Simplification of the tetracyclic SIRT1-selective inhibitor MC2141: Coumarin- and pyrimidine-based SIRT1/2 inhibitors with different selectivity profile

Article abstract of DOI:10.1016/j.bmc.2011.01.025

In this report we describe the synthesis and biological characterization of two series of sirtuins' inhibitors (SIRTi), designed as simplification products of the previously reported SIRT1-selective inhibitor MC2141 (4). In the first series (5a-t) we report a number of 2-substituted-1,2-dihydrobenzo[f]chromen-3- ones with a marked selectivity for the inhibition of SIRT2 over SIRT1. Some of such derivatives showed also high pro-apoptotic (5i and 5l) and/or cytodifferentiating (5d, 5i, and 5o) properties in a human leukemia cell line (U937). The second group of SIRTi (6a-q) is characterized by some analogues of cambinol (3), a well known SIRTi active against the Burkitt lymphoma. Such compounds, differently from the unselective prototype, are endowed with a selective inhibition of SIRT1 over SIRT2, and, in some cases (6j, 6k, and 6q), are more efficient than 3 to induce apoptosis in U937 cells.

Full text of DOI:10.1016/j.bmc.2011.01.025

Bioorganic & Medicinal Chemistry 19 (2011) 3659–3668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Simplification of the tetracyclic SIRT1-selective inhibitor MC2141:
Coumarin- and pyrimidine-based SIRT1/2 inhibitors with different
selectivity profile
Dante Rotili a, Vincenzo Carafa b, Domenico Tarantino a, Giorgia Botta a, Angela Nebbioso b,
a,
Lucia Altucci b, , Antonello Mai
⇑
⇑
a Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Università degli Studi di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this report we describe the synthesis and biological characterization of two series of sirtuins’ inhibitors
(SIRTi), designed as simplification products of the previously reported SIRT1-selective inhibitor MC2141
(4). In the first series (5a–t) we report a number of 2-substituted-1,2-dihydrobenzo[f]chromen-3-ones
with a marked selectivity for the inhibition of SIRT2 over SIRT1. Some of such derivatives showed also
high pro-apoptotic (5i and 5l) and/or cytodifferentiating (5d, 5i, and 5o) properties in a human leukemia
cell line (U937). The second group of SIRTi (6a–q) is characterized by some analogues of cambinol (3), a
well known SIRTi active against the Burkitt lymphoma. Such compounds, differently from the unselective
prototype, are endowed with a selective inhibition of SIRT1 over SIRT2, and, in some cases (6j, 6k, and
6q), are more efficient than 3 to induce apoptosis in U937 cells.
Received 15 November 2010
Revised 22 December 2010
Accepted 13 January 2011
Available online 19 January 2011
Keywords:
Sirtuins
Selective inhibition
Apoptosis
Cytodifferentiation
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
both deacetylase and mART activities.3 Moreover, to date, no ro-
bust enzymatic activity has been found for SIRT7 as yet.
Class III histone deacetylases (HDACs), also called sirtuins
(SIRTs) from their founding member, the Sir2 (silent information
regulator 2) protein of Saccharomyces cerevisiae, are seven enzymes
in humans (SIRT1-7) which have NAD+ as a co-substrate for their
catalytic activity, show no homology with the other classes of
HDACs, and are sensible to specific inhibitors.1 SIRTs catalyze the
removal of the acetyl group from the lysine residues of their sub-
strates, coupling this event with the hydrolysis of NAD+ to generate
nicotinamide, deacetylated protein, and O-acetyl-ADP-ribose.1,2
The ADP-ribosyl transferase activity of sirtuins was long time con-
sidered a low efficiency side-reaction due to the partial uncoupling
of intrinsic deacetylation and acetate transfer to ADP-ribose.
Recently, however, mono-ADP-ribosyl transferase (mART) activity
has been found to be the main enzymatic activity of at least two
human sirtuins, SIRT4 and SIRT6, whereas SIRT2 and SIRT3 display
Like class I, II, IV HDACs, besides histones sirtuins deacetylate
and/or ADP-ribosylate many other important proteins, including
transcription factors (FOXO1, NF-
etc.), enzymes (AceCS1/2, GDH, p300), nuclear receptors (androgen
receptor), and other regulatory (HIV Tat) and structural ( -tubulin)
jB, MEF2, HOXA10, PGC1-a,
a
proteins, so modulating their own activities.1–4 These evidences
can explain the multiple biological functions of sirtuins, that range
from repression of gene expression to regulation of cellular
cytodifferentiating and/or apoptotic processes, from control of
energetic metabolism to aging.1–4 In yeast, Sir2 is critical for tran-
scriptional silencing at three specific loci: the telomeres, ribosomal
DNA, and the silent mating type loci.5,6 Sir2 and its homologues
have gained considerable attention for their ability to mimic the
diet known as caloric restriction, which extends lifespan in a vari-
ety of organisms, including yeast,7 Caenorhabditis elegans,8 ro-
dents,9 and probably primates.10 Since their catalytic activity
depends on the intracellular levels of the co-substrate NAD+ and
of the vitamin nicotinamide, that at physiological conditions exerts
the classical non-competitive product inhibition, sirtuins have also
been proposed as sensors of the cell metabolic state.1–4 Moreover,
since these enzymes in humans regulate cell survival under stress
conditions as well as neuro/cardio protection, stimulate insulin
secretion and lipolysis, and inhibit adipogenesis, sirtuins’ activa-
tors (such as resveratrol) have been proposed for the treatment
Abbreviations: BCL6, B-cell lymphoma 6; FACS, fluorescence activated cell
sorting; FOXO, forkhead box class O; NAD+, nicotinamide adenine dinucleotide;
NF-j
B, nuclear factor-
jB; PGC-1
a, peroxisome proliferator-activated receptor c
coactivator 1
a; SIRT, silent information regulator 2 type enzyme.
⇑
Corresponding authors. Tel.: +39 081 5667569; fax: +39 081 450169 (L.A.); tel.:
+39 06 49913392; fax: +39 06 49693268 (A.M.).
