Novel 3,5-bis(bromohydroxybenzylidene)piperidin-4-ones as coactivator-associated arginine methyltransferase 1 inhibitors: Enzyme selectivity and cellular activity

Article abstract of DOI:10.1021/jm200453n

Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis(3-bromo-4-hydroxybenzylidene)piperidin-4-one (7g) and its analogues 8a-l. Among them, 7g, 8e, and 8l displayed high and selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity.

Full text of DOI:10.1021/jm200453n

BRIEF ARTICLE
pubs.acs.org/jmc
Novel 3,5-Bis(bromohydroxybenzylidene)piperidin-4-ones as
Coactivator-Associated Arginine Methyltransferase 1 Inhibitors:
Enzyme Selectivity and Cellular Activity
||
||
Donghang Cheng,*,†, Sergio Valente,‡, Sabrina Castellano,§ Gianluca Sbardella,§ Roberto Di Santo,‡
Roberta Costi,‡ Mark T. Bedford,*,† and Antonello Mai*,‡
†The University of Texas MD Anderson Cancer Center, Science Park—Research Division, Smithville, Texas 78957, United States
‡Istituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universitꢀa di Roma,
P.le A. Moro 5, 00185 Roma, Italy
§Dipartimento di Scienze Farmaceutiche e Biomediche, Universitꢀa degli Studi di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy
S Supporting Information
b
ABSTRACT: Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent
tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis-
(3-bromo-4-hydroxybenzylidene)piperidin-4-one (7g) and its analogues 8aꢀl. Among them, 7g, 8e, and 8l displayed high and
selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g
showed a significant dose-dependent reduction of the PSA promoter activity.
rginine methylation of mainly nuclear proteins is a reversible
post-translational modification process involved in structural
Some pyrazole-containing compounds (1ꢀ4) and the benzo-
[d]imidazole (5) have been reported as inhibitors of CARM1,11ꢀ15
and the plant-derived ellagic acid (6)16 has been recently
shown to selectively block methylation at Arg17 of histone H3
(H3R17),16 the CARM1 histone site for methylation (Chart S1
in Supporting Information).
Despite the fact that all of these compounds showed sub-
micromolar inhibitory activity against CARM1, no inhibitor has
been demonstrated to exhibit cellular effects to date.
Pursuing our searches on design, synthesis, and biological
validation of small molecule modulators of epigenetic targets,17
in 2008 we prepared and tested some bis(3-bromo-4-hydroxy-
and 3,5-dibromo-4-hydroxyphenyl) compounds and their
analogues against PRMT1,18 CARM1,18 SET7 (an histone
lysine methyltransferase, HKMT),18 p300/CBP (an HAT
enzyme),18,19 SIRT1, and SIRT2.18 Depending on the extent
of bromination of the molecule (presence of four bromine
atoms) and on the nature of the linker connecting the two
dibromophenol moieties (penta-1,4-dien-3-one, 2,6-dimethylene-
(hetero)cycloalkanone, 1,1-(1,3-phenylene)diprop-2-en-1-one,
and hepta-1,6-diene-3,5-dione), some of such compounds be-
haved as epigenetic multiple ligands (epi-MLs), they being active
against all the tested enzymes.18 In contrast, compounds carry-
ing two or three bromine atoms in their structure or featuring
a bis(3,5-dibromo-4-hydroxybenzamide) or bis(3,5-dibromo-
4-hydroxyanilide) scaffold failed to be recognized as epi-MLs
and showed some degree of selectivity against a particular
epigenetic target.
A
remodeling of chromatin.1,2 Protein arginine methyltransferase
(PRMT) enzymes remove the methyl group from the donor
molecule S-adenosyl-L-methionine (AdoMet), generating the
product S-adenosyl-L-homocystein (AdoHcy), and transfer this
methyl residue to the terminal nitrogen atom(s) of the guanidi-
nium side chain of an individual arginine residue in the target
protein.3 PRMTs are ubiquitously expressed in most cell types
and tissues of the human body with the unique exception of
PRMT8, which appears to be restricted to neurons in the brain.4
Moreover, they differ in their substrate specificities and are
therefore probably involved in different physiological processes.
