Epigenetic multiple ligands: Mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (Sirtuin) inhibitors

Article abstract of DOI:10.1021/jm701595q

A number of new compounds bearing two ortho-bromo- and ortho,ortho- dibromophenol moieties linked through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1-9) were prepared as simplified analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA, PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a substrate. They were also screened against HAT and SIRTs, because they are structurally related to some HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds (1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line, the epi-MLs induced high apoptosis levels [i.e., 40.7% (4l) and 42.6% (7b)] and/or massive, dose-dependent cytodifferentiation [i.e., 95.2% (1c) and 96.1% (4j)], whereas the single-target inhibitors eosin, curcumin, and sirtinol were ineffective or showed a weak effect.

Full text of DOI:10.1021/jm701595q

J. Med. Chem. 2008, 51, 2279–2290
2279
Epigenetic Multiple Ligands: Mixed Histone/Protein Methyltransferase, Acetyltransferase, and
Class III Deacetylase (Sirtuin) Inhibitors
Antonello Mai,*,† Donghang Cheng,‡ Mark T. Bedford,*,‡ Sergio Valente,† Angela Nebbioso,§ Andrea Perrone,†
Gerald Brosch, Gianluca Sbardella,* Floriana De Bellis,§,O Marco Miceli,§ and Lucia Altucci*,§
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, UniVersità degli Studi di Roma “La Sapienza”, P.le A.
Moro 5, 00185 Roma, Italy, The UniVersity of Texas M.D. Anderson Cancer Center, Science Park-Research DiVision, SmithVille, Texas 78957,
Dipartimento di Patologia Generale, Seconda UniVersità degli Studi di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy, DiVision of
Molecular Biology, Biocenter, Innsbruck Medical UniVersity, Fritz-Preglstrasse 3, 6020 Innsbruck, Austria, Dipartimento di Scienze
Farmaceutiche, UniVersità degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy, and Department of Cancer Biology,
IGBCM, Strasbourg, France
ReceiVed December 19, 2007
A number of new compounds bearing two ortho-bromo- and ortho,ortho-dibromophenol moieties linked
through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1–9) were prepared as simplified
analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine
methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA,
PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a
substrate. They were also screened against HAT and SIRTs, because they are structurally related to some
HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds (1b, 1c, 4b, 4f, 4j, 4l,
7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as
multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line,
the epi-MLs induced high apoptosis levels [i.e., 40.7% (4l) and 42.6% (7b)] and/or massive, dose-dependent
cytodifferentiation [i.e., 95.2% (1c) and 96.1% (4j)], whereas the single-target inhibitors eosin, curcumin,
and sirtinol were ineffective or showed a weak effect.
Introduction
sirtuins (SIRTs,a class III HDACs), and by histone lysine
methyltransferases (HKMTs), protein arginine methyltrans-
ferases (PRMTs), and histone demethylases (such as the lysine-
specific demethylase LSD1 and the JmjC domain-containing
demethylases).4,5 In addition to histone substrates, such families
of enzymes can also act on nonhistone proteins, such as
transcription factors (i.e., GATA1, BCL6, STAT3, NF-κB,
MyoD, YY1), tumor suppressors (p21, p53), cell cycle regulators
(Rb, E2F), cytoskeletal proteins (R-tubulin), the chaperone heat
shock protein 90 (Hsp90), and others.7 All histone/nonhistone
protein modifications, either directly or through the recruitment
of regulatory protein complexes, can modulate a number of
specific DNA-based processes such as transcription, DNA
replication, DNA repair, cell cycle progression, and chromosome
stability. Histone acetylation generally leads to activation of gene
For many years, tumorigenesis has been believed to be the
result of a multistep process involving genetic defects such as
gene mutations and deletions or chromosomal abnormalities
leading to either loss or gain of function of tumor suppressor
genes or oncogenes, respectively. Such multistep process can
involve an imbalance in the molecular signaling programs
responsible for differentiation and proliferation. In addition,
distinct gene expression programs are switched on or off during
development, growth, and differentiation. Recently it has been
demonstrated that epigenetic modifications play a key role in
these processes.1–3 Post-translational modification of core his-
tones include serine/threonine phosphorylation, lysine/arginine
methylation, lysine acetylation/deacetylation, ubiquitylation, and
sumoylation. These covalent modifications crosstalk with each
others, thus forming a complex network of signals that allows
gene expression to be finely tuned to the requirements of the
cell.4–6
a Abbreviations: BCL6, B cells lymphoma 6; BL21, Escherichia coli B
cells lack the Lon protease; CARM1, coactivator-associated arginine
methyltransferase 1; CD11c, cluster differentiation 11c; E2F, gene E2
promoter specific factor; ELISA, enzyme linked immunosorbent assays;
GATA1, erythroid transcription factor 1 or globin transcription factor 1;
GST-RmtA, glutathione-S-transferase-fungal arginine methyltransferase A;
HIV-1, human immunodeficiency virus 1; HSV1, herpes simplex virus 1;
IgG1, immunoglobulin G1; IPTG, isopropyl-ꢀ-D-thiogalactopyranoside;
JmjC, transcription factor jumonji domain-containing protein; LSD1, human
histone lysine specific demethylase 1; Myo-D, myogenic differentiation
factor; NAD, nicotinamide adenine dinucleotide; NAM, nicotinamide; NF-
κB, nuclear factor κB; Npl3, nuclear shuttling protein; p300/CBP, CREB
(cAMP-response element-binding protein) binding protein; PABP1, poly(A)
binding protein 1; PBS, phosphate buffer saline; PBS-MLK, phosphate
buffer saline-nonfat dry milk; PCAF, p300/CBP-associated factor; PVDF,
polyvinylidene fluoride; Rb, retinoblastoma protein; RPMI medium, Roswell
Park Memorial Institute medium; SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; SET7, three Drosophylia proteins that
arbor the domain 7; su(var.), enhancer of zeste and trithorax; SIRT, silent
mating type information regulation; STAT3, signal transducer and activator
of transcription 3; TCA, trichloroacetic acid; YY1, ying yang transcription
factor 1.
Histone tails are modified by a wide group of chromatin-
associated enzymes, including histone acetyltransferases (HATs)
and the counteracting enzymes, histone deacetylases (HDACs),
* To whom correspondence should be addressed. (A.M.) Tel.: +3906-
4991-3392. Fax: +3906-491491. E-mail: antonello.mai@uniroma1.it. Bio-
chemistry (M.T.B.), Tel.: +1-512-237-9539. Fax: +1-512-237-2475. E-mail:
mtbedford@mdanderson.org. Biology (L.A.), Tel.: +39081-566-7569. Fax:
+39081-2144840. E-mail: lucia.altucci@unina2.it.
