heated at
100 ꢁC for 12 h under nitrogen. After cooling, the reaction was
filtered off through a Celite pad and washed with EtOAc. Evapora-
tion of the solvent gave a residue which was purified by column
chromatography, using PE/EtOAc 3:7 and then EtOAc to give 2 as
a white solid after crystallization with EtOAc (0.18 g, 64%). Mp
187e192 ꢁC. 1H NMR (300 MHz, CD3OD)
d 9.03 (s, 1-H), 8.51 (d,
J ¼ 3.7 Hz, 1-H), 8.29e8.27 (m, 2-H), 7.54e7.50 (m, 1-H); 13C NMR
(75 MHz, CD3OD)
d 148.3, 146.1, 143.1, 133.9, 127.3, 124.3; MS (ESI)
Fig. 3. Global mitochondrial acetylation in the presence or absence of compound 2 in
HeLa cells pre-treated for the indicated times. No toxicity was evident during the
incubation period. The data is representative of 3 separate experiments that yielded
similar results.
m/z 147 (M þ H)þ; IR (KBr) 3467, 3118, 1637, 1560, 1434, 1311,
1134 cmꢀ1. Anal. Calcd for C7H6N4: C, 57.53; H, 4.14; N, 38.34.
Found: C, 57.67; H, 4.25; N, 38.10.
FlukaeAldrich or Lancaster. Tetrahydrofuran (THF) was distilled
immediately before use from Na/benzophenone under a slight
positive atmosphere of N2. Dicholoromethane was dried by distil-
lation from P2O5 and stored on activated molecular sieves (4 Å).
Dimethylformamide was distilled under vacuum from KOH and
stirred on activated molecular sieves (4 Å). Methanol was dried by
distillation from magnesium. When needed the reactions were
performed in flame- or oven-dried glassware under a positive
pressure of dry N2.
4.1.1.2. 3-Azidopyridine. To a solution of 3.6 mL of H2SO4 98% wt and
21.0 mL of water heated at 55 ꢁC, 3-aminopyridine (1.89 g,
20.10 mmol,1 eq) was added. After 5 min, the solution was cooled at
0
ꢁC and a solution of sodium nitrite (1.66 g, 2.41 mmol, 1.2 eq in
14.0 mL of water) was added dropwise. After 20 min, urea was added
(0.24 g, 4.00 mmol, 0.2 eq) and after 20 min a solution of NaN3 (1.65 g,
24.00 mmol, 1.2 eq in 15 mL of water) was added dropwise. The
reaction was then stirred at room temperature for 1.5 h and then
quenched with saturatedaqueous NaHCO3 and extractedwithdiethyl
ether (ꢃ3). Evaporation of the solvent gave a brown oil which was
enough pure to be used for the next step without purification (95%).
Melting points were determined in open glass capillary with
a
Stuart scientific SMP3 apparatus and are uncorrected. All
1H NMR (300 MHz, CDCl3)
13C NMR (75 MHz, CDCl3)
d
8.23e8.19 (m, 2-H), 7.17e7.12 (m, 2-H);
compounds were checked by IR (FT-IR THERMO-NICOLET AVATAR),
1H and 13C APT (JEOL ECP 300 MHz), and mass spectrometry
(Thermo Finningan LCQ-deca XP-plus) equipped with an ESI source
and an ion trap detector. Chemical shifts are reported in part per
million (ppm). Column chromatography was performed on silica gel
(Merck Kieselgel 70e230 mesh ASTM) or neutral alumina oxide
d
145.9, 141.2, 137.0, 125.8, 124.1.
4.1.1.3. 3-(4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl)pyridine. To
a
solution of 3-azidopyridine (0.60 g, 5.00 mmol, 1 eq) in DMF/MeOH
9:1 (10.0 mL), CuI (0.05 g, 0.25 mmol, 0.05 eq) and
Fig. 4. Effect of compound 2 on ATP and superoxide levels. Cells were either maintained in growth medium (DMEM þ 10% serum) or in a balanced saline solution (Locke) in the
absence of glucose. (A) ATP levels in SK-MEL-28 cells treated with compound 2 (100
m
M) or antimycin (as a positive control) for 24 h. Data are mean þ S.E.M. of 10e14 replicates
from 3 separate experiments. (B) DHE fluorescence (indicative of superoxide) in SK-MEL-28 cells treated with compound 2 (100
Data are mean þ S.E.M. of 3e4 separate experiments in which 10000 cells were read by FACS analysis.
mM) or antimycin (as a positive control) for 24 h.

U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
63
trimethylsilylacetylene (0.74 mL, 7.50 mmol, 1.5 eq) were added.
