Preparation of phenylethylbenzamide derivatives as modulators of DNMT3 activity

Article abstract of DOI:10.1039/c3md00214d

DNA-methyltransferases (DNMTs) are a class of epigenetic enzymes that catalyze the transfer of a methyl moiety from the methyl donor S-adenosyl-l-methionine onto the C5 position of cytosine in DNA. This process is dysregulated in cancers and leads to the hypermethylation and silencing of tumor suppressor genes. The development of potent and selective inhibitors of DNMTs is of utmost importance for the discovery of new therapies for the treatment of cancer. We report herein the synthesis and DNMT inhibitory activity of 29 analogues derived from NSC 319745. The effect of selected compounds on the methylation level in the MDA-MB-231 human breast cancer cell line was evaluated using a luminometric methylation assay. Molecular docking studies have been conducted to propose a binding mode for this series.

Full text of DOI:10.1039/c3md00214d

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CONCISE ARTICLE
Preparation of phenylethylbenzamide derivatives as
modulators of DNMT3 activity
Cite this: Med. Chem. Commun., 2013,
4, 1562
Anzhelika Kabro,a Hugo Lachance,ab Iris Marcoux-Archambault, Valerie Perrier,
a
a
´
a
c
c
c
´
´
Vicky Dore, Christina Gros, Veronique Masson, Jean-Marc Gregoire,
c
d
e
be
´ ´
Frederic Ausseil, David Cheishvili, Nathalie Bibens Laulan, Yves St-Pierre,
d
c
ab
*
*
Moshe Szyf, Paola B. Arimondo and Alexandre Gagnon
DNA-methyltransferases (DNMTs) are a class of epigenetic enzymes that catalyze the transfer of a methyl
moiety from the methyl donor S-adenosyl-L-methionine onto the C5 position of cytosine in DNA. This
process is dysregulated in cancers and leads to the hypermethylation and silencing of tumor suppressor
genes. The development of potent and selective inhibitors of DNMTs is of utmost importance for the
discovery of new therapies for the treatment of cancer. We report herein the synthesis and DNMT
inhibitory activity of 29 analogues derived from NSC 319745. The effect of selected compounds on the
methylation level in the MDA-MB-231 human breast cancer cell line was evaluated using a luminometric
methylation assay. Molecular docking studies have been conducted to propose a binding mode for
this series.
Received 24th July 2013
Accepted 20th September 2013
DOI: 10.1039/c3md00214d
www.rsc.org/medchemcomm
tRNAAsp
.
DNMT3L is catalytically inactive since it lacks the
5
Introduction
necessary catalytic domain. However, DNMT3L has been found
to associate with DNMT3A/B to modulate its activity. DNMT3A/
B are involved in de novo methylation of DNA, initiating meth-
ylation on a strand of DNA that is complementary to either the
unmethylated or methylated DNA template. Thus, DNMT3A/B
can generate new DNA methylation sites irrespective of the
pattern of methylation of template DNA.6 DNMT1 is implicated
in the maintenance of methylation patterns during cell division
and methylates hemimethylated DNA. Therefore, DNMT1
copies the pattern of methylation from the parental strand to
the nascent strand.7
In cancer cells, abnormal DNA methylation of the genome is
observed with both hypomethylation and hypermethylation of
critical genes8 as well as global reduction in the level of DNA
methylation across the genome9 and hypermethylation of CpG
islands in the promoter regions of some specic genes such as
tumor suppressor genes. This process leads to silencing of
those genes due to poor recognition by transcription factors and
recruitment of proteins involved in the chromatin remodeling
such as the methyl-binding proteins (MBD).10 Contrary to
Epigenetics is dened as the changes that occur on chromo-
somes without alteration of the DNA sequence and that lead to a
stable phenotype.1 Epigenetic information is regulated by
chromatin modications, which involve histones and DNA.2
DNA methylation is a stable epigenetic modication leading to
the installation of a methyl group at the C5 position of cytosine.3
This reaction is catalyzed by DNA methyltransferase (DNMT)
enzymes, which use S-adenosyl-L-methionine (SAM) as methyl
donor. The products of the reaction are 5-methylcytosine in
DNA and S-adenosyl-L-homocysteine (SAH).4 Five DNMTs have
been identied: DNMT1, DNMT2, DNMT3A, DNMT3B, and
DNMT3L. However, only DNMT1 and DNMT3A/B are catalyti-
cally active on DNA. In fact, DNMT2 has a very weak DNA
methylation activity and rather performs the methylation of
a
´
´
`
´
´
Universite du Quebec a Montreal, Departement de chimie, Case Postale 8888,
´
´
Succursale Centre-Ville, Montreal, Quebec, H3C 3P8, Canada. E-mail: gagnon.