†
Università degli Studi di Roma “La Sapienza”.
University of Texas M.D. Anderson Cancer Center.
Seconda Università degli Studi di Napoli.
‡
§
Innsbruck Medical University.
* Università degli Studi di Salerno.
O
IGBCM, Strasbourg.
10.1021/jm701595q CCC: $40.75
2008 American Chemical Society
Published on Web 03/19/2008

2280 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Mai et al.
Chart 1. Small-Molecule Modulators of Epigenetic Targets
expression, whereas deacetylation is related to gene silencing.8
The role of histone methylation in regulating gene expression
can be either positive or negative, depending on the context
and the types of enzymes involved. According to the site and
the extent of lysine methylation, this modification leads to either
active or repressed chromatin. Thus, methylation of Lys9 and
Lys27 in H3 and Lys20 in H4 are associated primarily with
transcriptional silencing, whereas Lys4, Lys36, and Lys79
methylation of H3 correlate with transcriptional activation.9,10
Differently from normal cells, cancer cells show aberrant
epigenetic features (such as CpG island hypermethylation, global
genomic hypomethylation, loss of acetylation at Lys16, and loss
of trimethylation at Lys20 in H4) that play a key role in invasion,
metastasis, chemotherapy resistance, and immune response of
the tumor disease.11,12 The cancer epigenome is currently being
unravelled using the powerful approaches of ChIP-on-chip and
ChIP-seq technologies.13 Some epigenetic aberrations can be
reverted by inhibition of histone-modifying enzymes (i.e.,
HDAC inhibitors (HDACi), HAT inhibitors (HATi), sirtuin
inhibitors (SIRTi), methyltransferase inhibitors (HMTi)), restor-
ing the normal epigenetic state of the cells.4,14–16
compounds) reported as regulators of PRMT activity.58 Previ-
ously we had observed that eosin (reported as AMI-5, Chart 1)
efficiently inhibited both Arg and Lys methyltranferases,59 thus
we designed some simplified eosin analogues bearing two
pharmacophoric ortho-bromo- and ortho,ortho-dibromophenol
moieties linked through a saturated/unsaturated, linear/(poly)-
cyclic spacer (compounds 1–9, Figure 1). We tested their
activities against a panel of HMTs (PRMTs: fungal RmtA,
human PRMT1, human CARM1; HKMTs: SET7). Western blot
analyses were performed on histone treated with selected
derivatives to determine the H3K4 and the H4R3/H3R17
methylation extents as markers of HKMT and PRMT inhibition,
respectively, in the human leukemia U937 cell line. Preliminary
results on the 1,5-diphenyl-1,4-pentadien-3-ones 160 highlighted
the key role of bromo and hydroxy substituents at the phenyl
rings to obtain low-micromolar inhibiting activity, with the
number and the position of the bromine atoms that discriminated
for PRMT1 versus CARM1 or PRMT1 versus SET7 selectivity.
In this article, we describe in detail the synthesis and biological
evaluation of compounds 1, and we report the synthesis and
anti-HMT activities of the novel series of compounds 2–9.
The chemical strategy used for designing compounds 1–9 led
to a merger, into the structures of 1–9, of some chemical features
that are common to curcumin (Chart 1), a component of turmeric
(Curcuma longa), which has been recently reported as HAT
inhibitor,61,62 and to resveratrol (Chart 1), a polyphenolic
phytoalexin able to activate SIRT1.63–65 Moreover, the ortho,o-
rtho-dibromophenol moiety is shown by the indolinone GW5074
(Chart 1), recently reported to be endowed with SIRT2 inhibiting
activity.66 Thus, we tested selected derivatives 1–9 in human
p300/CBP HAT assay and in human SIRT1 and SIRT2 assays
to determine if such bromo- and dibromophenol-containing
compounds could be multiple ligands for epigenetic targets (epi-
MLs). In addition, the effects of selected compounds on cell
cycle, apoptosis induction, and granulocytic differentiation on
U937 cells were evaluated.
In cell-based studies, HDACi exhibited interesting antipro-
liferative, apoptotic, and/or cytodifferentiating properties. In
preclinical studies, some of them have been found to have potent
anticancer activities.17–23 However, such studies have also
highlighted the complexity of the molecular mechanism(s)
involved in the antitumor action(s) of HDACi.22,23 Actually,
highly complex diseases, such as cancer and central nervous
system disorders, easily involved a wide number of altered
cellular pathways and signals, and many “reductionist” single-
target chemotherapy approaches have proven to be largely
fruitless.24,25 Thus, the use of HDACi in combination with other
anticancer agents (epi-drugs such as 5-aza-2′-deoxycytidine and
retinoic acid, death-receptor–ligands, kinase inhibitors, regulators
of proteasomal degradation, and conventional chemotherapeutic
agents) seems to be a more promising application.19,23 In
particular, phase 1/2 studies of the combination of a DNA
hypomethylating agent with a HDACi (and the eventual addition
of all-trans retinoic acid) in patients with acute myeloid
leukemia or high-risk myelodysplastic syndrome showed that
this combination of epigenetic therapy was safe and active and
was associated with transient reversal of aberrant epigenetic
marks.26–28
An alternative to combination therapy is the development of
a strategy based on “smart” drugs simultaneously able to
modulatemultipletargets(designedmultipleligands,DMLs).25,29,30
This multitarget-directed drug design strategy has been suc-
cessfully proposed for the treatment of neoplastic disorders24,31–33
as well as neurodegenerative diseases.34–38 The overall goal of
the DML approach is to enhance the efficacy and/or improve
the safety of the therapy, with respect to the drug combination.
An advantage of the use of DMLs is the higher predictability
of pharmacokinetic and pharmacodynamic parameters during
therapy due to the administration of a single compound, as well
as an improved patient compliance.
Chemistry
The eosin analogues 1, 2, and 4–7 were prepared by
condensation of the appropriate (methoxymethoxy)- or hydroxy-
benzaldehydes with the suitable ketones or diketones in alkaline
or acidic medium, followed by eventual hydrolysis of the
methoxymethyl protection. Reaction between 3,5-dibromo-4-
(methoxymethoxy)benzoic acid and the opportune amines
followed by cleavage of the protecting ether furnished the
amides 8, while the anilides 9 were obtained by reaction of the
reported dicarboxylic acyl chlorides with 3,5-dibromo-4-hy-
droxyaniline.