Among PRMTs, PRMT4/CARM1 (coactivator-associated argi-
nine methyltransferase 1) was the first to be identified as a
transcriptional regulator.5 CARM1 methylates a number of
proteins that are involved in transcription and RNA processing,
including histone H3 (H3R17 and H3R26), amplified in breast
cancer 1 (AIB1), p300/CBP (cAMP-responsive element binding
protein [CREB] binding protein), poly(A)-binding protein 1
(PABP1), and coactivator of 150 kDa (CA150).1 CARM1
requires its enzymatic activity for all its in vivo functions.6 In
cancer, CARM1 has been shown to regulate estrogen-stimulated
MCF-7 breast cancer cell cycle progression through E2F1 up-
regulation.7 Moreover, CARM1 has been found up-regulated in
castration-resistant prostate cancer8 and in grade-3 breast
tumors,9 and CARM1 knockdown by siRNA completely inhib-
ited prostate cancer LNCaP cell proliferation by induction of
apoptosis.10
All these findings prompted researchers to develop molecules
able to inhibit CARM1 activity, as potential anticancer agents.
Received: April 19, 2011
Published: May 26, 2011
r
2011 American Chemical Society
4928
dx.doi.org/10.1021/jm200453n J. Med. Chem. 2011, 54, 4928–4932
|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 2. Inhibitory activities of 8aꢀl against CARM1 using PABP1 as
a substrate, PRMT1 using NPL3 as a substrate, and SET7 using histone
H3 as a substrate. The concentrations of the compounds used in each in
vitro methylation assay are shown.
Table 1. IC50 of 7g and 8aꢀf,jꢀl against CARM1, PRMT1,
and SET7a
IC50 (μM)
compd
CARM1/PABP1
PRMT1/NPL3
SET7/H3
7g
8a
8b
8c
8d
8e
8f
8.6 ( 0.8
>667
>667
>667
>667
>667
>667
>667
>667
>667
>667
>667
Figure 1. CARM1-selective inhibitors used in this study.
10.3 ( 3.3
15.2 ( 0.9
11.9 ( 2.3
12.5 ( 6.1
8.1 ( 2.2
>667
>667
Thus, to identify CARM1-selective inhibitors among them
and taking into account the fluorograph data previously reported,
we determined the IC50 values for selected bis(bromo- and
dibromophenol) compounds 7aꢀm (see Figure S1 and Table
S1 in Supporting Information) against PRMT1, CARM1, and
the HKMT SET7. Among the tested compounds, 7b showed
high potency and selectivity in inhibiting PRMT1, whereas 7c,
d,g,h,l,m preferably inhibited CARM1, 7g being the most
potent (IC50 = 7.1 μM). With the exception of 7a,b, all the
tested compounds displayed very low (if any) inhibition
against SET7.
Accordingly, we chose 7g as our lead compound for selective
CARM1 inhibition and prepared some 3,5-bis(3-bromo-4-hy-
droxybenzylidene)-1-benzylpiperidin-4-one analogues 8aꢀl by
insertion of a chlorine atom or a methyl or methoxy group at the
ortho, meta, or para position of the N1-benzyl moiety or by
replacing such benzyl group with a 2-phenylethyl, 3-phenylpro-
pyl, or 2-oxo-2-phenylethyl moiety at N1 (Figure 1). These new
compounds were tested as CARM1-selective inhibitors, and two
of them together with 7g were investigated in more detail in vitro
and in vivo.
>333
>667
174 ( 28
>600
12.2 ( 3.0
14.8 ( 2.5
16.0 ( 3.8
14.4 ( 1.7
8j
>667
8k
8l
>667
149 ( 26
a Values were determined from at least two separate experiments. The
mixture contained 0.1 μM GST-CARM1 and 0.5 μM GST-PABP1,
0.15 μM GST-PRMT1 and 0.5 μM GST-NPL3, or 0.15 μM GST-SET7
and 1.1 μM histone H3 with 0.22 μM [3H]AdoMet and different
concentrations of each compound for IC50 determinations. The soft-
ware used for fitting curves and determining IC50 is SigmPlot. The
equation used for fitting is y = y0 þ a/1 þ (x/x0)b.