E-mail addresses: lucia.altucci@unina2.it (L. Altucci), antonello.mai@uniroma1.it
(A. Mai).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.025

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D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
of some age-related diseases (type 2 diabetes, neurodegeneration,
heart failure, etc.) and specific metabolic disorders (obesity,
atherosclerosis, etc.).11,12 Since sirtuins have been found upregu-
lated in many tumor types (SIRT1/3/7), are able to inactivate at
transcriptional and posttranslational level some tumor suppressor
proteins (p53, p73, etc.) and to activate the oncoprotein BCL6
(SIRT1), exert anti-apoptotic and anti-differentiation activities
(SIRT1) through deacetylation of specific transcription factors
(E2F1, FOXO3a, Ku70, etc.), and regulate DNA repair (SIRT1/6),
cell-cycle progression, and chromosomal stability (SIRT1/2), the
inhibitors of such proteins (SIRT1 in particular) have been
proposed as potential anti-cancer agents.13–15 Recently, a SIRT2
specific inhibitor (AGK-2) has also been proposed as a useful agent
carba-NAD+, NADH), coumarin derivatives (dihydrocoumarin, A3,
splitomicin (1), HR-73 (2)), and 2-hydroxynaphthaldehyde deriva-
tives (2-OH-naphthaldehyde, sirtinol, para-sirtinol, M15, cambinol
(3), salermide).18 Recently, some compounds bearing different
scaffolds (JFD00244, TRIPOS 360702, Ro-31-8220) have been iden-
tified as SIRT inhibitors through virtual screening methods,19–21
and the suramin derivative NF-67522 as well as the tetrahydrocar-
bazole EX-52723 displayed high and selective SIRT1 inhibitory
activity. A number of SIRTi from natural sources has been also re-
ported, such as (+)-guttiferone G and its structural analogues
hyperforin and artisoforin,24 endowed with antiproliferative activ-
ity on human umbilical vein endothelial cells (HUVEC), the SIRT2
inhibitor tanikolide dimer,25 and the SIRT1 inhibitor amurensin
G,26 able to restore doxorubicin responsiveness in doxorubicin-
resistant breast cancer MCF-7/ADR cells. Cambinol (3), active at
micromolar level against both SIRT1 and SIRT2, when tested on
BCL6-expressing Burkitt lymphoma cells as well as on Burkitt lym-
phoma mouse xenografts, was able to induce apoptosis with
concomitant hyperacetylation of BCL6 and p53. Moreover, it was
well tolerated and inhibited the tumor growth in the animal
for protection against
a-synuclein-induced toxicity in different
models of Parkinson’s disease.16
When compared with the great number of class I/II/IV HDAC
inhibitors reported so far, a small number of sirtuin inhibitors
(SIRTi) have been described, but this field is in constant expansion
(Fig. 1).17 By a medicinal chemist point of view, the first SIRTi
can roughly be grouped into NAD+ derivatives (nicotinamide,
H
H
N
N
S
O
O
NH
O
N
O
O
O
OH
OH
splitomicin (1)
Br
sirtinol
HR-73 (2)
cambinol (3)
H
SO3Na
SO3Na
N
NaO3S
SO3Na
O
Cl
NaO3 S HN
O
O
NH SO3Na
N
NH
O
O
N
H
2
OH
O
N
H
N
H
EX-527
salermide
NH2
NF-675
OH
HO
OH
C11H23
O
H
OH
H
HO
HO
OH
HO
O
O
O
O
O
H
H
OH
OH
HO
OH
O
C11H23
H
O
H
tanikolide dimer
(+)-guttiferone G
HO
amurensin G
H
N
N
O
S
CN
Cl
Cl
O
O
N
O
N
H
N
H
O
HN
N
tenovin-6
O
N
O
AGK-2
MC2141 (4)
Figure 1. Chemical structures of known SIRTi.

D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
3661
SIRTi such as splitomicin40 (1) and HR-7341 (2). Then, we opened
and reduced the central pyran ring of the tetracyclic core of 4
producing a series of variously substituted 6-(hetero)aryl or
6-arylalkyl substituted 5-(2-hydroxynaphthalen-1-ylmethyl)-2-
thioxo-2,3-dihydro-1H-pyrimidin-4-ones (6a–q). Such derivatives,
easily prepared from the proper benzo[f]chromenones 5, can be
also considered analogues of cambinol (3) (Fig. 2). All the synthe-
sized compounds (5 and 6) were tested for their ability to inhibit
the enzymatic activity of human recombinant SIRT1 and SIRT2
and for their effects on human leukemia U937 cells.
O
O
5a-t
n = 0-2
O
n
a
b
O
N
Het/
Ar
b
N
a
O
O
2. Results and discussion
2.1. Chemistry
HO
O
HN
MC2141 (4)
6a-q
The 2-substituted-3H-benzo[f]chromen-3-ones (8a–t) were
prepared by Knoevenagel condensation between the commercially
available 2-hydroxy-1-naphthaldheyde and the proper b-ketoest-
ers (7a–t),38,39,42 prepared by a standard previously reported
procedure.43 The 2-(hetero)aroyl or 2-arylalkanoyl-1,2-dihydro-
benzo[f]chromen-3-ones (5a–t) were then obtained by reduction
of the intermediates 8a–t with sodium borohydride in dry pyridine
(Scheme 1).38,39,42 The 6-substituted-5-(2-hydroxynaphthalen-
1-ylmethyl)-2-thioxo-2,3-dihydro-1H-pyrimidin-4-ones 6a–q
were finally synthesized upon treatment of the 1,2-dihydro-
benzo[f]chromen-3-ones (5a–n, p, r, s) with thiourea in dry etha-
nol in the presence of sodium ethoxide (Scheme 1).42,43
The reference compounds splitomicin (1), HR-73 (2), and camb-
inol (3) were prepared following the procedures reported in the
literature.40–42 Chemical and physical data of the novel final com-
pounds 5g–i, k–s and 6a, g–q are listed in Table 1. Chemical and
physical properties of the already reported final compounds 5a–f,
j, t and 6b–f are in agreement with the literature data.39,42
Chemical and physical properties of the novel intermediates 8g–i,
k, m–s are reported in Supplementary data, while chemical and
physical data of the known intermediates 8a–f, j, l, t are in
agreement with the literature.39,42,44
S
N
H
n
Het/
Ar
Figure 2. MC2141 (4) structural simplification plan.