Since 1999, we have been engaged in design, synthesis, and
biological evaluation of small molecule modulators of epigenetic
targets. Up to now, we have described several series of
HDACi,39–54 a group of sirtinol analogues as SIRTi,55 and some
quinoline-based HATi,56,57 evaluating their effects on cell cycle,
proliferation, apoptosis, and cytodifferentiation on several
leukemia cell lines. In an effort to discover chemical entities
active against HMT enzymes, we undertook molecular modeling
studies of a series of dyes and dye-like compounds (AMI-

Epigenetic Multiple Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2281
Figure 1. Novel simplified AMI-5 analogues.
The chemistry section is reported in detail as Supporting
Information (see Schemes S1–S6, Tables S1–S3, and Experi-
mental Procedures).
potent compounds (IC50s: 69 and 40 µM, respectively). It is
noteworthy that the 3-carboxy-4-hydroxy substitution at the 1,5-
diphenyl-1,4-pentandien-3-one scaffold furnished also a highly
active compound (the new compound 1j, IC50 ) 55 µM). In
the chalcone series 2, the tribromo- and tetrabromo-containing
compounds 2c and 2d showed the highest inhibitory activity
(IC50s: 40 and 29 µM, respectively), they being as potent as
(2c) or 2.4-fold more potent than (2d) the corresponding 1,5-
diphenyl-1,4-pentadien-3-ones 1c and 1b. On the other hand,
the compound 3 was totally inactive against RmtA. Among the
compounds 4 bearing a cyclic ketone as a linker between the
two bromo-hydroxy- or dibromo-hydroxyphenyl moieties,
the observed trend of activity was fully respected: the bis(di-
bromo-hydroxyphenyl) derivatives 4b,f,h,j,l were always more
potent than the corresponding bis(bromo-hydroxyphenyl) coun-
terparts 4a,d,g,i,k against the fungal RmtA. The highest activity
was recorded with the introduction of cyclohexanone as a spacer
(compound 4b, IC50 ) 19 µM, showing the same activity as
AMI-5 used as reference drug). The insertion of heteroatom-
containing cyclic ketones (i.e., N-methyl-4-piperidone, tetrahy-
dro-4H-pyran-4-one, and tetrahydro-4H-thiopyran-4-one) led to
a slight decrease of the potency (see compounds 4f, IC50 ) 39
µM; 4j, IC50 ) 29 µM; and 4l, IC50 ) 45 µM), whereas with
the N-benzyl-4-piperidone, a barely active compound (4h, IC50
) 210 µM) was obtained. Compounds carrying bicyclic rings
such as ꢀ-tetralone (5a,b) and 8-methyl-8-azabicyclo[3.2.1]octan-
3-one (6a,b) as a connection between the two pharmacophore
moieties showed up to 2-fold decrease of the RmtA inhibitory
activity in comparison with the cyclohexanone-containing 4a,b
and the N-methyl-4-piperidone derivatives 4d,f, respectively.
The curcuminoids characterized by a benzene insertion at the
R3 position (compounds 7a-d) displayed high potencies against
RmtA, with the 1,1′-(1,3-phenylene)bis(3-(3,5-dibromo-4-hy-
droxyphenyl)prop-2-en-1-one) 7b being the most effective
compound (IC50 ) 10 µM, 2-fold more active than AMI-5).
The curcumin analogues 7e,f were 10- to 5-times less active
than 7b, whereas the 4,4-dimethyl derivatives 7g,h showed no
or slight RmtA inhibition. In the bis(benzamide) series 8, the
IC50 data reported in Table 1 showed that compounds with the
ethyl and n-propyl (8a,b) but not n-butyl (8c) chains connecting
the two amide functions were tolerated for enzyme inhibition,
and that the 1,3-substituted benzene spacer was 2-fold more
efficient than the related 1,4-substituted ring. About the bis(a-
Results and Discussion
Part 1. HMT Assays. As we started our work by designing
the compounds 1–9 as simplified analogues of AMI-5 (eosin),
a recently reported inhibitor of both Lys and Arg methyltrans-
ferases,59 we first investigated the activities of our compounds
against such enzymes, using histone as well as nonhistone
proteins as a substrate. Therefore, we tested compounds 1–9
against RmtA, a fungal PRMT acting on histone H4 substrate
that was shown to be a useful, predictive model for studying
PRMT inhibition in mammals.58 Selected compounds were
tested (50 µM) against human recombinant PRMT1 in vitro
using histone H4 as a substrate to confirm their inhibitory
activity. Afterward, we screened compounds 1–9 (100 µM)
against two human PRMTs (PRMT1 and CARM1) by using
nonhistone proteins as a substrate [the RNA-binding nuclear
shuttling protein (Npl3) and the poly(A)binding protein 1
(PABP1), respectively], to observe the influence of substrates
different from histones on the inhibiting activity. In addition,
we tested our compounds against the HKMT SET7 using histone
H3 as a substrate to assess their capability to also inhibit this
Lys methyltransferase. Finally, to study the in vivo efficacy of
our compounds to inhibit Lys and Arg methylation reactions,
we performed Western blot analyses on human leukemia U937
cells using monomethyl-H3K4, monomethyl-H4R3, and di-
methyl-H3R17 antibodies, and the methylation extent on such
residues in U937 cells after treatment with selected compounds
1–9 (50 µM, 24 h) was determined.
A. Inhibitory Activity against RmtA. Compounds 1–9 were
tested against Aspergillus nidulans RmtA, a fungal PRMT with
significant sequence similarity to human PRMT1 and specific
for methylation at Arg3 of histone H4.67 The percent values of
inhibition at a fixed dose (nearly 100 µM) were first determined
(data not shown), and then the IC50 values for the active
compounds were established (Table 1).