’ RESULTS AND DISCUSSION
The new compounds were tested by fluorograph at 50 and
20 μM against CARM1 using PABP1 as a substrate20 and at
50 μM against PRMT1 (substrate, the heterogeneous nuclear
ribonucleoprotein NPL3)21 and SET7 (substrate, histone H3) to
assess their potency and selectivity (Figure 2). At 20 μM, the
4-methyl- and the 2-, 3-, and 4-methoxybenzyl analogues of 7g
(8c and 8gꢀi) as well as the 3-phenylpropylpiperidone 8k showed
no effect against the PABP1 methylation; thus, the methoxy-
containing compounds were excluded by IC50 calculation.
IC50 values for 7g and 8aꢀf,jꢀl were determined against
CARM1 using PABP1 as a substrate and against PRMT1 and
SET7 using NPL3 and histone H3 as substrates, respectively
(Table 1). The corresponding IC50 curves are reported in
Supporting Information. All the tested compounds displayed
low micromolar activity against CARM1, the insertion of methyl
and chloro substituents at the N1-benzyl moiety having only
modulator effects on enzyme inhibition. The preferred position
for introduction of a methyl group at the benzyl portion seems to
be the ortho position (8a), while for chlorine insertion the benzyl
meta position afforded the highest inhibitory activity (8e),
similar to that of the lead 7g.
’ CHEMISTRY
3,5-Bis(3-bromo-4-(methoxymethoxy)benzylidene)piperidin-4-
one 9, the key intermediate of the title compounds, was prepared
by condensation of 3-bromo-4-(methoxymethoxy)benzaldehyde18
with 4-piperidone in alkaline medium (barium hydrate). Alkyla-
tion reactions of 9, carried out at 60 °C with the appropriate alkyl
bromide in the presence of dry potassium carbonate in acetoni-
trile, furnished the N-arylalkyl-3,5-bis(3-bromo-4-(methoxy-
methoxy)benzylidene)piperidin-4-ones 10aꢀl that were sub-
jected to acidic hydrolysis in methanolic 3 N HCl at 60 °C to
afford the desired bis(3-bromo-4-hydroxybenzylidene) analogues
8aꢀl (Scheme S1 in Supporting Information).
Experimental procedures for 9 and 10 and chemical and
physical data (Tables S2ꢀS4) for 8ꢀ10 are reported as Support-
ing Information.
4929
dx.doi.org/10.1021/jm200453n |J. Med. Chem. 2011, 54, 4928–4932

Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 4. Inhibitory activities of 7g, 8e, and 8l against a panel of
HKMTs (SET7, DOTL1, Suv39H1, and G9a) using the indicated
histone and/or non-histone substrates.
efficacy of potential PRMT inhibitors in cell may require days of
treatment while waiting for the methylated substrates to turn-
over. Under these conditions, compounds with pleiotropic
effects would be difficult to investigate in a cell-based assay. To
reduce the exposure time of the compound to cells and bypass
this problem, we developed a Flag-tagged PABP1 inducible cell
line obtained by engineering a tetracycline-controlled transre-
pressor protein (TetR) in human embryonic kidney HEK293
cells.24 The TetR protein binds to tet operator (tetO) sequences
in the absence but not in the presence of tetracycline, silencing
the transcriptional activities at the promoter.
We can thus easily distinguish between the endogenous
PABP1 and the induced Flag-tagged PABP1 because of its slower
migration by SDSꢀPAGE. We tested 7g, 8e, and 8l in this
reporter system. Upon addition of tetracycline, Flag-PABP1 is
induced in HEK293 cells in the presence of the indicated
compound, and its methylation status can be detected by the
use of a methyl-specific PABP1 antibody generated in our lab.24
In this reporter system, only 7g was able to inhibit Flag-PABP1
methylation (Figure S4 in Supporting Information).