The reaction was heated at 100 ꢁC for 2.5 h under nitrogen. After
cooling, the reaction was filtered off through a Celite pad and
washed with EtOAc. Evaporation of the solvent gave a residue
which was purified by column chromatography, using PE/EtOAc 4:6
to give the desired product as a white solid after crystallization with
J ¼ 15.9 Hz, 1-H); 13C NMR (75 MHz, CD3OD)
d
168.6, 149.5, 148.2,
137.1, 135.0, 131.5, 124.3, 123.1; MS (ESI) m/z 149 (M þ H)þ; IR (KBr)
3151, 3036, 2370, 1683, 1604, 1400, 980 cmꢀ1. Anal. Calcd for
C8H8N2O: C, 64.85; H, 5.44; N, 18.91. Found: C, 64.54; H, 5.12; N,
19.20.
PE (0.56 g, 51%). Mp 136e138 ꢁC. 1H NMR (300 MHz, CDCl3)
d
9.00
4.1.1.8. 4-Methylnicotinamide (6). To a solution of 4-methyl nico-
tinic acid hydrochloride (0.40 g, 2.30 mmol, 1 eq) in DMF dry
(4.0 mL), ammonium chloride (0.74 g, 13.90 mmol, 6 eq), HOBt
(0.45 g, 3.30 mmol, 1.44 eq), EDCI (0.64 g, 3.30 mmol, 1.44 eq) and
N-methylmorpholine (0.63 mL, 5.60 mmol, 2.44 eq) were subse-
quently added. The reaction was stirred at room temperature,
under nitrogen for 3 h. Evaporation of the solvent gave a residue
which was purified by column chromatography (neutral alumine
oxide, Brockmann grade III) using EtOAc as eluant to give 6 as white
solid after crystallization with EtOAc (0.25 g, 79%). Mp. 157e162 ꢁC.
(d, J ¼ 2.2 Hz, 1-H), 8.67 (dd, J ¼ 4.9/1.5 Hz, 1-H), 8.13 (dt, J ¼ 8.3/
1.5 Hz, 1-H), 7.95 (s, 1-H), 7.48 (dd, J ¼ 7.9/4.6 Hz, 1-H), 0.38 (s, 9-H);
13C NMR (75 MHz, CDCl3)
d 149.7, 148.1, 141.8, 133.8, 128.4, 127.1,
124.3, ꢀ1.1; MS (ESI) m/z 219 (M þ H)þ.
4.1.1.4. 3-(1H-1,2,3-Triazol-1-yl)pyridine (3). To a cooled (0 ꢁC)
solution of the silylated triazole (0.56 g, 2.55 mmol, 1 eq) in THF
(7.8 mL), TBAF 1 M in THF (3.06 mL, 3.06 mmol, 1.2 eq) were added
dropwise. The reaction was stirred at room temperature, under
nitrogen for 4 h. Evaporation of the solvent gave a residue which
was purified by column chromatography using EtOAc as eluant to
give 3 as a white solid after crystallization with PE/EtOAc (0.37 g,
1H NMR (300 MHz, CD3OD)
d
8.52 (s, 1-H), 8.43 (d, J ¼ 5.2 Hz, 1-H),
7.34 (d, J ¼ 5.2 Hz, 1-H), 2.49 (s, 3-H); 13C NMR (75 MHz, CD3OD)
d
170.8, 149.6, 146.8, 146.7, 132.9, 126.1, 18.2; GCeMS m/z 136 (M)þ;
99%). Mp 94e97 ꢁC. 1H NMR (300 MHz, CDCl3)
d
9.10 (d, J ¼ 2.4 Hz,
IR (KBr) 3335, 3046, 2800, 1685, 1597, 1430, 1175, 842 cmꢀ1. Anal.
Calcd for C7H8N2O: C, 61.75; H, 5.92; N, 20.58. Found: C, 62.10; H,
6.25; N, 20.60.
1-H), 8.67 (d, J ¼ 1.5 Hz, 1-H), 8.65 (d, J ¼ 0.9 Hz, 1-H), 8.24 (ddd,
J ¼ 8.2/2.4/1.2 Hz, 1-H), 7.95 (d, J ¼ 1.2 Hz, 1-H), 7.67 (ddd, J ¼ 8.2/
4.9/0.6 Hz, 1-H); 13C NMR (75 MHz, CDCl3)
d 149.3, 141.2, 134.3,
134.1, 128.7, 124.7, 123.2; MS (ESI) m/z 147 (M þ H)þ; IR (KBr) 3105,
2434, 1777, 1586, 1467, 1028 cmꢀ1. Anal. Calcd for C7H6N4: C, 57.53;
H, 4.14; N, 38.34. Found: C, 57.56; H, 4.14; N, 38.20.