Structure–activity relationship on the effect of compounds 1
on the fungal RmtA has been recently reported by us as a
Communication.60 Briefly, the IC50 data (Table 1) highlighted
the role of ortho-bromo- and ortho,ortho-dibromophenol moi-
eties in inhibiting the enzyme, with 1b and 1c being the most

2282 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Table 1. RmtA Inhibitory Activity of Compounds 1–9a
Mai et al.
cmpd
1a
1b
1c
1d
1e
1f
1g
1h
1i
R
R1
R2
R3
R4
R5
IC50 (µM) or % inhbtn
3-Br-4-OH
3-Br-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
2-Br-4-OH
2,6-Br2-4-OH
2,4-Br2-6-OH
3,5-Me2-4-OH
3-F-4-OH
162
40
69
114
215
238
206
169.4
249
55
190
115
40
29
0 at 90 µM
90
14
0 at 89.4 µM
161.6
123.1
39
0 at 77.0 µM
210
132
3,5-Br2-4-OH
2-Br-4-OH
2,6-Br2-4-OH
2,4-Br2-6-OH
3,5-Me2-4-OH
3-F-4-OH
3-NO2-4-OH
3-COOH-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3-NO2-4-OH
3-COOH-4-OH
H
1j
2a
2b
2c
2d
3
4a
4b
4c
4d
4e
4f
H
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3-Br-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
CH2
CH2
CH2CH2
NCH3
NCH3
NCH3
NCH2Ph
NCH2Ph
O
3-Br-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
4g
4h
4i
4j
4k
4l
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3-Br-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
3,5-Br2-4-OH
O
S
S
29
0 at 88.5 µM
45
92
37
0 at 84.6 µM
84
59
10
47
37
109
48
0 at 91.8 µM
156.4
89.7
75.4
616
61.4
122.3
32.6
101.7
156.1
172.6
13.8
5a
5b
6a
6b
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
1,3-Ph
1,3-Ph
1,4-Ph
1,4-Ph
CH2
CH2
C(CH3)2
C(CH3)2
(CH2)2
(CH2)3
(CH2)4
1,3-Ph
1,4-Ph
none
CH2
(CH2)2
(CH2)3
1,3-Ph
1,4-Ph
0 at 70.4 µM
18
AMI-5
a Values are means determined for at least two separate experiments.
nilides) 9, the oxalyl derivative 9a was the most potent
compound among those with aliphatic spacers (9a-d), and the
increasing insertion of methylene units (compounds 9b-d) led
to a constant decrease of the inhibiting activity. In the phthalic
anilide series (compounds 9e,f), the benzene 1,3-substitution
assured the highest RmtA inhibiting activity (see compound 9e,
IC50 ) 13.8 µM).
B. Human PRMT1/H4 Assay. Selected compounds were
tested at 50 µM against human recombinant PRMT1, using
histone H4 as a substrate (Figure S1 in Supporting Information).
Data depicted in Figure S1 confirmed that the PRMT1 inhibitory
potency of the derivatives depends on the extension of bromi-
nation at the phenyl rings (the more bromine atoms the more
potent the compound, see 1a-c and 2c,d), and the feasibility
of the chalcone scaffold as an alternative to the 1,4-diphenyl-
1,5-pentadien-3-one for the design of new PRMTi. Among
compounds 4 bearing a cyclic ketone as a spacer connecting
the two (di)bromo-hydroxyphenyl moieties, the tetrahydro-4H-
pyran-4-one derivative 4j showed the highest inhibitory activity
(91.1% of inhibition), followed by the thiopyranone and the
N-methyl-4-piperidone analogues 4f and 4l (72.8 and 72.7% of
inhibition). The cyclohexanone-containing 4b, which was the
most effective in inhibiting the fungal RmtA, inhibited the
human PRMT1 activity of 44.2% at 50 µM. The 1,3-disubsti-
tuted-benzene curcuminoid 7b was 2-fold more efficient than
the 1,4-disubstituted counterpart 7d in inhibiting PRMT1, and
the bromo-analogue of curcumin 7f showed 70.2% of inhibition
in this assay. It is noteworthy that curcumin has been reported
inactive against G9a and other HMTs,61 and in our hand it
displayed millimolar inhibiting activity against both RmtA and
PRMT1.58 Among the bis(benzamide) and bis(anilide) series 8
and 9, compound 8d was highly potent in inhibiting PRMT1
(86.1% of inhibition), whereas the corresponding bis(anilide)
9e displayed a drop of activity.
C. PRMT1/Npl3p, CARM1/PABP1, and SET7/H3
Inhibitory Activity. Compounds 1, 2, and 4–9 were screened

Epigenetic Multiple Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2283
Figure 2. Inhibitory activities of compounds 1, 2, 4–9 (100 µM) against
PRMT1 using Npl3p as a nonhistone substrate, CARM1 using PABP1
as a nonhistone substrate, and SET7 using histone H3 as a substrate.
at 100 µM (fluorograph analysis) against human PRMT1, using
as a substrate the heterogeneous nuclear ribonucleoprotein
(hnRNP) Npl3p, an in vivo substrate of HMT1 from Saccha-
romyces cereVisiae,68 against human CARM1 with the poly-
(A)binding protein 1 (PABP1) as a substrate69 and against the
HKMT SET7 using histone H3 as a substrate (Figure 2). As a
rule, the tetrabromo-substituted compounds endowed with
inhibitory activity displayed high potency against all three tested
enzymes. Exceptions are 4j, which showed low activity against
PRMT1, 7d, which was less active against SET7, and 8e, 9a,
and to a lesser extent 9b and 9f, which exhibited a degree of
selectivity toward CARM1. In the bis(phenyl)pentadienone
series, the dibromo compound 1a was particularly active against
CARM1 and, to a lesser extent, PRMT1, and the addition of a
third bromine atom increased the potency of the compound (1b)
against SET7. The 1,4-bis(2,6-dibromo-4-hydroxyphenyl) and
the 1,4-bis(2,4-dibromo-6-hydroxyphenyl) analogues 1e and 1f
showed inhibitory activities against the PRMTs but not against
SET7, while the dinitro-derivative 1i was to some extent
CARM1-selective. Compounds belonging to the chalcone series
2 were inactive in this assay, with 2c,d showing low inhibition
against only SET7. In the 4, 5, and 7 series, the termini bearing
two bromine atoms in their structures (4a, 4c, 4g, 4i, 4k, 5a,
7a, 7c, and 7e) generally displayed low or no activity against
PRMT1 and SET7, whereas they were able to inhibit CARM1.
When the spacer is represented by the N-methylpiperidone, the
dibromo-derivative 4d failed in inhibiting the tested enzymes,
and a third bromine atom (4e) is required to furnish a CARM1-
inhibitory activity. The 8-methyl-8-azabicyclo[3.2.1]octan-3-one
derivatives 6a,b, as well as the 4,4-dimethylhepta-1,6-diene-
3,5-diones 7g,h, were inactive in this fluorograph assay. Among
the bis(benzamides) 8, while those with alkyldiamino spacers
(8a-c) showed no inhibiting activity, the 1,3-phenylenediamino
derivative 8d displayed a slight effect against SET7, and the
1,4-phenylenediamino counterpart 8e displayed high CARM1
inhibition. In the bis(anilide) series 9, the oxalyl (9a), malonyl
Figure 3. Western blot analyses performed with selected compounds
1–9 (at 50 µM for 24 h) on H3K4 (A) and H4R3/H3R17 (B)
methylation. The strong signal corresponds to methylation of the
appropriate lysine (A) or arginine (B), whereas a weaker signal shows
a decrease of the methylation in comparison with the control. As control
for equal loading total histone H4 (A) or the Ponceau Red staining of
histones (B) have been used, respectively.