There is increasing evidence of the involvement of CARM1 in
hormone responsive cancers such as prostate cancer. Thus, we
determined the effect of 7g, 8e, and 8l on prostate-specific
antigen (PSA) promoter in human prostate adenocarcinoma
LNCaP cells by using PSA luciferase assay, relative to a CMV-
Renilla control (Figure 5, top). In particular, we transfected PSA
reporter into LNCaP cells and then we treated the cells with
increasing concentrations of 7g, 8e, or 8l for 2 days.
As seen in Figure 5, a dose-dependent decrease of the reporter
activity was observed with 7g and 8e up to 8ꢀ10 μM while 8l was
effective only at 30 μM. In parallel, we measured the cell viability
through Cell Titer-Glo (CTG), based on quantitation of the
ATP present (Figure 5, bottom). This was done to confirm that
the observed PSA effects were the results of CARM1 inhibition
and to rule out involvement of other targets and/or cell death. 7g
and 8l displayed no or little effect on cell viability, at concentra-
tions that impacted the luciferase assay.
Figure 3. Inhibitory activity of 7g, 8e, and 8l against CARM1 using
PABP1, CA150, SMB, and histone H3 as substrates and against a panel
of PRMTs (PRMT1, PRMT3, PRMT5, and PRMT6) using indicated
histone and/or non-histone substrates. The fluorographs are shown in
the left panels, and the tritium count for each band is depicted in the right
panels.
All of the tested compounds were selective toward CARM1,
showing very low (if any) activity against PRMT1 and SET7.
Among them, we selected 7g, 8e, and 8l for further experiments:
7g and 8e were the most potent inhibitors of CARM1 with
PABP1 as a substrate (see Table 1), while 8l was the only
analogue carrying a structural diversity, the carbonyl group at
the substituent at N1, that could influence its binding with the
enzyme and its inhibitory behavior.
First, we repeated the CARM1 assay, testing 7g, 8e, and 8l at
100 μM by fluorograph and using four different CARM1 sub-
strates: PABP1, CA150,22 the spliceosome protein SmB,22 and
histone H3 (Figure 3). All the three tested compounds strongly
inhibited the CARM1 activity on the various substrates. Among
these, CA150 was the most sensitive, whereas the use of histone
H3 yielded the lowest CARM1 inhibition. To check the real
selectivity of 7g, 8e, and 8l against various PRMTs, we tested
them at 100 μM against (i) PRMT1 using NPL3 and histone H4
as a non-histone and histone substrate, respectively, (ii) PRMT3
using NPL3 and the ribosome protein rpS223 as substrates, (iii)
PRMT5 and histone H4 as a substrate, (iv) PRMT6 using NPL3
and histone H3 as substrates (Figure 3). In addition, 7g, 8e,
and 8l were tested at 100 μM against a panel of HKMTs,
namely, SET7 (substrate, H3), DOTL1 (substrate, nucleosome),
Suv39H1 (substrate, H3), and G9a (substrate, H3) (Figure 4).
Against PRMTs, 7g and 8e were able to inhibit to some extent
PRMT3, and 8e and 8l showed high inhibition of PRMT5 at
100 μM. Nevertheless in all cases the observed inhibition values
were weaker than those observed with CARM1 when used at the
same concentration (see Figure 3). No significant activity at
100 μM was registered for 7g, 8e, and 8l against the tested
HKMTs (see Figure 4).
In conclusion, we have reported on the ability of the 1-sub-
stituted-3,5-bis(3-bromo-4-hydroxybenzylidene)piperidin-4-
ones 7g and 8aꢀl to selectively inhibit CARM1 activity. Com-
pounds 7g, 8e, and 8l were able to inhibit CARM1-mediated
methylation of different substrates (PABP1, CA150, SmB, and
H3) up to single-digit micromolar level, displaying low inhibitor
activity (if any) against a panel of different PRMTs or HKMTs.
In human prostate cancer LNCaP cells, 7g showed a significant
dose-dependent reduction of the PSA promoter activity at a
concentration that did not affect cell viability.
Known CARM1 substrates such as PABP1 are hypermethy-
lated in vivo, and this methylation is very stable. To test the
4930
dx.doi.org/10.1021/jm200453n |J. Med. Chem. 2011, 54, 4928–4932

Journal of Medicinal Chemistry
BRIEF ARTICLE
Plasmids and Antibodies. See Supporting Information.