model.27 Recently, tenovins have been reported to induce hyper-
acetylation of p53, to inhibit the deacetylating activities of SIRT1
and SIRT2, and to decrease tumor growth in vivo as single agents.28
Very recently, also the SIRT1/2 inhibitor salermide, developed by
our research group, showed very promising cancer-specific pro-
apoptotic properties in a panel of tumor cell lines.29
Pursuing our efforts for identifying epigenetic-disorder-modify-
ing agents30–35 and, in particular, sirtuins modulators,36–38 we
recently identified the 10-phenyl-7-oxa-8,10-diazabenzo[a]anth-
racene-9,11-dione (MC2141, 4) as the prototype of a novel series
of tri- and tetracyclic pyrimidinediones (benzodeazaoxaflavins,
BDF4s) as SIRTi endowed with high and selective SIRT1 inhibition
and a promising antiproliferative activity in human cancer cell
lines.39 Here we report a systematic med-chem structural simplifi-
cation approach on 4 aimed to improve its solubility and drug-like
properties (Fig. 2).
2.2. Sirtuin inhibitory activity
Firstly, we simplified the tetracyclic scaffold of the BDF4
prototype 4 by removal of the pyrimidinedione portion, and
prepared of a series of differently substituted 2-(hetero)aroyl or
2-arylalkanoyl-1,2-dihydrobenzo[f]chromen-3-ones (5a–t), which
are also structurally related to other benzochromene-containing
The two series of the 4-simplified derivatives, compounds 5a–t
and 6a–q, were tested at 50
(Table 2). Compounds 3 and 4 were included in the tests for
lM against hrSIRT1 and hrSIRT2
Het/
Ar
Het/
Ar
O
O
Het/
Ar
O
O
n
n
OEt
n
7a-t
+
O
O
a
b
O
O
CHO
5a-t
OH
8a-t
c
HO
O
HN
6a-q
S
N
H
n
Het/
Ar
Scheme 1. Reagents and conditions: (a) piperidine, acetic acid, dry ethanol, reflux, 4 h; (b) (1) NaBH4, pyridine, 0 °C 1 h, then rt 4 h; (2) 1 N HCl; (c) Na, dry ethanol, thiourea,
reflux, 18 h.

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D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
Table 1
Table 2
Chemical and physical data of compounds 5g–i, k–s and 6a, g–q
Inhibition of SIRT1/2 by the compounds 5a–t and 6a–qa
HO
HO
O
O
O
O
HN
HN
O
O
S
N
H
R
S
N
H
R
O
R
O
R
5g-i,k-s
6a,g-q
5a-t
6a-q
Compd
R
Mp (°C)
Recryst. systema Yield (%)
Compd
R
% Inhibition @ 50 lM
5g
5h
5i
5k
5l
5m
5n
5o
5p
5q
5r
1-Naphthyl
2-Naphthyl
40-Biphenyl
3-Furanyl
2-Thienyl
3-Thienyl
166–168
186–188
202–204
239–240
153–154
170–171
A
B
A
C
C
C
C
C
C
C
A
D
E
E
F
E
E
E
F
F
F
E
E
E
83
84
78
79
81
75
76
43
73
78
80
55
14
67
33
20
36
52
40
36
35
21
42
29
SIRT1
SIRT2
5a
5b
5c
5d
5e
5f
5g
5h
5i
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
1-Naphthyl
2-Naphthyl
40-Biphenyl
2-Furanyl
3-Furanyl
2-Thienyl
3-Thienyl
N-Methyl-1H-2-pyrrolyl
4-Pyridyl
2-Benzo[b]thienyl
2-Benzo[b]furanyl
PhCH2
PhCH2CH2
Ph
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
1-Naphthyl
2-Naphthyl
40-Biphenyl
2-Furanyl
3-Furanyl
2-Thienyl
3-Thienyl
N-Methyl-1H-2-pyrrolyl
2-Benzo[b]thienyl
PhCH2
28.3
19.2
17.1
25.0
19.1
11.3
25.4
5.2
90.1
88.2
89.5
88.2
88.1
89.8
89.5
91.1
88.8
83.3
89.9
28.2
78.9
76.7
94.5
38.2
35.6
81.2
85.8
20.2
29.8
36.1
35.9
38.4
36.1
28.5
45.8
47.9
52.4
45.9
43.8
42.7
43.4
46.2
51.2
47.4
49.8
51.4
20.1
N-Methyl-1H-2-pyrrolyl 196–198
4-Pyridyl
158–160
208–209
204–206
128–130
70–71
2-Benzo[b]thienyl
2-Benzo[b]furanyl
PhCH2
PhCH2CH2
2-Cl-Ph
1-Naphthyl
2-Naphthyl
40-Biphenyl
2-Furanyl
3-Furanyl
2-Thienyl
3-Thienyl
N-Methyl-1H-2-pyrrolyl 234–237
8.9
5j
5k
5l
28.9
18.0
33.2
19.1
37.3
69.3
35.1
30.4
11.3
24.6
32.1
82.3
82.6
79.9
82.6
83.2
80.2
81.7
83.0
81.9
82.3
78.2
77.8
77.3
81.3
82.1
82.4
83.0
44.2
86.5
5s
6a
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
>250
209–212
259–262
>250
245–248
>250
5m
5n
5o
5p
5q
5r
5s
5t
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
>250
>250
2-Benzo[b]thienyl
PhCH2
PhCH2CH2
>250
167–171
244–247
a
A: ethyl acetate; B: ethyl acetate/tetrahydrofuran; C: acetonitrile; D: petroleum
ether; E: chloroform; F: chloroform/methanol.