(9b), and to a lesser extent 1,4-benzenedicarboxylyl (9f)
derivatives were selectively active against CARM1, whereas
the 1,3-benzenedicarboxylyl analogue 9e highly inhibited all
three tested enzymes.
D. Western Blot Analyses. Selected compounds 1–9 were
subjected to Western blot analyses on human leukemia U937
cells (at 50 µM for 24 h), to study their effects on H3K4 and
H4R3/H3R17 methylation. Data depicted in Figure 3 show that
tested compounds from 1c to 7b were efficient in inhibiting
H3K4 methylation in U937 cells, with the bis(benzylidene)het-
erocycloalkanones 4f, 4j, and 4l being the most potent.
Compounds 7d, 7f, 8d, and 9e gave a less evident histone
hypomethylation, whereas 1a and 1b were inactive in the H3K4
assay. In the Arg methylation assays (part B of Figure 3), all
the tested compounds with the exception of 1a clearly inhibited
H4R3 and H3R17 methylation, showing somehow a different
extent of inhibition according to their chemical structure and/
or the specific antibody (anti-H4R3 or anti-H3R17) used in the
Western blots. Thus, the pentadienones 1b,c, the chalcones 2c,d,
and the bis(benzylidene)-cyclohexanone and -N-methyl-4-pip-
eridone 4b,f gave almost the same degree of inhibition against
both the Arg residues, whereas the N-benzyl-4-piperidone 4g,
the thiopyranone 4l, and the 1,1′-(1,3-phenylene)bis(prop-2-en-
1-one) 7b displayed the highest activity against the H4R3
methylation. The amides 8d and 9e selectively inhibited the
methylation of H3R17.

2284 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Mai et al.
Figure 4. p300/CBP HAT assay performed on selected compounds
1–9 (at 50 µM). An indirect ELISA assay has been performed for the
detection of acetyl residues on histone H3 substrate using 10 µg of
U937 cell nuclear extract per assay as a source of p300/CBP enzymes.
The incubations with DMSO alone (control) or with selected compounds
1–9, AMI-5, curcumin, and anacardic acid (AA; all at 50 µM) have
been carried out for 90 min. Data have been expressed as 3H-acetyl
substrate incorporation activity.
Figure 5. SIRT1 and SIRT2 assays performed on selected compounds
1–9 (at 25 µM). First (deacetylation phase), the SIRT1 or SIRT2
enzymes were incubated with the substrate Fluor de Lys-SIRT1 or
-SIRT2 in the presence of NAD+ and various concentrations of tested
compounds (sirtuins activators or inhibitors). The second stage is
initiated by the addition of the Developer II, including nicotinamide
(NAM), a sirtuin inhibitor that stops the SIRT1/2 activity, and the
fluorescent signal is produced. Data are expressed as % of activity.
Part 2. p300/CBP (HAT) Assay. Compounds bearing a
bis(phenyl)pentadienone, chalcone, bis(benzylidene)(hetero)cy-
cloalkanone, or bis(phenyl)eptandione structure (curcuminoids)
are strictly related to the natural spice curcumin and have been
widely investigated as antitumor (both in vitro and in vivo),70–74
antiangiogenic (vascular endothelial cell proliferation, capillary
tube formation, and growth inhibitors),75,76 and chemoprotective
(phase 2 enzyme inducers and radical scavengers) agents.77 In
addition, curcuminoids have been reported to inhibit HIV-1
integrase78 and to block HSV-1 infection.79 Curcumin has been
recently identified as a p300/CBP HAT specific inhibitor, also
able to repress the p300-mediated acetylation of p53 in vivo,
and of HIV-Tat protein in vitro, thus inhibiting the HIV
proliferation.61
As our bis(dibromophenol)-containing compounds resembled
some chemical features of curcumin and curcuminoids, we tested
some representative samples of 1–9 at 50 µM against p300/
CBP immunoprecipitate (IP) in U937 cells to determine their
potential anti-HAT effect. Curcumin and anacardic acid (AA),80
a well-known p300/CBP and PCAF inhibitor, were used as
reference drugs. As a result (Figure 4), the fully brominated
bis(phenyl)pentadienone 1c showed the highest HAT inhibitory
activity (100.0% of inhibition), whereas the dibromo analogue
1a was ineffective. The 1-(3-bromo-4-hydroxyphenyl)-5-(3,5-
dibromo-4-hydroxyphenyl)penta-1,4-dien-3-one 1b had an in-
termediate behavior (61.9% of inhibition). Chalcone derivatives
2c,d were either slightly or not active in this assay, whereas all
the bis(benzylidene)(hetero)cycloalkanone compounds 4 exhib-
ited 61.0% (4j) to 95.6% (4b) of inhibition of the p300/CBP
IP. Among the curcumin analogues 7b,d,f, the compounds 7b
and 7f inhibited the p300/CBP activity more efficiently than
curcumin, whereas 7d was ineffective. Finally, the bis(dibro-
mo)benzamide 8d and the bis(dibromo)anilide 9e were highly
active in this HAT inhibitory assay, whereas AMI-5 showed
only a weak inhibition.81
exhibited SIRT2 inhibiting activity,66 selected compounds 1–9
plus AMI-5 were tested at 25 µM against SIRT1 and SIRT2.
EX-527 (0.5 µM),82 a recently reported SIRT1-selective inhibitor
active at submicromolar level, and sirtinol (50 µM),55 more
efficient in inhibiting SIRT2 than SIRT1, were used as a control
for SIRT1 and SIRT2 inhibition, respectively, and resveratrol
(100 µM) was added as a control for SIRT1 activation. Data
depicted in Figure 5 clearly shows that in the bis(phenyl)pen-
tadienone series, the compound 1a, carrying two bromine atoms
at the phenyl rings, was totally inactive in inhibiting SIRT1
and SIRT2. The further introduction of one bromine atom
(compound 1b) elicited a fine inhibitory activity against both
of the enzymes (60% and 65% of inhibition against SIRT1 and
SIRT2 at 25 µM), and the compound with four bromine atoms
at the phenyl rings (1c) showed 61% inhibition against SIRT1
and totally inhibited the activity of SIRT2 at the tested
concentration. Compounds belonging to the cyclohexanone- and
cyclohexanone-like-containing series (4), as well as the cur-
cuminoids 7, showed in general >50% of inhibition against both
SIRT1 and SIRT2 at 25 µM, the most potent being the 1,1′-
(1,3-phenylene)bis(3-(3,5-dibromo-4-hydroxyphenyl)prop-2-en-
1-one) 7b against SIRT1 (73% of inhibition). AMI-5 and the
chalcones 2 displayed 30% of inhibition against the two
enzymes, whereas the bis(benzamide) 8d and its bis(anilide)
isomer 9e were totally ineffective in these assays.