In Vitro Methylation Assay and IC50 Determination. The
assays have been described in detail previously.25 Briefly, all methylation
reactions were performed in a final volume of 30 μL of PBS (137 mM
NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.4) and
in the presence of S-adenosyl-l-[methyl-3H]methionine ([3H]AdoMet,
85 Ci/mmol from a 0.5 mCi/mL in dilute HCl/ethanol 9:1, pH 2.0ꢀ2.5,
PerkinElmer Life Sciences). The mixture contained 0.5ꢀ1.5 μM sub-
strate and 0.1ꢀ0.2 μM recombinant enzyme with 100 μM of each
indicated compound for fluorograph (Figures 3 and 4) or different doses
of each compound for IC50 determination (Table 1). The mixture was
incubated at 30 °C for 90 min and then separated by SDSꢀPAGE,
transferred to a PVDF membrane, sprayed with Enhance (PerkinElmer
Life Sciences), and exposed to film overnight for fluorography. After
fluorography, the same PVDF membrane was stained by Ponceau S and
the visualized bands of substrate were cut to count dpm by using a liquid
scintillation analyzer (Tri-Carb, Packard) for graphic depiction or IC50
determination.
Cell Lines and Cultures. See Supporting Information.
Luciferase Assay. LNCaP cells were cultured in phenol-red-free
RPMI 1640 supplemented with 10% charcoal-stripped fetal calf serum.
Approximately 20 h before transfection, cells were seeded into each well
of 24-well culture dishes. The cells in each well were transfected with
Lipofectamine 2000 transfection reagent (Invitrogen) according to the
manufacturer’s protocol. For each transfection, 300 ng of PSA(ARE)-
LUC and 2 ng of humanized CMV-Renilla internal control were used.
After 12 h of transfection, cells were treated with 20 nM DHT to induce
PSA-firefly and to indicate the amount of compound. After 42ꢀ44 h, the
cells were washed twice with PBS and harvested. Five of six cells were
used to perform luciferase assay using the dual luciferase assay system
(Promega) (Figure 5 top), and one of six cells was used to determine cell
viability using CellTiter-Glo luminescent reagent (Promega) according
to the manufacturer’s protocol (Figure 5 bottom).
Figure 5. Effects of increasing concentrations of 7g, 8e, and 8l on PSA
promoter activity by luciferase assay in LNCaP cells relative to a CMV-
Renilla control (top) and on cell viability based on quantitation of the
ATP present, which is an indicator of metabolically active cells and is
used to determine the viability of cells in culture (bottom). The results
are presented as the mean ( SD calculated from triplicate luciferase
assays.
’ EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi 530 melting
’ ASSOCIATED CONTENT
1
point apparatus and are uncorrected. H NMR and 13C NMR spectra
S
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). EIMS spectra were recorded with a Fisons
Trio 1000 spectrometer; only molecular ions (Mþ) and base peaks are
given. All solvents were reagent grade and, when necessary, were purified
and dried by standard methods. Organic solutions were dried over
anhydrous sodium sulfate. Elemental analysis was used to determine
purity of the described compounds, which is >95%. Analytical results are
within (0.40% of the theoretical values (see Table S3 in Supporting
Information). All chemicals were purchased from Aldrich Chimica (Milan,
Italy) or from Alfa Aesar (Milan, Italy) and were of the highest purity.
General Procedure for the Synthesis of N-Substituted 3,
5-Bis(3-bromo-4-hydroxybenzylidene)piperidin-4-ones (8aꢀl).