4.1.1.9. 5H-Pyrrolo[3,4-b]pyridine-5,7(6H)-dione. Pyridine-2,3-dicar-
boxylic acid (8.40 g, 50.30 mmol, 1 eq) was suspended in acetic
anhydride (11.0 mL, 116.50 mmol, 2.3 eq) and the mixture was
heated at 120 ꢁC, distilling 9.5 mL of acetic acid. After cooling at
100 ꢁC the acetamide (6.70 g, 113.60 mmol, 2.2 eq) was added
portionwise in 5 min. The reaction was then heated at reflux for
2.5 h. After cooling at room temperature, the precipitate was
filtered off and washed with water (2 ꢃ 12 mL) to give the desired
product as grey solid after crystallization with EtOH/H2O 95:5
4.1.1.5. Pyridine-3-sulfonyl chloride. 3-Pyridine sulfonic acid (4.0 g,
25.1 mmol, 1 eq) and PCl5 (5.75 g, 27.70 mmol, 1.1 eq) were heated
at 130 ꢁC for 20 h. After cooling, ice was added and the mixture was
extracted with EtOAc (ꢃ3). The combined organic layers were
washed with saturated aqueous NaHCO3 (ꢃ1), brine (ꢃ1) and dried
over sodium sulphate. After filtration and evaporation of the
solvent a yellow oil is obtained. (2.94 g, 66%). 1H NMR (300 MHz,
(3.46 g, 47%). Mp. 230e231 ꢁC. 1H NMR (300 MHz, DMSO-d6)
d 11.63
(br s, NH), 8.93 (d, J ¼ 4.9 Hz, 1-H), 8.22 (d, J ¼ 7.7 Hz, 1-H), 7.74 (dd,
CDCl3)
d
9.17 (d, J ¼ 2.4 Hz, 1-H) 8.91 (dd, J ¼ 4.9/1.5 Hz, 1-H), 8.26
J ¼ 7.6/4.9 Hz, 1-H). IR (KBr) 3483, 3189, 3100, 1735, 1704 cmꢀ1
.
(ddd, J ¼ 8.3/2.5/1.8 Hz, 1-H), 7.57 (ddd, J ¼ 8.3/4.9/0.6 Hz, 1-H); 13C
NMR (75 MHz, CDCl3)
d
155.6, 147.5, 141.0, 139.7, 124.4; IR (KBr)
4.1.1.10. 7-Hydroxy-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one. To
a cooled solution (ꢀ20 ꢁC) of 5H-pyrrolo[3,4-b]pyridine-5,7(6H)-
dione (1.00 g, 6.76 mmol, 1 eq) in MeOH/CHCl3 1:1 (67.0 mL),
sodium borohydride (0.38 g, 10.13 mmol, 1.5 eq) was added por-
tionwise. After 30 min, the reaction was acidified with HCl 3 M
(pH ¼ 3) and after 10 min, the solution was basified with NaOH 2 M
(pH ¼ 9). Then, the reaction mixture was let reach room temper-
ature and the solvent evaporated. The residue was purified by
column chromatography, using MeOH/CH2Cl2 1:5 as eluant to give
a white solid formed by a mixture of the two isomers a and b with
a ratio of 3:1. (0.86 g, 84.6%). The solid was crystallized twice with
EtOH to give 0.44 g of the isomer a as white solid. Mp. 211e214 ꢁC.
3045, 1625, 1522, 1375, 1247, 1173 cmꢀ1
.
4.1.1.6. Pyridine-3-sulfonamide (4). Pyridine-3-sulfonyl chloride
(1.00 g, 6.13 mmol, 1 eq) and ammonium carbonate (1.77 g,
18.4 mmol, 3 eq) were heated at 100 ꢁC for 3 h. After cooling and
evaporation, the residue was purified by column chromatography,
using PE/EtOAc 4:6 as eluant to give 4 as yellowish solid. (0.16 g,
17%). Mp 106e108 ꢁC dec. 1H NMR (300 MHz, DMSO-d6)
d
8.96 (d,
J ¼ 2.4 Hz,1-H), 8.77 (dd, J ¼ 4.6/1.5 Hz,1-H), 8.16 (dd, J ¼ 8.3/1.8 Hz,
1-H), 7.60 (m, 3-H); 13C NMR (75 MHz, DMSO-d6)
d 153.1, 146.9,
140.7, 134.2, 124.6; MS (ESI) m/z 159 (M þ H)þ; IR (KBr) 3308, 2681,
1568, 1347, 1171 cmꢀ1. Anal. Calcd for C5H6N2O2S: C, 37.97; H, 3.82;
N, 17.71. Found: C, 38.15; H, 73.90; N, 18.10.
1H NMR (300 MHz, DMSO-d6)
d
9.10 (br s, NH), 8.74 (dd, J ¼ 4.8/
1.2 Hz, 1-H), 8.02 (dd, J ¼ 7.6/1.5 Hz, 1-H), 7.52 (dd, J ¼ 7.6/4.9 Hz, 1-
H), 6.51 (br s, eOH), 5.80 (br s, 1-H); 13C NMR (75 MHz, DMSO-d6)
4.1.1.7. (E)-3-(Pyridin-3-yl)acrylamide (5). To a solution of (E)-3-
(pyridin-3-yl)acrylic acid (0.40 g, 2.68 mmol, 1 eq) in DMF dry
(4.0 mL), ammonium chloride (0.85 g, 16.10 mmol, 6 eq), HOBt
(0.52 g, 3.86 mmol, 1.44 eq), EDCI (0.74 g, 4.20 mmol, 1.44 eq) and
N-methylmorpholine (0.43 mL, 3.90 mmol, 1.44 eq) were subse-
quently added. The reaction was stirred at room temperature,
under nitrogen for 3 h. After adding water, the reaction was filtered
off through a neutral alumine oxide pad, washing with EtOAc/
MeOH 9:1 (3 ꢃ 10 mL). The filtrate was evaporated and the residue
was purified by column chromatography, using EtOAc/MeOH 9:1 as
eluant to give 5 as white solid after crystallization with EtOAc
d
167.3, 166.1, 153.3, 131.7, 128.0, 125.0, 79.0; GCeMS m/z 150 (M)þ;
IR (KBr) 3270, 3000, 1740 cmꢀ1
.