Part 4. In-Cell Evaluation. Effects on Cell Cycle,
Apoptosis Induction, and Granulocytic Differentiation on
Human Leukemia U937 Cell Line. From the data reported
here on the activities of selected compounds 1–9 against HMTs
(Part 1), HAT (Part 2), and SIRT (Part 3), it seems feasible
that some of them could act as multiple ligands by inhibiting at
the same time and with similar potencies several epigenetic
targets involved in regulation of gene expression and transcrip-
tion (epigenetic multiple ligands, epi-MLs). To date, the most
studied application of epi-drugs is the treatment of cancer
Part 3. SIRT1 and SIRT2 Assays. Because the chemical
scaffolds used for compounds 1–9 resembled some characteristic
features showed by the plant phytoalexin resveratrol, a known
SIRT1 activator,63–65 and by the indolinone GW5074, which

Epigenetic Multiple Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2285
Figure 6. Apoptosis induction exerted by selected compounds 1–9
(at 25 µM) in human leukaemia U937 cell line. Apoptosis measured
as caspase 3 activity has been taken as readout of the action of selected
compounds.
Figure 7. Granulocytic differentiation showed by selected 1–9 on
human leukemia U937 cells. Granulocytic differentiation is represented
by the CD11c positive cells. PI positive cells, which represent dead
cells, have been excluded from the analysis. The concentrations are
indicated.
diseases. We thus tested our selected compounds 1–9 on human
leukemia U937 cells by determining their effects on cell cycle
progression, apoptosis induction, and granulocytic differentiation.
For a comparison purpose, the HMTi AMI-5, the HATi
curcumin, and the SIRTi sirtinol were added to the assays. All
the compounds were tested at 25 µM to study their effects on
cell cycle (the analysis was determined after 30 h of treatment)
and apoptosis induction (measured as caspase 3 activation by
FACS analysis and checked after 30 h of treatment). To evaluate
granulocytic differentiation on U937 cells, the CD11c expression
levels upon 30 h of stimulation were determined. In this assay,
the highest testable dose for the selected compounds 1–9 and
AMI-5 was 5 µM or lower because their deeply colored
solutions interfered with the assay at higher concentrations. In
the same assay, the reference drugs curcumin and sirtinol were
tested at 25 µM, as for cell cycle and apoptosis studies.
Figure S2 in Supporting Information shows the effect of the
selected compounds (tested at 25 µM) on cell cycle phases in
the U937 cells. Compounds 1b, 2c, 4f, 4l, and 7b showed a
high increase of percent of G2 phase cells, in two cases (4f and
7b) with a total absence of cells in S phase, whereas compounds
4b and 7f as well as sirtinol displayed a total lack of cells in
G2 phase.
The tetrahydro-4H-pyran-4-one 4j showed a high level of
apoptosis (28%) when tested in the U937 cell line to determine
the apoptosis induction (Figure 6) at 25 µM for 30 h of
treatment. More importantly, its thio-analogue 4l and the 1,1′-
(1,3-phenylene)bis(3-(3,5-dibromo-4-hydroxyphenyl)prop-2-en-
1-one) 7b were highly more effective (4l, 40.7%; 7b, 42.6% of
apoptosis). A 10.5% of apoptosis was detected after treatment
of U937 cells with 7f. The pentadienone 1a, as well as curcumin
and sirtinol, showed <10% of apoptosis, and AMI-5 was totally
inactive in this assay.
In these conditions, the 1,5-bis(3,5-dibromo-4-hydroxyphenyl)-
penta-1,4-dien-3-one 1c and the 3,5-bis(3,5-dibromo-4-hydroxy-
benzylidene)dihydro-2H-pyran-4(3H)-one 4j displayed a mas-
sive, dose-dependent differentiating effect, with nearly 100%
of CD11c positive/PI negative cells. When tested at 0.5 and 1
µM, 1c and 4j retained high values of cell differentiation activity
[77–81% (1c); 59–67% (4j)]. At the tested doses (5 µM), also
the 1c-related compound 1b and the 4j-analogues 4b and 4f
showed high cell differentiation (68, 71, and 53% CD11c
positive/PI negative cells, respectively), whereas the thiopyran-
4(3H)-one 4l was ineffective. Among the tested curcuminoids
7b,d,f the compounds 7b and 7f displayed a granulocytic
differentiation effect (39 and 33% CD11c positive/PI negative
cells, respectively), whereas 7d failed to yield such effect. The
bis(benzamide) 8d and the bis(anilide) 9e were also inactive in
this assay. Among the reference compounds, only curcumin (at
25 µM) showed a moderate differentiation effect on U937 cells
(25% CD11c positive/PI negative cells).
Conclusion
In this article we have described the synthesis of a number
of 1,5-diphenyl-1,4-pentadien-3-ones (1a-j), chalcones (2a-d),
benzophenone (3), bis(benzylidene)(hetero)cycloalkanones
(4a-l) and bicyclic analogues (5a,b and 6a,b), 1,1′-(1,3- and
1,4-phenylene)bis(prop-2-en-1-ones) (7a-d), 1,7-bis(phenyl)-
hepta-1,6-diene-3,5-ones (7e-h), bis(benzamides) (8a-e), and
bis(anilides) (9a-f) as simplified analogues of AMI-5 (eosin),
a recently reported HMTi. Such derivatives were tested against
a panel of HMTs, including both PRMTs (the fungal RmtA,
human PRMT1, and human CARM1) and HKMT (human
SET7), using histone and nonhistone proteins as a substrate.
They were also screened for studying their potential HAT and
SIRT modulating activities, because they resemble in their
structures some chemical features typical of curcumin, a known
p300 HAT inhibitor, resveratrol, recently reported as SIRT1
activator, and GW5074, able to inhibit SIRT2.
Granulocytic differentiation has been evaluated in human
leukemia U937 cells by determining the percent values of
CD11c positive/propidium iodide (PI) negative cells after 30 h
of treatment with selected compounds 1–9 at 5 µM (1a-c, 2c,d,
4b,f,j,l, 7d,f, 8d, and 9e), or 1 µM (7b and AMI-5) (Figure 7).