Example: 3,5-Bis(3-bromo-4-(hydroxybenzylidene)-1-
(3-chlorobenzyl)piperidin-4-one (8e). A solution of 10e (0.42
mmol, 0.3 g) in methanol (5 mL) and 3 N hydrochloric acid (5 mL) was
stirred at 60 °C for 3 h. Then the suspension was neutralized with 1 N
sodium hydrogen carbonate. The precipitated solid was filtered and
washed with water (3 ꢁ 10 mL) and diethyl ether (3 ꢁ 10 mL) to give
pure 8e as a yellow powder. 1H NMR (DMSO-d6, 400 MHz, δ, ppm) δ
4.43ꢀ4.48 (s, 6H, PhCH2 and CH2 piperidone), 7.05ꢀ7.74 (m, 12H,
PhCH and benzene protons), 11.13 (bs, 2H, OH) ppm; 13C NMR
(DMSO-d6, 400 MHz, δ, ppm) δ 53.4 (2C), 63.9, 113.7 (2C), 118.0
(2C), 126.0, 126.9, 127.3, 128.7 (2C), 129.6 (2C), 131.3 (2C), 132.2,
134.0, 136.9, 140.6 (2C), 145.9 (2C), 155.8 (2C), 186.0 ppm. MS (EI)
m/z: 588.95 (M)þ.
Supporting Information. Chemistry and experimental
b
details; IC50 curves for 7g and 8aꢀl against CARM1/PABP1,
PRMT1/NPL3, and SET7/H3. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For D.C.: phone, þ1-512-237-9328; fax, þ1-512-237-
2475; e-mail, dcheng@mdanderson.org. For M.T.B.: phone,
þ1-512-237-9539; fax, þ1-512-237-2475; e-mail, mtbedford@
mdanderson.org.ForA.M.:phone, þ3906-4991-3392; fax, þ3906-
49693268; e-mail, antonello.mai@uniroma1.it.
Author Contributions
These authors contributed equally to this work.
’ ACKNOWLEDGMENT
This work was partially supported by grants from Fondazione
Roma (A.M.) and COST Action TD09/05 Epigenetics (A.M.)
and by institutional NIEHS Grants ES007784 and ES015188
(M.T.B.).
’ ABBREVIATIONS USED
AdoHcy, S-adenosyl-L-homocystein; AdoMet, S-adenosyl-L-methio-
nine; AIB1, amplified in breast cancer 1; CA150, coactivator of
Experimental procedures for 9 and 10 and chemical and physical data
(Tables S2ꢀS4) for 8ꢀ10 are in Supporting Information.
4931
dx.doi.org/10.1021/jm200453n |J. Med. Chem. 2011, 54, 4928–4932

Journal of Medicinal Chemistry
BRIEF ARTICLE
150 kDa; CREB, cAMP-responsive element binding protein;
NPL3, heterogeneous nuclear ribonucleoprotein; PABP1, poly-
(A)-binding protein 1; rpS2, ribosome protein; PSA, prostate-
specific antigen; SmB, spliceosome protein; tetO, tet operator;
TetR, tetracycline-controlled transrepressor protein
(16) Selvi, B. R.; Batta, K.; Kishore, A. H.; Mantelingu, K.; Varier,
R. A.; Balasubramanyam, K.; Pradhan, S. K.; Dasgupta, D.; Sriram, S.;
Agrawal, S.; Kundu, T. K. Identification of a novel inhibitor of
coactivator-associated arginine methyltransferase 1 (CARM1)-mediated
methylation of histone H3 Arg-17. J. Biol. Chem. 2010, 285, 7143–7152.
(17) (a) Mai, A.; Massa, S.; Rotili, D.; Simeoni, S.; Ragno, R.; Botta,
G.; Nebbioso, A.; Miceli, M.; Altucci, L.; Brosch, G. Synthesis and
biological properties of novel, uracil-containing histone deacetylase
inhibitors. J. Med. Chem. 2006, 49, 6046–6056. (b) Mai, A.; Rotili, D.;
Tarantino, D.; Ornaghi, P.; Tosi, F.; Vicidomini, C.; Sbardella, G.;
Nebbioso, A.; Miceli, M.; Altucci, L.; Filetici, P. Small-molecule inhibi-
tors of histone acetyltransferase activity: identification and biological
properties. J. Med. Chem. 2006, 49, 6897–6907. (c) Pasco, M. Y.; Rotili,
D.; Altucci, L.; Farina, F.; Rouleau, G. A.; Mai, A.; Neri, C. Character-
ization of sirtuin inhibitors in nematodes expressing a muscular dystro-
phy protein reveals muscle cell and behavioral protection by specific
sirtinol analogues. J. Med. Chem. 2010, 53, 1407–1411. (d) Binda, C.;
Valente, S.; Romanenghi, M.; Pilotto, S.; Cirilli, R.; Karytinos, A.;
Ciossani, G.; Botrugno, O. A.; Forneris, F.; Tardugno, M.; Edmondson,
D. E.; Minucci, S.; Mattevi, A.; Mai, A. Biochemical, structural, and
biological evaluation of tranylcypromine derivatives as inhibitors of
histone demethylases LSD1 and LSD2. J. Am. Chem. Soc. 2010,
132, 6827–6833.