comparison. In the first series (5a–t), the introduction of chlorine
or methyl substituent at the ortho, meta or para position of the ben-
zene ring of the 2-benzoyl-1,2-dihydrobenzo[f]chromen-3-one (5t)
conferred a significant selectivity in the inhibition of SIRT2 (>85%
of inhibition at 50
l
M) over SIRT1 (11–28% of inhibition at
6m
6n
6o
6p
6q
3
50 M). The same degree of selectivity was obtained through sub-
l
stitution of the entire phenyl ring with bulkier aromatic groups
(5g–i), with furanyl (5j, k) and 3-thienyl (5m) rings, and with the
more flexible benzyl (5r) and phenethyl (5s) moieties. Moderate
SIRT2 selectivity was displayed by the N-methyl-1H-2-pyrrolyl
derivative 5n and the 4-pyridyl compound 5o. No SIRT2 selective
inhibition was observed with the 2-thienyl derivative (5l) and with
the heteroaromatic bicyclic compounds 5p and 5q that, similarly to
the prototype 5t, showed moderate inhibitory activities against
both the tested sirtuins. IC50 values against SIRT2 and/or SIRT1
were determined for selected 5 derivatives (Table 3). On the basis
of these enzymatic assays, we can conclude that the removal of the
pyrimidine portion of the tetracyclic scaffold of 4 leads to simpli-
fied derivatives (5a–t) with a reduced inhibitory activity against
SIRT1, while most of the substitutions at the 2 position of the
1,2-dihydrobenzo[f]chromen-3-one tricyclic system lead to selec-
tive SIRT2 inhibitors.
Compounds 6a–q, when compared with the lead structure 3,
showed in general higher SIRT1 and equal or lower SIRT2 inhibi-
tory activity. The introduction of substituents (2-, 3-, or 4-chloro;
2-, 3-, or 4-methyl; 4-phenyl) at the C-6 phenyl ring as well as
the replacement of the C-6 phenyl with either naphthyl or mono/
bicyclic heteroaromatic rings gave only modulator effects on the
inhibiting action of the derivatives. Also the increase of the dis-
tance between the pyrimidine C-6 position and the phenyl ring
with one or two methylene groups (compounds 6p, q) led to the
same results. Basically, 6a–q showed high inhibitory potency and
PhCH2CH2
4
a
Data are reported as the percent of inhibition at a concentration of 50 lM rela-
tive to the control reaction with no added inhibitor. Data are reported as the average
of three independent determinations; standard deviation of the average is 65%.
Table 3
IC50 values of selected 5 and 6 compounds
Compd
IC50 (lM) SD
SIRT1
SIRT2
9.4 1.2
8.9 1.1
10.0 1.3
178.7 24.6
49.9 9.5
50.8 11.2
40.7 10.4
191.2 25.6
5a
5h
5i
5t
6h
6q
3
89.8 9.4
NDa
ND
112.0 3.5
12.4 1.5
17.5 2.4
57.9 14.7
8.4 0.2
4
a
ND, not determined.
modest selectivity towards SIRT1, but they are devoid of struc-
ture-activity relationship. IC50 values calculated for the selected 6
derivatives 6h and 6q confirmed this trend of activity (Table 3).

D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
3663
Figure 3. (A) Cell-cycle effects, (B) apoptosis induction, and (C) cytodifferentiating activity exerted by compounds 15a–t (50
l
M, 30 h) in the human U937 leukemia cell line.

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D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
Figure 4. (A) Cell-cycle effects and (B) apoptosis induction exerted by compounds 16a–q (50 lM, 30 h) in the human U937 leukemia cell line.
2.3. Cell-based studies
at the C2 position of the coumarin scaffold, as well as the replace-
ment of this phenyl moiety with a 4-pyridyl residue seems to be
important (compare 5a, c, d, f, i, o with the unsubstituted proto-
type 5t). On the contrary, replacement of the same phenyl ring
with a five-member heterocycle such as furan, thiophene, and
pyrrole as well as with bulkier naphthyl or 2-benzo[b]thienyl/2-
benzo[b]furanyl rings, or introduction of methylene unit(s)
between the carbonyl group and the phenyl ring at the C2 position
of the coumarin base led to scarcely or no active compounds in this
test.
The two series of derivatives 5a–t and 6a–q were tested at
50 lM for 30 h in human U937 leukemia cells, together with spli-
tomicin (1), HR-73 (2), cambinol (3), and 4 as reference drugs at the
same concentration, to determine their effects on cell-cycle, apop-
tosis induction (measured as caspase 3–7 activation), and granulo-
cytic differentiation (measured as CD11c expression). In U937
cells, none of the tested compounds showed significant cytotoxic-
ity up to 50 lM.
As regards to their effect on U937 cell-cycle, most of com-
pounds 5 (in particular, 5j, h, j, m, o, r, t) elicited cell-cycle arrest
at the G1/S phase, while 5c displayed a block at the G2/M phase
similar to that exerted by 4 (Fig. 3A). The FACS analysis for apopto-
sis indicated that the majority of 5 were able to induce pro-
grammed cell death more efficiently than the related coumarin
derivatives 1 and 2, with compounds 5i and 5l being the most effi-
cient (18.4% and 19.5% of apoptosis, respectively; Fig. 3B) and even
stronger than the parent compound MC2141 (4). Nevertheless, at
least for 5l such apoptotic property seems to be unrelated to SIRT
inhibition. In the CD11c test to evaluate the effects on granulocytic
differentiation, six compounds (5a, c, d, f, i, o) displayed an induc-
tion of differentiation >30%, with the derivative 5d being the most
efficient (46.2% of CD11c positive/propidium iodide (PI) negative
cells) and much more active than the parent compound 4 and
the coumarin analogues 1 and 2 (Fig. 3C). To reach high differenti-
ation property in U937 cells, the insertion of a chloro or methyl or
phenyl substituent at the 2 or 4 position (not 3!) of the phenyl ring
Most of the compounds of the second series (6a–q) elicited a
significant cell-cycle arrest at the G1 phase, particularly evident
with the derivatives 6b–h, n (Fig. 4A). The FACS analysis for apop-
tosis showed that the majority of 6a–q were able to induce an
apoptosis level similar to that obtained by 3. Nevertheless, even
the most efficient derivatives 6j, k, q were less potent than the par-
ent compound 4 in this cell line at the described conditions. More-
over, no link to cellular sirtuin inhibition has been established. In
the CD11c test to evaluate the effects of compounds 6a–q on gran-
ulocytic differentiation, none of the compounds including refer-
ence compounds 3 and 4 showed a significant induction of
differentiation (Fig. S1 in the Supplementary data).