alexandre@uqam.ca; Fax: +1-514-987-4054; Tel: +1-514-987-3000 ext. 6856
b
´
Pharmaqam, Departement de chimie, Case Postale 8888, Succursale Centre-Ville,
´
´
Montreal, Quebec, H3C 3P8, Canada. E-mail: gagnon.alexandre@uqam.ca; Fax: +1-
sequence aberrations, DNA methylation is
a reversible
514-987-4054; Tel: +1-514-987-3000 ext. 6856
process.11 More importantly, DNA methyltransferases are aber-
rantly deregulated and were proposed two decades ago as novel
anticancer targets.12 In fact, experiments suggest that inhibition
of DNMTs can lead to demethylation and reactivation of
epigenetically silenced tumor suppressor genes.13 Moreover the
emergence of the eld of “behavioral epigenetics” points to
the possibility that DNA methylation inhibitors might be of
value in treating mental health diseases and psychopathology
cUSR CNRS-Pierre Fabre no 3388-ETaC Epigenetic Targeting of Cancer, CRDPF 3
avenue Hubert Curien, 31035 Toulouse Cedex 01, France. E-mail: paola.arimondo@
etac.cnrs.fr; Fax: +33 5 34 50 34 92; Tel: +33 5 34 50 64 92
dDepartment of Pharmacology and Therapeutics, McGill University, 3655 Sir William
´
´
Osler Promenade, Montreal, Quebec, H3G 1Y6, Canada. E-mail: moshe.szyf@mcgill.
ca; Fax: +1-514-398-6690; Tel: +1-514-398-7107
e
´
Centre INRS-Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Quebec,
H7V 1B7, Canada. E-mail: yves.st-pierre@iaf.inrs.ca; Fax: +1-450-686-5501; Tel: +1-
450-686-5354
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disorders.14 Since neurons are postmitotic, DNA methylation
In 2010, Medina-Franco and Lyko reported the discovery of
inhibitors that are not incorporated into DNA will be needed. NSC 319745, a selective DNMT1 inhibitor (Fig. 1).35 This
Consequently, DNMT1 and DNMT315 constitute highly valuable compound was identied following
targets for drug development.16,17
campaign of a 65 000 compound collection from the NCI
a virtual screening
The discovery of potent and selective inhibitors of DNMTs is database using a homology model of the catalytic domain of
of utmost importance for the development of novel therapies human DNMT1. Using a biochemical assay, a residual enzy-
for the treatment of cancer18 and for the generation of tool- matic activity of 66% and 128% was measured against DNMT1
compounds for studying the involvement of DNMTs in the and DNMT3B, respectively, in the presence of 100 mM of NSC
development of various diseases.19 Two inhibitors of DNMTs 319745. Because of its inhibitory activity against DNMT1 and its
have been approved by the FDA for the treatment of myelodys- structural features, NSC 319745 represents an attractive starting
plastic syndrome (MDS) and acute myeloid leukemia (AML): point for hit-to-lead programs. To the best of our knowledge,
5-azacytidine (Vidaza) and 5-aza-20-deoxycytidine (Dacogen).20 structure–activity-relationship (SAR) studies on this compound
Aer being phosphorylated, these suicide substrates are incor- have never been reported.
porated into the DNA where they trap the DNMT by forming a
We report herein the synthesis of NSC 319745 and the
covalent adduct thus preventing the regeneration of the cata- preparation of 29 analogues of this molecule. The inhibitory
lytic cysteine.21 This process leads to the inactivation of the activity of these compounds against human DNMT1 and
DNMT, which allows the demethylation of tumor suppressor DNMT3A was measured and for the most potent compounds,
genes and their reactivation. However, these drugs are poorly the apparent EC50 was calculated. The compounds were also
bioavailable, non-selective and are incorporated everywhere in tested against the histone methyltransferases EZH2 and DOT1L
the genome. As a consequence, there is an urgent need for new to evaluate their selectivity against other SAM-dependent
potent and selective inhibitors of DNMTs that possess good enzymes. The effect of the most potent inhibitors on the
pharmacokinetic proles and that show minimal toxicity.
methylation level in MDA-MB-231 cells was explored and
DNMT inhibitors that do not rely on incorporation into DNA molecular docking studies have been performed to propose a
might offer a larger therapeutic window and a viable alternative mode of binding for this series.