Higher doses of the chosen compounds gave interference with
the reading of the results because of the deeply colored solutions
obtained. Curcumin and sirtinol were tested at the same
concentration (25 µM) used in cell cycle and apoptosis assays.
Taken all together, the anti-HMT, anti-HAT, and anti-SIRT
data for selected compounds 1–9 (Table 2) showed that the

2286 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Mai et al.
Table 2. Summary of Activities of Selected Compounds 1–9 Toward the Investigated Epigenetic Targets
a See Part 1, A, B, and C. b See Part 1, C. c See Part 2. d See Part 3. e Y, yes; N, no. f NA, not active.
bis(dibromophenol) motif linked through an unsaturated, (di)-
oxo-containing spacer could be useful for designing new
epigenetic multiple ligands (epi-MLs) active against HMTs,
p300 HAT, SIRT1, and SIRT2 at the same time. To date, in
epigenetics, only the psammaplins83 were reported endowed
with dual anti-DNMT and anti-HDAC activities, and the
selective PRMTi AMI-1 has been recently found also active
against SIRT1.84 In particular, bis(dibromophenol) moieties
connected through a penta-1,4-dien-3-one (1c), a 2,6-dimeth-
ylene(hetero)cycloalkanone (4b, 4f, 4j, 4l), a 1,1′-(1,3-phe-

Epigenetic Multiple Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2287
Table 3. Summary of Cellular Activities Observed with Epi-MLs on
(Table 3). Further biological in vitro and in vivo studies are in
progress to better characterize the observed properties of these
epi-MLs.
Human Leukemia U937 Cellsa
compd
1b
1c
4b
4f
4j
4l
7b
7f
cell cycle
apoptosis induction differentiation
arrest in S phase
arrest in G2 phase
arrest in S phase
arrest in G2 phase
arrest in S phase
arrest in S/G2 phase
arrest in G1/G2 phase
arrest in S phase
no effect
1.0%
1.1%
2.0%
1.0%
27.9%
40.7%
42.6%
10.5%
0.5%
8.5%
7.5%
68.1%
95.2%
70.9%
53.2%
96.1%
9.1%
38.8%
33.0%
5.7%
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. 1H 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). EI MS spectra
were recorded with a Fisons Trio 1000 spectrometer; only molecular
ions (M+) and base peaks are given. All compounds were routinely
checked by TLC and 1H NMR. TLC was performed on aluminum-
AMI-5
curcumin no effect
sirtinol weak arrest in S phase
25.3%
7.5%
a See Part 4.
backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254
)
with spots visualized by UV light. All solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved
the use of a rotary evaporator operating at a reduced pressure of
about 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 Lancaster Synthesis GmbH, Milan (Italy), and were
of the highest purity.
General procedures as well as description of the reactions used
to obtain compounds 1–22 are reported in detail as Supporting
Information (Schemes S1–S6, Tables S1–S3, and Experimental
Section).
nylene)diprop-2-en-1-one (7b), and a hepta-1,6-diene-3,5-dione
linker (7f) showed RmtA inhibiting activity in the range 10 to
69 µM were able to inhibit >50% of activity of human PRMT1
at 50 µM, gave p300 HAT inhibition ranging from 60 to 100%
at 50 µM and were highly efficient (50 to 100% inhibition) in
inhibiting both SIRT1 and SIRT2 at 25 µM. Compounds
carrying less than four bromine atoms (i.e., 1a, 1b, and 2c) were
less or not active against the tested enzymes: only the 1c-related
1-(3-bromo-4-hydroxyphenyl)-5-(3,5-dibromo-4-hydroxyphenyl)-
penta-1,4-dien-3-one 1b among them showed significant inhibition
of all the tested enzymes. The tetrabromo-chalcone 2d was able
to inhibit the fungal RmtA and the human PRMT1 using a histone
substrate. Nevertheless, it was inactive against HAT and showed
only moderate SIRT inhibition. The 1,1′-(1,4-phenylene)diprop-
2-en-1-one derivative 7d displayed good HMT and SIRT inhibitory
activities, but was unable to inhibit HAT at 50 µM. The N,N′-
(1,3-phenylene)dibenzamide 8d well inhibited RmtA and PRMT1
tested with a histone substrate, but showed very low or no inhibition
against PRMT1 and CARM1 with nonhistone proteins and against
SET7. Conversely, the related N1,N3-diphenylisophthalamide 9e
gave high PRMT inhibition using nonhistone substrate and was
less potent in the PRMT1/H4 assay. When tested against HAT
and SIRTs, 8d and 9e showed high HAT inhibition but no action
toward the tested sirtuins. The reference compound AMI-5 in our
hands displayed a good inhibition of HMTs but only a slight effect
against HAT and SIRTs.
Biochemistry. Preparation of GST-RmtA Fusion Proteins.
The coding sequence of RmtA67 was cloned into a pGEX-5X-1
expression vector (Amersham Pharmacia Biotech). RmtA-Protein
was expressed in BL21 cells in LB-medium. 250 mL cultures with
an A600 of 0.4 were induced with a final concentration of 1 mM
IPTG and grown for 4 h at 37 °C. After centrifugation of cells at
4000g, the pellet was resuspended in 6 mL of GST-binding buffer
(140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4,
pH 7.3) containing one protease inhibitor tablet (Complete, Roche,
Mannheim, Germany) for 50 mL of buffer. For cell lysis, lysozyme
was added at a final concentration of 5 mg/mL binding buffer and
cells were passed through a french press with a pressure setting of
1000 psi. The resulting lysate was centrifuged at 20000g for 10
min at 4 °C. GST fusion protein was purified from soluble extracts
by binding to a GST-HiTrap column (Amersham Pharmacia
Biotech). Proteins were eluted with 50 mM Tris-HCl, 10 mM
reduced glutathione, pH 8.0, and assayed for histone methyltrans-
ferase activity.
RmtA Inhibitory Assay. For inhibition assays, affinity purified
GST-RmtA fusion proteins were used as the enzyme source. HMT
activities were assayed using chicken erythrocyte core histones as
substrates. GST-RmtA fusion proteins (500 ng) were incubated with
different concentrations of compounds for 15 min at room tem-
perature. A total of 20 µg of chicken core histones and 0.55 µCi of
[3H]-S-adenosyl-L-methionine ([3H]AdoMet) were added. This
mixture was incubated for 30 min at 30 °C. Reaction was stopped
by trichloroacetic acid (TCA) precipitation (25% final concentration)
and samples were kept on ice for 20 min. Whole sample volumes
were collected onto glass fiber filters (Whatman GF/F) preincubated
with 25% TCA. Filters were washed three times with 3 mL of 25%
TCA and then three times with 1 mL of ethanol. After drying the
filters for 10 min at 70 °C, radioactivity was measured by liquid
scintillation spectrophotometry (3 mL scintillation cocktail). In the
IC50s determination, the SD values were within (5%.