3. Conclusion
In the present study, we describe the development of two series
of simplification products of the previously reported SIRT1 selec-
tive inhibitor MC2141 (4).39

D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
3665
The first series comprises several coumarin derivatives (5a–t),
also related to the well known SIRTi splitomicin (1)40 and HR-73
(2),41 that depending on the profile of substitution at the 2-position
of the 1,2-dihydrobenzo[f]chromen-3-one common scaffold
showed a selectivity for inhibition of SIRT2 over SIRT1. Two of
these compounds (5i and 5l) displayed promising pro-apoptotic
properties in a human leukemia cell line (U937), that seems (at
least for 5l) to be unrelated to their SIRT inhibition. Some of them
(5d, 5i, and 5o) were endowed with high cytodifferentiation prop-
erties in the same cell line (U937). The second group of derivatives
consists of a series of 3-analogues, characterized by various substi-
tutions at the C-6 position of the pyrimidine ring of the prototype
and that, differently from the unselective SIRTi 3, displayed a selec-
tive inhibition of SIRT1 over SIRT2, similarly to the parent tetracy-
clic compound 4. In U937 cells, selected compounds (6j, 6k, and
6q) of this series were more efficient than 3 but less than 4 to in-
duce apoptosis.
and purified by recrystallization yielding the desired pure com-
pound 5n. 1H NMR (CDCl3) d 3.59 (m, 1H, HCHCH(CO)COO), 3.76
(m, 1H, HCHCH(CO)COO), 3.93 (s, 1H, NCH3), 4.54 (m, 1H,
CH2CH(CO)COO), 6.17 (m, 1H, H pyrrole ring), 6.89 (m, 1H, H pyr-
role ring), 7.02 (m, 1H, H pyrrole ring), 7.25 (d, 1H, H naphthalene
ring), 7.45 (t, 1H, H naphthalene ring), 7.54 (t, 1H, H naphthalene
ring), 7.76–7.87 (m, 3H, H naphthalene ring).
4.1.2.1.
2-(1-Naphthoyl)-1,2-dihydro-benzo[f]chromen-3-one
(5g). 1H NMR (CDCl3) d 3.59 (m, 1H, HCHCH(CO)COO), 3.85 (m,
1H, HCHCH(CO)COO), 4.84 (m, 1H, CH2CH(CO)COO), 7.25 (m, 1H,
H naphthalene rings), 7.40–7.53 (m, 5H, H naphthalene rings),
7.73–8.02 (m, 6H, H naphthalene rings), 8.19 (d, 1H, H naphthalene
ring).
4.1.2.2.
2-(2-Naphthoyl)-1,2-dihydro-benzo[f]chromen-3-one
(5h). 1H NMR (CDCl3) d 3.61 (m, 1H, HCHCH(CO)COO), 3.86 (m,
1H, HCHCH(CO)COO), 4.82 (m, 1H, CH2CH(CO)COO), 7.29 (d, 1H,
H naphthalene ring), 7.44–7.62 (m, 4H, H naphthalene rings),
7.79–7.96 (m, 6H, H naphthalene rings), 8.03 (m, 1H, H naphtha-
lene ring), 8.51 (s, 1H, H naphthalene ring).
4. Experimental section
4.1. Chemistry
4.1.2.3. 2-(40-Biphenylcarbonyl)-1,2-dihydro-benzo[f]chromen-
3-one (5i). 1H NMR (CDCl3) d 3.61 (m, 1H, HCHCH(CO)COO), 3.83
(m, 1H, HCHCH(CO)COO), 4.82 (m, 1H, CH2CH(CO)COO), 7.27 (d,
1H, H naphthalene ring), 7.37–7.62 (m, 7H, H biphenyl and naph-
thalene rings), 7.70 (m, 2H, H biphenyl and naphthalene rings),
7.78–7.88 (m, 3H, H biphenyl and naphthalene rings), 8.06 (m,
2H, H biphenyl and naphthalene rings).
Melting points were determined on a Buchi 530 melting point
apparatus and are uncorrected. 1H NMR spectra were recorded at
400 MHz on a Bruker AC 400 spectrometer; chemical shifts are re-
ported in ppm units relative to the internal reference tetramethyl-
silane (Me4Si). All the compounds were routinely checked by TLC
and 1H NMR. TLC was performed on aluminum backed silica gel
plates (Merck DC, Alufolien Kieselgel 60 F254) with spots visual-
ized by UV light. All of the 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 a reduced pressure of ca.
20 Torr. Organic solutions were dried over anhydrous sodium sul-
fate. Analytical results are within 0.40% of the theoretical values.
All the chemicals were purchased from Aldrich Chimica, Milan
(Italy) or from Lancaster Synthesis GmbH, Milan (Italy), and were
of the highest purity.
4.1.2.4. 2-(Furan-3-carbonyl)-1,2-dihydro-benzo[f]chromen-3-
one (5k). 1H NMR (CDCl3) d 3.55 (m, 1H, HCHCH(CO)COO), 3.82
(m, 1H, HCHCH(CO)COO), 4.35 (m, 1H, CH2CH(CO)COO), 6.80 (m,
1H, H furan ring), 7.23 (d, 1H, H naphthalene ring), 7.46–7.50 (m,
2H, H furan and naphthalene rings), 7.56 (t, 1H, H naphthalene
ring), 7.76–7.90 (m, 3H, H naphthalene ring), 8.17 (s, 1H, H furan
ring).