4.1.1.11. 6,7-Dihydro-5H-pyrrolo[3,4-b]pyridin-5-one (7). To a solu-
tion of 7-hydroxy-6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one
(0.32 g, 2.13 mmol, 1 eq) in acetic acid (8.6 mL), zinc (0.56 g,
8.50 mmol, 4 eq) was added and the suspension was heated at
reflux for 24 h. After cooling at room temperature, the mixture was
filtered off through a Celite pad. The filtrate was evaporated and the
residue was taken up with CH2Cl2. To this solution CaCl2 was added
and the suspension was filtered. The filtrate was evaporated and
the residue was purified by column chromatography using EtOAc
and then EtOAc/MeOH 95/5 as eluant to give 7 as white solid after
crystallization with EtOH (0.11 g, 38%). Mp. 206 ꢁC (dec). 1H NMR
(0.34 g, 85%). Mp 154e156 ꢁC. 1H NMR (300 MHz, CD3OD)
d 8.71 (d,
J ¼ 2.1 Hz, 1-H), 8.51 (dd, J ¼ 4.9/1.5 Hz, 1-H), 8.05 (dt, J ¼ 7.9/1.8 Hz,
1-H), 7.57 (d, J ¼ 15.9 Hz, 1-H), 7.47 (dd, J ¼ 7.9/4.9 Hz, 1-H), 6.77 (d,

64
U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
(300 MHz, DMSO-d6)
d
8.66 (br s, 1-H), 8.63 (d, J ¼ 4.9 Hz, NH), 7.95
1232, 1068 cmꢀ1. Anal. Calcd for C8H8N2O: C, 64.85; H, 5.44 N, 18.91.
Found: C, 65.10; H, 5.80; N, 18.72.
(d, J ¼ 7.05 Hz, 1-H), 7.38 (dd, J ¼ 7.6/4.9 Hz, 1-H), 4.3 (s, 2-H); 13C
NMR (75 MHz, DMSO-d6)
d 168.7, 164.9, 153.0, 131.9, 126.4, 123.6,
47.1; GCeMS m/z 134 (M)þ; IR (KBr) 3352, 3199, 1707, 1607, 1411,
1138 cmꢀ1. Anal. Calcd for C7H6N2O: C, 62.68; H, 4.51; N, 20.88.
Found: C, 62.73; H, 4.50; N, 20.50.
4.1.1.16. 2-Methylnicotinamide (9). To a solution of acid 2-
methylpyridin-3-carboxylic (0.40 g, 2.90 mmol, 1 eq) in DMF dry
(4.0 mL), ammonium chloride (0.93 g, 17.50 mmol, 6 eq), HOBt
(0.57 g, 4.20 mmol, 1.44 eq), EDCI (0.81 g, 4.20 mmol, 1.44 eq) and
N-methylmorpholine (0.46 mL, 4.20 mmol, 1.44 eq) were subse-
quently added. The reaction was stirred at room temperature,
under nitrogen for 2.5 h. Evaporation of the solvent gave a residue
which was purified by column chromatography (neutral alumine
oxide, Brockmann grade III) using EtOAc as eluant to give 9 as white
solid after crystallization with EtOAc (0.27 g, 69%). Mp. 163e166 ꢁC.
4.1.1.12. Methyl 2-((trimethylsilyl)ethynyl)nicotinate. To a solution
of methyl-2-chloro nicotinate (3.00 g, 17.40 mmol, 1 eq) in DIPEA
(40.0 mL) under nitrogen, Pd/C 5% (2.50 g), PPh3 (0.68 g, 2.62 mmol,
0.15 eq), CuI (0.10 g, 0.52 mmol, 0.03 eq) and trimethylsilylacety-
lene (4.17 mL, 29.70 mmol, 1.7 eq) were subsequently added. The
mixture was then heated at 80 ꢁC for 8 h. After cooling, the reaction
was filtered off through a Celite pad and the filtrate was diluted
with water and extracted with diethyl ether (ꢃ3). The collected
organic layers were washed with brine (ꢃ1) and dried over sodium
sulphate. After filtration and evaporation of the solvent, the residue
was purified by column chromatography using PE/EtOAc 95:5 as
eluant to give the desired product as a brown oil. (2.03 g, 50%). 1H
1H NMR (300 MHz, CD3OD)
d
8.45 (dd, J ¼ 4.9/1.8 Hz, 1-H), 7.83 (dd,
J ¼ 7.6/1.5 Hz, 1-H), 7.30 (dd, J ¼ 7.6/4.9 Hz, 1-H), 2.61 (s, 3-H); 13C
NMR (75 MHz, CD3OD) d 172.0, 155.4, 149.2, 135.7, 132.2, 121.2, 21.2;
GCeMS m/z 136 (M)þ; IR (KBr) 2768, 2389, 1926, 1692, 1448,
1365 cmꢀ1. Anal. Calcd for C7H8N2O: C, 61.75; H, 5.92; N, 20.58.