’ REFERENCES
(1) Bedford, M. T.; Clarke, S. G. Protein arginine methylation in
mammals: who, what, and why. Mol. Cell 2009, 33, 1–13.
(2) Bedford, M. T.; Richard, S. Arginine methylation an emerging
regulator of protein function. Mol. Cell 2005, 18, 263–272.
(3) Smith, B. C.; Denu, J. M. Chemical mechanisms of histone lysine
and arginine modifications. Biochim. Biophys. Acta 2009, 1789, 45–57.
(4) Taneda, T.; Miyata, S.; Kousaka, A.; Inoue, K.; Koyama, Y.; Mori,
Y.; Tohyama, M. Specific regional distribution of protein arginine
methyltransferase 8 (PRMT8) in the mouse brain. Brain Res. 2007,
1155, 1–9.
(5) Chen, D.; Ma, H.; Hong, H.; Koh, S. S.; Huang, S. M.; Schurter,
B. T.; Aswad, D. W.; Stallcup, M. R. Regulation of transcription by a
protein methyltransferase. Science 1999, 284, 2174–2177.
(6) Kim, D.; Lee, J.; Cheng, D.; Li, J.; Carter, C.; Richie, E.; Bedford,
M. T. Enzymatic activity is required for the in vivo functions of CARM1.
J. Biol. Chem. 2010, 285, 1147–1152.
(18) Mai, A.; Cheng, D.; Bedford, M. T.; Valente, S.; Nebbioso, A.;
Perrone, A.; Brosch, G.; Sbardella, G.; De Bellis, F.; Miceli, M.; Altucci,
L. epigenetic multiple ligands: mixed histone/protein methyltransferase,
acetyltransferase, and class III deacetylase (sirtuin) inhibitors. J. Med.
Chem. 2008, 51, 2279–2290.
(7) Frietze, S.; Lupien, M.; Silver, P. A.; Brown, M. CARM1
regulates estrogen-stimulated breast cancer growth through up-regula-
tion of E2F1. Cancer Res. 2008, 68, 301–306.
(19) Costi, R.; Di Santo, R.; Artico, M.; Miele, G.; Valentini, P.;
Novellino, E.; Cereseto, A. Cinnamoyl compounds as simple molecules
that inhibit p300 histone acetyltransferase. J. Med. Chem. 2007,
50, 1973–1977.
(20) Lee, J.; Bedford, M. T. PABP1 identified as an arginine methyl-
transferase substrate using high-density protein arrays. EMBO Rep.
2002, 3, 268–273.
(21) McBride, A. E.; Cook, J. T.; Stemmler, E. A.; Rutledge, K. L.;
McGrath, K. A.; Rubens, J. A. Arginine methylation of yeast mRNA-
binding protein Npl3 directly affects its function, nuclear export, and
intranuclear protein interactions. J. Biol. Chem. 2005, 280, 30888–30898.
(22) Cheng, D.; Cote, J.; Shaaban, S.; Bedford, M. T. The arginine
methyltransferase CARM1 regulates the coupling of transcription and
mRNA processing. Mol. Cell 2007, 25, 71–83.
(8) Hong, H.; Kao, C.; Jeng, M. H.; Eble, J. N.; Koch, M. O.;
Gardner, T. A.; Zhang, S.; Li, L.; Pan, C. X.; Hu, Z.; MacLennan, G. T.;
Cheng, L. Aberrant expression of CARM1, a transcriptional coactivator
of androgen receptor, in the development of prostate carcinoma and
androgen-independent status. Cancer 2004, 101, 83–89.