4.1.2.5. 2-(Thiophene-2-carbonyl)-1,2-dihydro-benzo[f]chromen-
3-one (5l). 1H NMR (CDCl3) d 3.60 (m, 1H, HCHCH(CO)COO),
3.83 (m, 1H, HCHCH(CO)COO), 4.61 (m, 1H, CH2CH(CO)COO), 7.16
(m, 1H, H thiophene ring), 7.24 (d, 1H, H naphthalene ring),
7.46 (t, 1H, H naphthalene ring), 7.55 (t, 1H, H naphthalene
ring), 7.72 (m, 1H, H naphthalene ring), 7.78 (m, 1H, H naphthalene
4.1.1. General procedure for the synthesis of 2-substituted-3H-
benzo[f]chromen-3-ones (8a–s). Example: 2-(3-thienyl)-3H-
benzo[f]chromen-3-one (8m)
Catalytic amounts of piperidine and acetic acid were added to a
mixture of 2-hydroxy-1-naphthaldehyde (2.15 g, 12.5 mmol)
and 3-oxo-3-thiophen-3-yl-propionic acid ethyl ester (2.97 g,
14.9 mmol) in dry ethanol (66 mL). The solution was then stirred
at reflux temperature for 5 h. After completion of the reaction,
the cooled suspension was filtered off and the crude yellow solid
was purified by recrystallization. 1H NMR (CDCl3) d 7.35 (m, 1H,
H thiophene ring), 7.48 (d, 1H, H naphthalene ring), 7.57–7.63
(m, 2H, H thiophene and naphthalene rings), 7.70 (t, 1H, H naph-
thalene ring), 7.92 (d, 1H, H naphthalene ring), 8.07 (d, 1H, H naph-
thalene ring), 8.13 (m, 1H, H thiophene ring), 8.23 (d, 1H, H
naphthalene ring), 8.88 (s, 1H, @CH–).
ring), 7.82–7.88 (m, 3H,
rings).
H
thiophene and naphthalene
4.1.2.6.
2-(Thiophene-3-carbonyl)-1,2-dihydro-benzo[f]chro-
men-3-one (5m). 1H NMR (CDCl3) d 3.56 (m, 1H, HCHCH(CO)COO),
3.81 (m, 1H, HCHCH(CO)COO), 4.59 (m, 1H, CH2CH(CO)-
COO), 7.23 (d, 1H, H naphthalene ring), 7.34 (m, 1H, H thiophene
ring), 7.46 (t, 1H, H naphthalenering), 7.53–7.57 (m, 2H, H thiophene
and naphthalene rings), 7.78 (m, 1H, H naphthalene ring), 7.83–7.88
(m, 2H, H naphthalene ring), 8.19 (m, 1H, H thiophene ring).
4.1.2.7. 2-(Pyridine-4-carbonyl)-1,2-dihydro-benzo[f]chromen-
3-one (5o). 1H NMR (CDCl3) d 3.63 (m, 1H, HCHCH(CO)COO), 3.77
(m, 1H, HCHCH(CO)COO), 4.71 (m, 1H, CH2CH(CO)COO), 7.24 (m,
1H, H naphthalene ring), 7.43–7.61 (m, 4H, H pyridine and naph-
thalene rings), 7.74–7.88 (m, 3H, H pyridine and naphthalene
rings), 8.84 (m, 2H, pyridine ring).
4.1.2. General procedure for the synthesis of 2-substituted-1,2-
dihydrobenzo[f]chromen-3-ones (5a–t). Example: 2-(1-methyl-
1H-pyrrole-2-carbonyl)-1,2-dihydro-benzo[f]chromen-3-one
(5n)
Compound 8n (1.0 g, 3.29 mmol, 1 equiv) was dissolved in dry
pyridine (25 mL) at 0 °C, and NaBH4 (125 mg, 3.29 mmol, 1 equiv)
was added to the solution. The mixture was stirred at 0 °C for 1 h
and then at room temperature for 4 h. Afterward, the reaction
was poured into 1 N HCl (125 mL), and the obtained precipitate
was filtered off and subsequently washed with H2O (3 ꢀ 30 mL)
4.1.2.8. 2-(Benzo[b]thiophene-2-carbonyl)-1,2-dihydro-benzo-
[f]chromen-3-one (5p). 1H NMR (CDCl3)
d
3.67 (m, 1H,
HCHCH(CO)COO), 3.86 (m, 1H, HCHCH(CO)COO), 4.74 (m, 1H,

3666
D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
CH2CH(CO)COO), 7.24–7.27 (m, 1H, H naphthalene ring), 7.40 (t,
1H, H benzothiophene ring), 7.46–7.49 (m, 2H, H benzothiophene
and naphthalene rings), 7.56 (t, 1H, H benzothiophene ring), 7.80
(d, 1H, H naphthalene ring), 7.84–7.90 (m, 4H, H benzothiophene
and naphthalene rings), 8.10 (s, 1H, H benzothiophene ring).
4.1.3.2. 5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-(naphthalen-
1-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6g). 1H NMR
(DMSO) d 3.77 (d, 1H, CHH at C-5), 3.94 (d, 1H, CHH at C-5), 6.56
(d, 1H, H naphthalene ring), 7.07–7.44 (m, 10H, H naphthalene
rings), 7.78 (m, 2H, H naphthalene ring), 9.07 (s, 1H, OH), 12.36
(s, 1H, NH), 12.68 (s, 1H, NH).
4.1.2.9. 2-(Benzofuran-2-carbonyl)-1,2-dihydro-benzo[f]chro-
men-3-one (5q). 1H NMR (CDCl3) d 3.65 (m, 1H, HCHCH(CO)-
COO), 3.83 (m, 1H, HCHCH(CO)COO), 4.76 (m, 1H, CH2CH(CO)-
COO), 7.27 (d, 1H, H naphthalene ring), 7.31–7.33 (m, 1H, H
benzofuran ring), 7.44-7.57 (m, 4H, H benzofuran and naphtha-
lene rings), 7.67 (d, 1H, H benzofuran ring), 7.71 (m, 1H, H naph-
thalene ring), 7.78–7.88 (m, 3H, H benzofuran and naphthalene
rings).