Found: C, 61.91; H, 6.20; N, 20.10.
NMR (300 MHz, CDCl3)
7.29e7.24 (m, 1-H), 3.90 (s, 3-H), 0.26 (s, 9-H); 13C NMR (75 MHz,
CDCl3) 165.7, 152.4, 142.0, 138.1, 129.0, 122.5, 102.3, 100.4,
52.4, ꢀ0.31.
d 8.66e8.63 (m, 1-H), 8.18e8.13 (m, 1-H),
4.1.1.17. 1H-Pyrrolo[3,4-c]pyridin-3(2H)-one (10). To a solution of
3,4-pyridindicarboxamide (0.40 g, 2.7 mmol, 1 eq) in acetic acid
(12 mL), zinc (0.78 g, 11.9 mmol, 4.4 eq) was added and the
suspension was heated at reflux for 24 h. After cooling at room
temperature, the mixture was filtered off through a Celite pad. The
filtrate was evaporated and the residue was taken up with CH2Cl2.
To this solution CaCl2 was added and the suspension was filtered.
The filtrate was evaporated and the residue was purified by column
chromatography using EtOAc/MeOH 95/5 as eluant to give 10 as
yellowish solid after crystallization with EtOH (0.061 g, 17%). Mp.
d
4.1.1.13. Methyl 2-ethynylnicotinate. To a cooled (0 ꢁC) solution of
methyl 2-((trimethylsilyl)ethynyl)nicotinate (1.00 g, 4.29 mmol, 1
eq) in THF dry (10.0 mL), TBAF 1 M in THF (4.30 mL, 4.29 mmol, 1
eq) were added dropwise. The reaction was stirred at room
temperature for 20 min. Evaporation of the solvent gave a residue
which was purified by column chromatography using PE/EtOAc
95:5 as eluant to give the product as an orange oil. (0.27 g, 56%). 1H
190e193 ꢁC dec. 1H NMR (300 MHz, DMSO-d6)
d 8.84 (s, 1-H), 8.70
NMR (300 MHz, CDCl3)
J ¼ 8.3/1.8 Hz, 1-H), 7.29 (dd, J ¼ 8.0/4.6 Hz, 1-H), 3.86 (s, 3-H), 3.43
(s, 1-H); 13C NMR (75 MHz, CDCl3)
165.2, 152.5, 141.7, 138.0, 129.0,
123.0, 82.1, 52.6; GCeMS m/z 161 (M)þ; IR (KBr) 3267, 2953, 2111,
1731, 1561, 1425, 1281, 1080 cmꢀ1
d
8.64 (dd, J ¼ 4.9/1.8 Hz, 1-H), 8.15 (dd,
(br d, 2-H), 7.62 (d, J ¼ 5.5 Hz, 1-H), 4.42 (s, 2-H); 13C NMR (75 MHz,
DMSO-d6) d 169.1, 153.3, 151.6, 145.3, 129.0, 119.9, 45.2; GCeMS m/z
d
134 (M)þ; IR (KBr) 2856, 1728, 1692, 1455, 1206, 1026 cmꢀ1. Anal.
Calcd for C7H6N2O: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.75; H,
4.50; N, 20.63.
.
4.1.1.14. Methyl 2-vinylnicotinate. To
a
solution of methyl 2-
4.1.1.18. Ethyl 4-((trimethylsilyl)ethynyl)nicotinate. To a solution of
ethyl 4-chloro nicotinate (1.30 g, 7.00 mmol, 1 eq) in DIPEA
(17.0 mL) under nitrogen, Pd(PPh3)4 (1.62 g, 1.40 mmol, 0.2 eq),
CuI (0.27 g, 1.40 mmol, 0.2 eq) and trimethylsilylacetylene
(1.66 mL, 11.87 mmol, 1.7 eq) were subsequently added. The
mixture was then heated at 80 ꢁC for 8 h. After cooling, the
reaction was filtered off through a Celite pad and the filtrate was
diluted with water and extracted with diethyl ether (ꢃ3). The
collected organic layers were washed with brine (ꢃ1) and dried
over sodium sulphate. After filtration and evaporation of the
solvent, the residue was purified by column chromatography
using PE/EtOAc 95:5 as eluant to give the desired product as an
ethynylnicotinate (0.43 g, 2.68 mmol, 1 eq) in EtOAc (12.0 mL), Pd
Lindlar (0.13 g) was added. Then the reaction was equipped with
a balloon of hydrogen and stirred at room temperature. After 4 h,
the reaction mixture was filtered through a pad of Celite washing
with EtOAc. The filtrate was evaporated and the residue (as a brown
oil) was used as such for the next step. (0.37 g, 84%). 1H NMR
(300 MHz, CDCl3)
d
8.68 (dd, J ¼ 4.6/1.8 Hz, 1-H), 8.14 (dd, J ¼ 7.9/
1.8 Hz, 1-H), 7.60 (dd, J ¼ 16.8/10.7 Hz, 1-H), 7.21 (dd, J ¼ 7.6/4.6 Hz,
1-H), 6.48 (dd, J ¼ 16.8/2.2 Hz, 1-H), 5.58 (dd, J ¼ 10.7/2.2 Hz, 1-H),
3.93 (s, 3-H); 13C NMR (75 MHz, CDCl3)
d 167.0, 155.1, 138.5, 133.9,
134.0, 132.1, 124.3, 122.0, 121.7, 52.5; GCeMS m/z 163 (M)þ; IR (KBr)
2952, 1724, 1559, 1431, 1264, 1078 cmꢀ1
.