Tested in the human leukemia U937 cell line to determine
their effects on apoptosis induction and granulocytic differentia-
tion, only the epi-MLs 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f showed
interesting apoptotic and/or differentiating properties, much more
evident than those displayed by the single HMT (AMI-5), HAT
(curcumin), and SIRT (sirtinol) inhibitors (Table 3). In particular,
4j, 4l, and 7b induced up to 43% apoptosis at 25 µM, and 1c
and 4j increased the CD11c levels of >90% at 5 µM, thus
showing a massive, dose-dependent cytodifferentiating effect
in the U937 cells. In this latter assay, 1b, 4b, 4f, 7b, and 7f
also showed high differentiating activities ranging from 33%
to 71% of CD11c positive PI negative cells. Thus, it is feasible
that the epi-MLs 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f, by acting at
the same time against several epigenetic targets such as PRMTs,
HKMTs, HAT, and SIRTs that interplay each other in modula-
tion of gene expression and transcription, could show high
important effects such as apoptosis and differentiation in the
U937 cells. Indeed, in the same assays the single-target inhibitors
curcumin and sirtinol gave <10% of apoptosis at 25 µM, and
only curcumin (25 µM) showed a slight cytodifferentiating effect
PRMT1, CARM1/PRMT4, and SET7 Inhibitory Assays. In
vitro methylation reactions have been described in detail previ-
ously.59 Briefly, all methylation reactions were carried out in the
presence of [3H]AdoMet (79 µCi from a 12.6 µM stock solution
in dilute HCl/ethanol 9:1, pH 2.0–2.5, Amersham Biosciences) and
PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, pH 7.4). To determine the specificity of the small
molecules, compounds were incubated with GST-PRMT1 and
Npl3p, GST-PRMT4 and PABP1, GST-SET7 and histone H3,

2288 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Mai et al.
respectively. Substrates (0.5 µg) were incubated with recombinant
enzymes (0.2 µg) in the presence of 0.5 µM [3H]AdoMet and 100
µM concentration of each of the compounds for 90 min at 30 °C
in a final volume of 30 µL PBS. Reactions were run on a 10%
SDS-PAGE, transferred to a PVDF membrane, sprayed with
Enhance, and exposed to film overnight. Reactions were performed
in the presence of DMSO at 3.3% v/v.
Western Blot Analysis. Histone Extraction Protocol. U937
cells were harvested and washed twice with ice-cold PBS 1× .
Then cells were lysed in Triton Extraction Buffer (TEB: PBS
containing 0.5% Triton X 100 (v/v), 2 mM phenylmethylsulfonyl
fluoride (PMSF), and 0.02% (w/v) NaN3) at a cellular density of
107 cells per mL for 10 min on ice, with gentle stirring. After a
brief centrifugation at 2000 rpm at 4 °C, the supernatant was
removed and the pellet was washed in half the volume of TEB and
centrifugated as before. The pellet was resuspended in 0.2 M HCl
at a cell density of 4 × 107 cells per mL and acid extraction was
proceeded overnight at 4 °C on a rolling table. The day after the
samples were centrifugated at 2000 rpm for 10 min at 4 °C, the
supernatant was removed and its protein content was established
using the Bradford assay.
Immunoblot Protocol. About 3–10 µg of acid-extracted proteins
were loaded on 15% SDS-polyacrylamide gel electrophoresis (SDS-
PAGE) and transferred to nitrocellulose. The blotted nitrocellulose
was washed twice with water and then blocked in freshly prepared
PBS, containing 3% nonfat dry milk (PBS-MLK) for one hour at
room temperature with constant agitation. The nitrocellulose was
incubated with 1:500 dilution of anti monomethyl-H3K4 (Abcam),
monomethyl-H4R3 (Abcam) or dimethyl-H3R17 (Abcam) antibod-
ies in freshly prepared PBS-MLK, overnight at 4 °C in agitation.
The day after, the nitrocellulose was washed three times with water
and incubated in the secondary reagent of choice in PBS-MLK for
1.5 h at room temperature in agitation. The nitrocellulose was
washed with water three times and once in PBS-0.05% Tween 20
for 5 min and then rinsed 4–5 times with water. At the end, the
ECL detection method (Amersham) was used.
FACS Analysis of Apoptosis on U937 Cells. Apoptosis was
measured by caspase 3 activation detection (B-BRIDGE) as
recommended by the suppliers; samples were analyzed by FACS
with Cell Quest technology (Becton Dickinson) as previously
reported.
Granulocytic Differentiation on U937 Cells. Granulocytic
differentiation was carried out as previously described.86 Briefly,
U937 cells were harvested and resuspended in 10 µL phycoeryth-
rine-conjugated CD11c (CD11c-PE). Control samples were incu-
bated with 10 µL PE conjugated mouse IgG1, incubated for 30
min at 4 °C in the dark, washed in PBS, and resuspended in 500
µL PBS containing PI (0.25 µg/mL). Samples were analyzed by
FACS with Cell Quest technology (Becton Dickinson). PI positive
cells have been excluded from the analysis.
Acknowledgment. This work was partially supported by
grants from AIRC 2007 (A.M.), PRIN 2006 (A.M.), European
Union (CancerDip project Number 200620; (L.A.), PRIN 2006
(L.A.), Regione Campania, L.5 annualità 2005 (L.A.), Welch
Foundation G-1495 (M.T.B.), and NCI CA114116 (M.T.B.).
The authors thank Fabrizio del Piaz, Salerno University, Italy,
for the technical assistance. Thanks are due to Maurizio
Recanatini and Maria Laura Bolognesi for the critical reading
of the manuscript and the useful suggestions.
Supporting Information Available: Detailed chemistry section
with synthetic procedures used for obtaing the title compounds
(Schemes S1–S6), characterization data for compounds 1–22
(Tables S1-S3), PRMT1/H4 assay (Figure S1), cell cycle effect
of selected compounds 1–9 on the U937 cells (Figure S2), and
experimental procedures (chemistry, PRMT1/H4 assay, cell cycle
analysis). This material is available free of charge via the Internet
at http://pubs.acs.org.
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