(9) El Messaoudi, S.; Fabbrizio, E.; Rodriguez, C.; Chuchana, P.;
Fauquier, L.; Cheng, D.; Theillet, C.; Vandel, L.; Bedford, M. T.; Sardet,
C. Coactivator-associated arginine methyltransferase 1 (CARM1) is a
positive regulator of the cyclin E1 gene. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 13351–13356.
(10) Majumder, S.; Liu, Y.; Ford, O. H., 3rd; Mohler, J. L.; Whang,
Y. E. Involvement of arginine methyltransferase CARM1 in androgen
receptor function and prostate cancer cell viability. Prostate 2006,
66, 1292–1301.
(23) Swiercz, R.; Cheng, D.; Kim, D.; Bedford, M. T. Ribosomal
protein rpS2 is hypomethylated in PRMT3-deficient mice. J. Biol. Chem.
2007, 282, 16917–16923.
(24) Deuschle, U.; Meyer, W. K.; Thiesen, H. J. Tetracycline-
reversible silencing of eukaryotic promoters. Mol. Cell. Biol. 1995, 15,
1907–1914.
(25) Mai, A.; Valente, S.; Cheng, D.; Perrone, A.; Ragno, R.;
Simeoni, S.; Sbardella, G.; Brosch, G.; Nebbioso, A.; Conte, M.; Altucci,
L.; Bedford, M. T. Synthesis and biological validation of novel synthetic
histone/protein methyltransferase inhibitors. ChemMedChem 2007,
2, 987–991.
(11) Purandare, A. V.; Chen, Z.; Huynh, T.; Pang, S.; Geng, J.;
Vaccaro, W.; Poss, M. A.; Oconnell, J.; Nowak, K.; Jayaraman, L.
Pyrazole inhibitors of coactivator associated arginine methyltransferase
1 (CARM1). Bioorg. Med. Chem. Lett. 2008, 18, 4438–4441.
(12) Huynh, T.; Chen, Z.; Pang, S.; Geng, J.; Bandiera, T.; Bindi, S.;
Vianello, P.; Roletto, F.; Thieffine, S.; Galvani, A.; Vaccaro, W.; Poss,
M. A.; Trainor, G. L.; Lorenzi, M. V.; Gottardis, M.; Jayaraman, L.;
Purandare, A. V. Optimization of pyrazole inhibitors of coactivator
associated arginine methyltransferase 1 (CARM1). Bioorg. Med. Chem.
Lett. 2009, 19, 2924–2927.
(13) Allan, M.; Manku, S.; Therrien, E.; Nguyen, N.; Styhler, S.;
Robert, M. F.; Goulet, A. C.; Petschner, A. J.; Rahil, G.; Robert Macleod,
A.; Deziel, R.; Besterman, J. M.; Nguyen, H.; Wahhab, A. N-Benzyl-1-
heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibi-
tors of co-activator associated arginine methyltransferase 1 (CARM1).
Bioorg. Med. Chem. Lett. 2009, 19, 1218–1223.
(14) Therrien, E.; Larouche, G.; Manku, S.; Allan, M.; Nguyen, N.;
Styhler, S.; Robert, M. F.; Goulet, A. C.; Besterman, J. M.; Nguyen, H.;
Wahhab, A. 1,2-Diamines as inhibitors of co-activator associated argi-
nine methyltransferase 1 (CARM1). Bioorg. Med. Chem. Lett. 2009,
19, 6725–6732.
(15) Wan, H.; Huynh, T.; Pang, S.; Geng, J.; Vaccaro, W.; Poss,
M. A.; Trainor, G. L.; Lorenzi, M. V.; Gottardis, M.; Jayaraman, L.;
Purandare, A. V. Benzo[d]imidazole inhibitors of coactivator associated
arginine methyltransferase 1 (CARM1)-hit to lead studies. Bioorg. Med.
Chem. Lett. 2009, 19, 5063–5066.
4932
dx.doi.org/10.1021/jm200453n |J. Med. Chem. 2011, 54, 4928–4932