orange oil. (0.88 g, 51%). 1H NMR (300 MHz, CDCl3)
d 9.06 (s, 1-H),
8.58 (d, J ¼ 4.9 Hz, 1-H), 7.36 (dd, J ¼ 6.2/4.3 Hz, 1-H), 4.38 (q,
4.1.1.15. 7,8-Dihydro-1,6-naphthyridin-5(6H)-one (8). To a solution
of methyl 2-vinylnicotinate (0.40 g, 2.47 mmol, 1 eq) in acetic acid/
water 1:1 (4.6 mL), ammonium chloride was added (1.78 g,
33.58 mmol, 14 eq) and the reaction mixture was heated at reflux
for 4 h. After cooling, the reaction was filtered through a pad of
Celite and washed with EtOAc and EtOAc/MeOH 9:1. The filtrate
was evaporated and the residue was purified by column chroma-
tography using EtOAC and EtOAc/MeOH 95:5 as eluants to give 8 as
a white solid after crystallization with EtOAc. (0.10 g, 25%). Mp.
J ¼ 7.0 Hz, 2H), 1.39 (t, J ¼ 7.0 Hz, 3-H), 0.23 (s, 9-H); 13C NMR
(75 MHz, CDCl3)
d 164.8, 151.9, 151.5, 131.3, 127.8, 127.1, 106.1, 100.7,
61.6, 14.3, ꢀ0.35.
4.1.1.19. Ethyl 4-ethynylnicotinate. To a cooled (0 ꢁC) solution of
ethyl 4-((trimethylsilyl)ethynyl)nicotinate (0.79 g, 3.20 mmol, 1 eq)
in THF dry (8.0 mL), TBAF 1 M in THF (3.2 mL, 3.20 mmol, 1 eq) were
added dropwise. The reaction was stirred at room temperature
under nitrogen for 1 h. Evaporation of the solvent gave a residue
which was purified by column chromatography using PE/EtOAc 9:1
as eluant to give the product as a yellow oil. (0.39 g, 70%). 1H NMR
168e171 ꢁC. 1H NMR (300 MHz, CDCl3)
d
8.61 (dd, J ¼ 4.9/1.8 Hz, 1-
H), 8.29 (dd, J ¼ 7.9/1.8 Hz, 1-H), 7.30 (dd, J ¼ 7.6/4.9 Hz, 1-H), 7.04
(br s, NH), 3.65 (td J ¼ 6.7/2.7 Hz, 2-H), 3.18 (t, J ¼ 6.7 Hz, 2-H); 13C
(300 MHz, CDCl3)
d
9.14 (s, 1-H), 8.67 (d, J ¼ 5.2 Hz, 1-H), 7.45 (d,
NMR (75 MHz, CDCl3)
d
165.9, 158.8, 152.4, 135.8, 124.8, 122.7, 39.4,
J ¼ 5.2 Hz, 1-H), 4.42 (q, J ¼ 7.0 Hz, 2-H), 3.62 (s, 1-H), 1.40 (t,
31.0; MS (ESI) m/z 149 (M þ H)þ; IR (KBr) 2924, 1679, 1590, 1460,
J ¼ 7.0 Hz, 3-H); 13C NMR (75 MHz, CDCl3)
d 164.6, 152.2, 151.6,

U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
65
130.9, 128.1, 127.3, 87.5, 79.8, 61.8, 14.2; GCeMS m/z 175 (M)þ; IR
(KBr) 3260, 2926, 2105, 1720, 1276, 1109 cmꢀ1
deacetylated by sirtuins. Samples were read in a microplate-
reading fluorimeter (excitation 355e390 nm, emission 460 nm).
.