to current treatments. A number of non-nucleoside inhibitors of
DNMTs have been reported in the literature.22 Natural products
such as EGCG,23 curcumin,24 caffeic acid,25 parthenolide,
nanaomycin A, mahanine, and genistein,26 and drugs that are
approved for other indications such as hydralazine,27
procaine,28 and procainamide29 have also been shown to inhibit
DNMTs.30
The discovery of small-molecule non-nucleoside inhibitors
of DNMTs has been accomplished using virtual31 or medium
throughput screening32 on various compound collections. This
process has led to the identication of a limited number of
compounds that inhibit DNMTs in the micromolar or sub-
micromolar range, such as the NSC series (Fig. 1). Two other
potent inhibitors of DNMTs, RG10833 and SGI1027,34 are
commonly used as reference compounds in screening assays.
Results and discussion
Chemistry
To initiate our medicinal chemistry efforts, we began by resyn-
thesizing NSC 319745. This compound was easily prepared from
bromophenethylamine 11 using the synthetic sequence illus-
trated in Scheme 1. Protection of the amine 11 followed by Heck
reaction with methyl acrylate and hydrogenation of the alkene
furnished phenylpropionate 12. Removal of the BOC protecting
group followed by amide formation using benzoyl chloride 14
and ester saponication gave NSC 319745 (10).
We then utilized this synthetic route to introduce different
le-hand side aryl groups on the molecule (Scheme 2,
compounds 20 to 27), to shorten the le-hand side alkyl chain
(compounds 28 and 29) and to install a methyl group at the b
Scheme 1 Synthesis of NSC 319745. Reagents and conditions: (a) BOC2O, Et3N, i-
PrOH, r.t., 2 h, 93%; (b) methyl acrylate, (PPh3)2PdCl2, LiCl, Et3N, DMF, microwave,
130 ꢀC, 3 h, 58%; (c) H2, Pd/C, MeOH, r.t., 3 h, 98%; (d) CF3CO2H, CH2Cl2, r.t., 3 h,
95%; (e) 5-chloro-2-methoxybenzoyl chloride (14), Et3N, CH2Cl2, r.t., 3 h, 87%; (f)
LiOH, THF, r.t., o.n., 87%.
Fig. 1 Structure of selected non-nucleoside small-molecule inhibitors of DNMTs.
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Scheme 5 Synthesis of amides 34–37. Reagents and conditions: (a) RR'NH,
TBTU, i-Pr2NEt, DMF, r.t., 60–94%.
Scheme 2 General synthetic sequence used for the preparation of analogues 20
to 31. Reagents and conditions: (a) BOC2O, Et3N, i-PrOH, r.t., 2 h, 79–93%; (b)
acrylate 17a–c, (PPh3)2PdCl2, LiCl, Et3N, DMF, microwave, 130 ꢀC, 3 h, 29–60%; (c)
H2, Pd/C, MeOH, r.t., 3 h; (d) CF3CO2H, CH2Cl2, r.t., 3 h, 79–94% after 2 steps; (e)
ArCOCl, Et3N, CH2Cl2, r.t., 53–94%; (f) LiOH, THF, r.t., o.n., 70–95%.
Scheme 6 Synthesis of compounds 38–43. Reagents and conditions: (a) 3-(3-(2-
aminoethyl)phenyl)propanoic acid methyl ester (44), Et3N, CH2Cl2, r.t., 83%; (b)
LiOH, THF, r.t., o.n., 61–98%; (c) 3-(4-(2-aminoethyl)phenyl)propan-1-ol (45), Et3N,
CH2Cl2, r.t., 27%; (d) (2-aminoethyl)phenylacetic acid ethyl ester (46), Et3N,
CH2Cl2, r.t., 83%; (e) 4-ethylphenethylamine (47), Et3N, CH2Cl2, r.t., 64%; (f)
phenethylamine, Et3N, CH2Cl2, r.t., 62%; (g) 4-(2-aminoethyl)benzoic acid
hydrochloride, NaOH, THF, r.t., 56%.
and a positions of the right hand side carboxylic acid group
(compounds 30 and 31).
The unsaturated analogue of NSC 319745 (compound 32)
was then prepared by simply omitting the hydrogenation step
from the general synthetic sequence (Scheme 3).
To study the importance of the amide N–H, we next prepared
the N-methyl amide 33 by performing a chemo-selective meth-
ylation reaction on 13 followed by saponication of the ester
(Scheme 4).
To evaluate potential acid surrogates, compounds 34 to 37
were prepared by amidation reaction of 10 using TBTU as the
coupling agent (Scheme 5).