4.1.3.3. 5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-(naphthalen-
2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6h). 1H NMR
(DMSO) d 3.98 (s, 2H, CH2 at C-5), 6.81 (d, 1H, H naphthalene ring),
7.09 (m, 2H, H naphthalene rings), 7.28 (d, 1H, H naphthalene ring),
7.37–7.43 (m, 2H, H naphthalene rings), 7.53–7.60 (m, 3H, H naph-
thalene rings), 7.80 (d, 1H, H naphthalene ring), 7.87–7.90 (m, 3H,
H naphthalene rings), 9.41 (s, 1H, OH), 12.39 (s, 1H, NH), 12.59 (s,
1H, NH).
4.1.2.10. 2-(1-Hydroxy-2-phenylethylidene)-1H-benzo[f]chro-
men-3(2H)-one (5r). 1H NMR (CDCl3) d 3.84 (s, 2H, CH2Ph), 4.00
(s, 2H, CH2), 7.19 (m, 1H, H benzene ring), 7.26 (m, 1H, H naphtha-
lene ring), 7.33–7.38 (m, 4H, H benzene ring), 7.46 (t, 1H, H naph-
thalene ring), 7.56 (t, 1H, H naphthalene ring), 7.70–7.74 (m, 2H, H
naphthalene ring), 7.81 (d, 1H, H naphthalene ring), 13.70 (s, 1H,
OH).
4.1.3.4. 6-(Biphenyl-4-yl)-5-[(2-hydroxynaphthalen-1-yl)methyl]-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(6i). 1H
NMR
(DMSO) d 3.98 (s, 2H, CH2 at C-5), 6.86 (d, 1H, H biphenyl ring),
7.13–7.17 (m, 1H, H naphthalene ring), 7.21–7.26 (m, 3H, H biphe-
nyl and naphthalene rings), 7.38–7.51 (m, 7H, H biphenyl and
naphthalene rings), 7.62–7.64 (m, 3H, H biphenyl and naphthalene
rings), 9.44 (s, 1H, OH), 12.29 (s, 1H, NH), 12.57 (s, 1H, NH).
4.1.2.11.
2-(3-Phenylpropanoyl)-1H-benzo[f]chromen-3(2H)-
4.1.3.5. 6-(Furan-2-yl)-5-[(2-hydroxynaphthalen-1-yl)methyl]-
one (5s). 1H NMR (CDCl3) d 2.81 (m, 2H, COCH2), 2.94 (m, 2H,
COCH2CH2), 3.62 (m, 1H, HCHCH(CO)COO), 3.82 (m, 1H,
HCHCH(CO)COO), 4.56 (m, 1H, CH2CH(CO)COO), 7.13–7.29 (m,
6H, H benzene and naphthalene rings), 7.45–7.48 (m, 1H, H naph-
thalene ring), 7.53–7.58 (m, 1H, H naphthalene ring), 7.67–7.88 (m,
3H, H benzene and naphthalene rings).
2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(6j). 1H
NMR
(DMSO) d 4.22 (s, 2H, CH2 at C-5), 6.56 (m, 1H, H furan ring),
7.00 (d, 1H, H naphthalene ring), 7.04 (d, 1H, H furan ring), 7.22
(t, 1H, H naphthalene ring), 7.32 (t, 1H, H naphthalene ring), 7.56
(d, 1H, H naphthalene ring), 7.68-7.71 (m, 2H, H naphthalene ring),
7.77 (m, 1H, H furan ring), 9.49 (s, 1H, OH), 12.07 (s, 1H, NH), 12.51
(s, 1H, NH).
4.1.3. General procedure for the synthesis of 6-substituted-5-(2-
hydroxynaphthalen-1-ylmethyl)-2-thioxo-2,3-dihydro-1H-pyri-
midin-4-ones (6a–q). Example: 6-benzyl-5-(2-hydroxynaphtha-
len-1-ylmethyl)-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (6p)
Sodium metal (56 mg, 2.45 mmol, 2.5 equiv) was dissolved in
6 mL of absolute ethanol, and thiourea (149 mg, 1.96 mmol,
2 equiv) and 2-phenylacetyl-1,2-dihydro-benzo[f]chromen-3-one
(5r) (393 mg, 0.98 mmol, 1 equiv) were added to the clear solution.
The mixture was heated at reflux for 2 h. After the mixture was
cooled, the solvent was distilled in vacuo at 40–50 °C until dry
and the residue was dissolved in a little amount of water (20 mL)
and made acidic with 2 N HCl. The resulting mixture was then ex-
tracted with ethyl acetate (3 ꢀ 30 mL). The combined organic solu-
tions were washed with brine (2 ꢀ 20 mL), dried and evaporated to
dryness. The residual crude was finally purified by column chro-
matography on silica gel eluting with a mixture ethyl acetate/n-
hexane 1:1. The obtained solid was recrystallized from chloroform
to give pure 6p. 1H NMR (DMSO) d 3.83 (s, 2H, CH2 at C-6), 4.14 (s,
2H, CH2 at C-5), 6.86 (m, 2H, H benzene ring), 7.01 (d, 1H, H naph-
thalene ring), 7.09 (m, 3H, H benzene ring), 7.26 (t, 1H, H naphtha-
lene ring), 7.37 (t, 1H, H naphthalene ring), 7.54 (d, 1H, H
naphthalene ring), 7.72 (d, 1H, H naphthalene ring), 7.89 (d, 1H,
H naphthalene ring), 9.88 (s, 1H, OH), 12.04 (s, 1H, NH), 12.54 (s,
1H, NH).