4.1.1.20. Ethyl 4-vinylnicotinate. To
a
solution of ethyl 4-
4.2.3. Measurement of hSirt1 and hSirt2 in vitro activity
ethynylnicotinate (0.06 g, 0.33 mmol, 1 eq) in EtOAc (1.7 mL), Pd
Lindlar (0.17 g) was added. Then the reaction was equipped with
a balloon of hydrogen and stirred at room temperature. After
40 min, the reaction mixture was filtered through a pad of Celite
washing with EtOAc. The filtrate was evaporated and the residue as
an orange oil was used as such for the next step. (0.054 g, 91%). 1H
Compound’s activity on hSirt1 and hSirt2 was tested in vitro
using SIRT1/SIRT2 Fluorimetric Drug Discovery Kit (BIOMOL). This
kit is based on Fluor-de-Lys substrate, a peptide comprising ami-
noacids 379e382 of human p53 (Arg-His-Lys-Lys(Ac)). The assay’s
fluorescence signal is generated in proportion of the amount of
deacetylation of the lysine-382, a known in vivo target of sirtuins. In
the first step of the assay, Fluor-de-lys substrate was incubated with
human Sirt1 or Sirt2, NADþ and compounds at a fixed concentra-
tion (1 mM). In a second step, treatment with a Developer II
produces a fluorophore. Samples were read in a microplate-reading
fluorimeter (excitation 350e380 nm, emission 450e480 nm) and
enzymatic activity of hSirt1 and hSirt2 was calculated as % of
control.
NMR (300 MHz, CDCl3)
d
9.05 (s, 1-H), 8.62 (d, J ¼ 5.2 Hz, 1-H), 7.48
(dd, J ¼ 17.4/11.0 Hz, 1-H), 7.42 (d, J ¼ 5.5 Hz, 1-H), 5.83 (dd, J ¼ 17.4/
0.9 Hz, 1-H), 5.52 (dd, J ¼ 11.0/0.9 Hz, 1-H), 4.37 (q, J ¼ 7.0 Hz, 2-H),
1.39 (t, J ¼ 7.0 Hz, 3-H); 13C NMR (75 MHz, CDCl3)
d 165.9, 152.5,
151.8, 146.8, 133.7, 124.2, 120.8, 120.4, 61.5, 14.3; GCeMS m/z 177
(M)þ; IR (KBr) 2926, 1720, 1588, 1556, 1384, 1277, 1108 cmꢀ1
.
4.1.1.21. 3,4-Dihydro-2,7-naphthyridin-1(2H)-one (11). To a solution
of ethyl 4-vinylnicotinate (0.22 g, 1.22 mmol, 1 eq) in acetic acid/
water 1: 1 (5.0 mL), ammonium chloride was added (0.96 g,
16.62 mmol, 14 eq) and the reaction mixture was heated at reflux
for 4 h. After cooling, the reaction was filtered through a pad of
Celite and the filtrate was evaporated. The residue was purified by
column chromatography using EtOAC and EtOAc/MeOH 95:5 as
eluants to give 11 as a white solid after crystallization with EtOAc.
4.2.4. Mitochondrial isolation and western blotting
For mitochondrial isolation, HeLa cells were grown in 150 cm [2]
flasks in the presence or absence of compound 2 (1 mM). After 1, 3
and 6 h, cells were trypsinized and washed once with ice-cold PBS.
Pellets were re-suspended in Buffer A (250 mM sucrose, 20 mM
HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM DTT,
protease inhibitors and phosphatase). After 15 min in ice, cells were
lysed with a syringe (27G) and centrifuged at 3300 rpm 4 ꢁC for
5 min. Supernatant, containing mitochondria and cytoplasmic
fraction, were centrifuged at 16,000 g 4 ꢁC for 15 min. Mitochon-
drial fraction (pellet) were lysed with Buffer B (20 mM Tris pH 7.5,
100 mM NaCl, 1% Triton, 1 mM DTT, protease inhibitors and phos-
phatase). Lysated were then corrected for protein amounts (Brad-
(0.035 g, 20%). Mp. 146e148 ꢁC. 1H NMR (300 MHz, CDCl3)
d 9.18 (s,
1-H), 8.63 (d, J ¼ 4.9 Hz, 1-H), 7.17 (d, J ¼ 4.9 Hz, 1-H), 6.91 (br s, 1-
H), 3.59 (td, J ¼ 6.4/2.8 Hz, 2-H), 3.00 (t, J ¼ 6.7 Hz, 2-H); 13C NMR
(75 MHz, CDCl3)
d 165.0, 152.6, 149.4, 147.6, 124.7, 122.1, 39.7, 27.7;
MS (ESI) m/z 149 (M þ H)þ; IR (KBr) 3415, 2944, 1658, 1601, 1481,
1335 cmꢀ1. Anal. Calcd for C8H8N2O: C, 64.85; H, 5.44; N, 18.91.
Found: C, 64.67; H, 5.34; N, 18.54.
ford assay, Sigma Aldrich) and 30
mg of protein were
electrophoresed on 10% v/v polyacrylamide SDS-PAGE gels. After
transfer of protein to nitrocellulose and blocking in TBS-Tween-20
with 5% BSA, acetylated-lysine were identified with a primary
antibody (1:500, Cell Signalling), anti-mouse secondary antibody
(1:5000, Biorad) and visualized by chemiluminescence (Amer-
sham, ECL Western Blotting System, GE Healthcare) either by film
exposure or by ChemiDoc hardware. The bands were normalized to
prohibitin, as an internal control.