Using this approach, the chain was moved to the meta
position (compound 38), shortened by one and two methylene
units (compounds 40 and 43), or completely removed
(compound 42). Compounds where the acid is reduced to the
alcohol (compound 39) or deleted (compound 41) were also
prepared.
We next proceeded with the introduction of an oxygen atom
in the propionic acid chain by coupling 4-hydroxyphenethyl-
amine 48 with benzoyl chloride 14 (Scheme 7). Compound 49
was then subjected to an alkylation reaction with methyl bro-
moacetate to provide the corresponding ester which was
saponied to afford the acid 50.
Introduction of modications on the right-hand side pro-
pionic acid chain was performed by reacting benzoyl chloride
14 with different amines (Scheme 6).
A simple variation of the previous synthetic sequence
allowed the introduction of a carboxylic acid group to the
central linker (compound 54, Scheme 8).
We next focused our attention on the benzamide moiety of
NSC 319745 and prepared the reverse amide 58 (Scheme 9). This
compound was accessed starting from acid 55 by formation of a
benzyl ester followed by a Heck reaction with methyl acrylate to
give the cinnamate 56. Saturation of the double bond and
Scheme 3 Synthesis of cinnamic acid 32. Reagents and conditions: (a) CF3CO2H,
CH2Cl2, r.t., 3 h, 85%; (b) 5-chloro-2-methoxybenzoyl chloride, Et3N, CH2Cl2, r.t.,
60%; (c) LiOH, THF, r.t., o.n., 95%.
Scheme 7 Synthesis of compounds 49 and 50. Reagents and conditions: (a) 14,
Et3N, CH2Cl2, r.t., 57%; (b) methyl bromoacetate, K2CO3, MeCN, reflux, 48%; (c)
LiOH, THF, r.t., o.n., 93%.
Scheme 4 Synthesis of N-methyl amide 33. Reagents and conditions: (a) NaH,
DMF, 0 ꢀC, 1 h then MeI, r.t., o.n., 80%; (b) LiOH, THF, r.t., o.n., 84%.
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Scheme 8 Synthesis of compound 54. Reagents and conditions: (a) MeI, K2CO3,
DMF, r.t., o.n., 88%; (b) methyl bromoacetate, K2CO3, MeCN, reflux, 2 h, 63%; (c)
CF3CO2H, CH2Cl2, r.t., 3 h, 98%; (d) 14, Et3N, CH2Cl2, r.t., 88%; (e) LiOH, THF, r.t.,
o.n., 80%.
Fig. 2 Inhibitory activity against DNMT1 expressed as % inhibition of methyla-
tion in the presence of 100 mM of inhibitors. SGI-1027 was used as a reference
compound.
reports, the resynthesized NSC 319745 (compound 10) showed
no signicant inhibitory activity against DNMT1 (ca. 2.9%). To
conrm this result, we retested compound 10 at 50 mM, 100 mM,
and 200 mM and observed no detectable inhibition of DNMT1
activity. These results indicate that NSC 319745 is not an
inhibitor of DNMT1 in our enzymatic test.
Scheme 9 Synthesis of reverse amide 58. Reagents and conditions: (a) BnOCOCl,
Et3N, DMAP, CH2Cl2, 0 ꢀC, 30 min, 70%; (b) methyl acrylate, (PPh3)2PdCl2, LiCl,
Et3N, DMF, microwave, 130 ꢀC, 3 h, 54%; (c) H2, Pd/C, MeOH, r.t., 3 h, 99%; (d)
5-chloro-2-methoxyaniline (59), TBTU, i-Pr2NEt, DMF, r.t., o.n., 36%; (e) LiOH, THF,
r.t., o.n., 74%.
The activity of the compounds was then measured against
DNMT3A at 10 mM and 100 mM using a nonradioactive assay
based on uorescence detection using the human catalytic
DNMT3A (Fig. 3).36 Interestingly, compounds 24, 26, 28, 29, 49,
50, and 54 showed greater than 60% inhibition at 100 mM. All of
these compounds, except 26, also showed more than 13%
inhibition at 10 mM. Resynthesized NSC 319745 (10) showed
negligible inhibitory activity against DNMT3A at both concen-
trations, in agreement with previous literature reports indi-
cating that NSC 319745 does not inhibit DNMT3B.
deprotection of the benzyl ester were performed simultaneously
under hydrogenation conditions to give the intermediate 57,
which was then coupled with aniline 59 to provide the nal
compound 58.