4.1.3.6. 6-(Furan-3-yl)-5-[(2-hydroxynaphthalen-1-yl)methyl]-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(6k). 1H
NMR
(DMSO) d 4.03 (s, 2H, CH2 at C-5), 6.53 (m, 1H, H furan ring),
7.01 (d, 1H, H naphthalene ring), 7.22 (t, 1H, H naphthalene ring),
7.29 (t, 1H, H naphthalene ring), 7.56–7.60 (m, 2H, H naphthalene
ring), 7.65 (m, 1H, H furan ring), 7.70 (d, 1H, H naphthalene ring),
7.99 (s, 1H, H furan ring), 9.52 (s, 1H, OH), 12.17 (s, 1H, NH), 12.48
(s, 1H, NH).
4.1.3.7. 5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-(thiophen-2-
yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6l). 1H NMR
(DMSO) d 4.03 (s, 2H, CH2 at C-5), 6.96–7.03 (m, 2H, H thiophene
and naphthalene rings), 7.18–7.22 (t, 1H, H naphthalene ring),
7.27–7.29 (m, 2H, H thiophene and naphthalene rings), 7.54–7.58
(m, 2H, H naphthalene ring), 7.66–7.70 (m, 2H, H thiophene and
naphthalene rings), 9.47 (s, 1H, OH), 12.28 (s, 1H, NH), 12.53 (s,
1H, NH).
4.1.3.8. 5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-(thiophen-3-
yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (6m). 1H NMR
(DMSO) d 3.98 (s, 2H, CH2 at C-5), 6.98 (d, 1H, H naphthalene ring),
7.07 (m, 1H, H thiophene ring), 7.19–7.25 (m, 2H, H naphthalene
ring), 7.38 (d, 1H, H naphthalene ring), 7.55 (m, 2H, H thiophene
and naphthalene rings), 7.69 (m, 1H, H naphthalene ring), 7.81 (s,
1H, H thiophene ring), 9.57 (s, 1H, OH), 12.28 (s, 1H, NH), 12.52
(s, 1H, NH).
4.1.3.1. 6-(2-Chlorophenyl)-5-[(2-hydroxynaphthalen-1-yl)methyl]-
2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(6a). 1H
NMR
(DMSO) d 3.86 (d, 1H, CHH at C-5), 3.97 (d, 1H, CHH at C-5), 6.86
(d, 1H, H benzene ring), 6.96 (d, 1H, H naphthalene ring), 7.01
(m, 1H, H benzene ring), 7.12–7.20 (m, 2H, H benzene and naph-
thalene rings), 7.24–7.30 (m, 2H, H benzene and naphthalene
rings), 7.46–7.50 (m, 2H, H naphthalene ring), 7.65 (d, 1H, H
naphthalene ring), 9.18 (s, 1H, OH), 12.31 (s, 1H, NH), 12.66 (s,
1H, NH).
4.1.3.9.
5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-(1-methyl-
1H-pyrrol-2-yl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(6n). 1H NMR (DMSO) d 3.34 (s, 3H, CH3), 3.99 (s, 2H, CH2 at C-5),
6.08 (m, 1H, H pyrrole ring), 6.35 (m, 1H, H pyrrole ring), 6.88 (m,
1H, H pyrrole ring), 7.01 (d, 1H, H naphthalene ring), 7.14-7.26 (m,
3H, H naphthalene ring), 7.57 (d, 1H, H naphthalene ring), 7.70 (d,

D. Rotili et al. / Bioorg. Med. Chem. 19 (2011) 3659–3668
3667
1H, H naphthalene ring), 9.68 (s, 1H, OH), 12.32 (s, 1H, NH), 12.59
(s, 1H, NH).
for 30 min at 4 °C in the dark, washed in PBS and resuspended in
500 L PBS containing PI (0.25 g/mL). Samples were analyzed
l
l
by FACS with Cell Quest technology (Becton Dickinson). PI positive
4.1.3.10. 6-(Benzo[b]thiophen-2-yl)-5-[(2-hydroxynaphthalen-
1-yl)methyl]-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
cells have been excluded from the analysis.
(6o). 1H NMR (DMSO) d 4.08 (s, 2H, CH2 at C-5), 6.86 (d, 1H, H
naphthalene ring), 7.12 (t, 1H, H naphthalene ring), 7.20 (t, 1H, H
naphthalene ring), 7.37–7.39 (m, 2H, H benzothiophene and naph-
thalene rings), 7.48 (d, 1H, H naphthalene ring), 7.54 (s, 1H, H ben-
Acknowledgments
This work was supported by grants from Fondazione Roma
(A.M.), Associazione Italiana per la Ricerca contro il Cancro (AIRC
to L.A.), HEALTH-F4-2007-200767 ‘Apo-Sys ’ (L.A.), HEALTH-F4-
2009-221952 ‘ATLAS’ (L.A.).
zothiophene ring), 7.63 (m, 2H,
H
benzothiophene and
naphthalene rings), 7.81 (m, 1H, H benzothiophene ring), 7.90
(m, 1H, H benzothiophene ring), 9.43 (s, 1H, OH), 12.40 (s, 1H,
NH), 12.57 (s, 1H, NH).
Supplementary data
4.1.3.11. 5-[(2-Hydroxynaphthalen-1-yl)methyl]-6-phenethyl-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one (6q). 1H NMR (DMSO)
d 1.96 (m, 2H, PhCH2CH2), 2.52 (m, 2H, PhCH2CH2), 4.10 (s, 2H,
CH2 at C-5), 6.91 (m, 2H, H benzene and naphthalene rings),
7.13–7.28 (m, 5H, H benzene and naphthalene rings), 7.38 (m,
1H, H naphthalene ring), 7.70 (d, 1H, H naphthalene ring), 7.78–
7.83 (m, 2H, H naphthalene ring), 9.93 (s, 1H, OH), 12.24 (s, 1H,
NH), 12.52 (s, 1H, NH).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.01.025.
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2.5 ꢀ 105 cells were collected and resuspended in 500
l
L of an
hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate, 50
lg/
mL PI, RNAse A). Cells were incubated in the dark for 30 min. Sam-
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lL
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l

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