4.2. Pharmacology
4.2.1. Cell culture and cytotoxicity assay
HeLa cells were cultured in MEM supplemented with 10% foetal
bovine serum, 2 mM
streptomycin (100 g/mL). SK-MEL-28 cells were cultured in DMEM
supplemented with 10% foetal bovine serum, 2 mM -glutamine,
penicillin (100 g/mL) and streptomycin (100 g/mL).
L-glutamine, penicillin (100 mg/mL) and
m
L
m
m
4.2.5. ATP measurement
For cytotoxicity assay cells were plated in 24-well plates and
grown for 48 h in the presence or absence of compound 2. On the
experimental day, cells were washed in Locke’s solution (134 mM
NaCl, 5 mM KCl, 4 mM NaHCO3, 10 mM HEPES [pH 7.6], 2.3 CaCl2,
1 mM MgCl2, 5 mM sucrose) and incubated at 37 ꢁC for 90 min with
ATP levels were determined by using the ATP bioluminescent
somatic cell assay kit (Sigma Aldrich). SK-MEL-28 cells were plated
in 96-well and grown for 24 h, in medium or in Locke’s solution, in
the presence or absence of compound 1, compound 2 and anti-
mycin as a positive control. On the experimental day, cellular ATP
was released using a somatic cell ATP releasing reagent. In each
well, the ATP assay mix solution, containing luciferase and luciferin,
was added. The amount of light emitted was immediately
measured with a luminometer.
MTT (250
mg/mL in Locke’s solution). The crystals formed were
solubilized in isopropyl alcohol/HCl before being read at 570 nm in
a spectrophotometer.
4.2.2. Protein expression and measurement of hSirt3 in vitro
activity
4.2.6. Measurement of intracellular superoxide levels
E. coli BL21 were transformed with hSirt3-pQE80 kindly
provided by the Prof. John Denu’s laboratory.
Transformed bacteria were grown to an A600 nm ¼ 0,6, and
The fluorescent dye dihydroethidium (DHE), obtained from
Sigma Aldrich, was used to estimate superoxide production. SK-
MEL-28 cells were plated in 6-well plates and grown for 24 h, in
medium or Locke’s solution, in the presence or absence of
compound 1, compound 2 and antimycin as a positive control. Cells
were washed once with PBS and labelled on culture plates at 37 ꢁC
in Locke’s solution (containing 5 mmol/L pyruvate) with DHE
induced with 0.15 mM isopropyl-
b-D-thiogalactopyranoside (IPTG)
at 20 ꢁC overnight.
For sirtuins in vitro activity assay, Z-(Ac)Lys-AMC (Z-MAL) were
synthesized. This substrate is formed by a fluorescent moiety, 7-
amino-4-methyl-coumarin, and by Z-
L
-lysine.
(10
m
mol/L; 1% DMSO).
All compounds were incubated for 6 h at 37 ꢁC with hSirt3,
extracted from bacteria, with a fixed concentration (1 mM) and in
the presence of NADþ and Z-MAL. The reaction was terminated by
adding nicotinamide and trypsin/that cuts Z-MAL only if it was
After 40 min, culture plates were placed on ice to stop the
reaction, cells were trypsinized and re-suspended in ice-cold PBS.
Samples were analyzed using a FACSVantage SE DiVa (Becton
Dickinson) cytofluorimeter (excitation 488 nm, emission 585 nm).

66
U. Galli et al. / European Journal of Medicinal Chemistry 55 (2012) 58e66
B.M. Dancy, W.J. Drury, R.J. Cotter, S.D. Taverna, P.A. Cole, Azalysine analogues as
probes for protein lysine deacetylation and demethylation, J. Am. Chem. Soc. 134
(2012) 5138e5148;
The mean fluorescence intensity (MFI) of 10,000 cells was analyzed
in each sample and corrected for autofluorescence from unlabelled
cells.
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N(epsilon)-acetyl lysine analogs, Mol. Biosyst. 7 (2011) 16e28.
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H.C. Kolb, K.B. Sharpless, The growing impact of click chemistry on drug
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Acknowledgements
(b) G.C. Tron, T. Pirali, R.A. Billington, P.L. Canonico, G. Sorba, A.A. Genazzani,
Click chemistry reactions in medicinal chemistry: applications of the 1,3-
dipolar cycloaddition between azides and alkynes, Med. Res. Rev. 28 (2008)
278e308.
Financial support from Università del Piemonte Orientale and
AIRC (IG10509)
[10] T. Jin, S. Kamijo, Y. Yamamoto, Copper-Catalyzed synthesis of N-unsubstituted
1,2,3-triazoles from nonactivated terminal alkynes, Eur. J. Org. Chem. 18
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