Finally, the analogue of NSC 319745 having a reduced amide
was prepared (compound 60, Scheme 10) via a reductive ami-
nation reaction between amine 19a and 5-chloro-2-methoxy-
benzaldehyde 61.
A
complete inhibition curve was measured for the
compounds that showed greater than 60% inhibitory activity
DNMT1 and DNMT3A inhibitory activity
Since NSC 319745 was reported to inhibit DNMT1, we started by
evaluating the inhibitory activity of the resynthesized NSC
319745 (compound 10) and its 29 analogues against full-length
DNMT1 using a 3H-methyl group incorporation assay (Fig. 2).
The results show that all the compounds give less than 20%
inhibition against DNMT1 compared to 100% inhibition by
reference compound SGI1027. The compounds that gave the
highest inhibition are the 4-nitrobenzamide 24 (14%), the
phenol 49 (16%), and the acid 50 (15%). Contrary to previous
Scheme 10 Synthesis of secondary amine 60. Reagents and conditions: (a)
5-chloro-2-methoxybenzaldehyde (61), NaBH3CN, MeOH, AcOH, r.t., o.n., 18%;
(b) LiOH, THF, r.t., o.n., 69%.
Fig. 3 Inhibitory activity against DNMT3A expressed as % inhibition of meth-
ylation in the presence of 100 mM (gray bars) and 10 mM (white bars) of inhibitors.
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The ortho methoxy group is found to be oriented in the same
direction as the amide carbonyl group oxygen. When the
binding of 49 is compared with that of SAH (magenta in Fig. 5),
we observe an overlap of the phenol moiety of 49 with the SAH
adenine group. The exible chain of the inhibitor extends
inside the binding pocket in the same orientation as the
homocysteine chain of SAH. These similarities in binding
mode seem to suggest that inhibitor 49 binds in the SAM/SAH
binding site.
Cellular activity
Fig. 4 Complete inhibition curve for compound 49 against DNMT3A. The
experiment was run twice in duplicate. The mean value is reported with the
standard error. A confidence interval of 95% and r2 ¼ 0.97 were calculated.
The impact of the most potent DNMT3A inhibitors on the
methylation level was evaluated in MDA-MB-231 breast cancer
cells using a luminometric methylation assay. The global
methylation level was determined in the presence of 100 mM
and 200 mM inhibitors using a methylation sensitive restriction
assay (HpaII/Msp1) followed by luminometric (LUMA) deter-
mination of cleavage by HpaII (sensitive to methylation at
CCmGG) versus Msp1 (insensitive to CCmGG methylation).
Compounds that were not sufficiently soluble in aqueous media
(compounds 28 and 29) or that were cytotoxic (compounds 24,
26 and 49) were not tested. Compounds 50 and 54 were thus
selected for cell studies based on potency, solubility and
absence of cytotoxicity (Fig. 6).
To our great disappointment, compound 50 induced no
signicant reduction of the methylation level at 100 mM and
200 mM. Similarly, when the cells were incubated in the presence
of compound 54, no effect on the methylation level could be
detected at 100 mM while only a minor reduction was observed at
200 mM. Although compounds 50 and 54 were effective in
inhibiting DNA methyltransferase activity in vitro, they did not
show signicant activity in cells as far as global DNA measure-
ments are concerned in contrast to the classical DNA methylation
inhibitor 5-azaC which dramatically reduced DNA methylation in
the same experiment. There could be several explanations for the
discrepancy between in vitro and in vivo potencies of these agents.
First, there might be permeability issues that reduce the
against DNMT3A at 100 mM. An apparent EC50 of 36 mM was
calculated for compound 49 (Fig. 4). Complete inhibition could
not be reached for the other compounds at concentrations up to
100 mM, thus making the determination of EC50 not possible.
In order to verify the selectivity of the prepared compounds
against other SAM-dependent methyltransferases, we tested
them against EZH2 and DOT1L. EZH2 (Enhancer of Zeste
homolog 2) catalyzes the methylation on lysine 27 of histone 3
(H3K27).37 Vire et al. have demonstrated that EZH2 serves as a
´
recruitment platform for DNMTs, thus suggesting a connection
between these two epigenetic enzymes in gene silencing.38
DOT1L (Disruptor of Telomeric silencing) is an enzyme that
performs the methylation on lysine 79 of histone 3 (H3K79).39
Using a 3H-SAM assay, no inhibition of EZH2 or DOT1L could be
observed at concentrations up to 80 mM, indicating the speci-
city of the compounds against DNA methylation.
Molecular modeling
Molecular docking of 49 was performed using the Forecasterꢀ
platform40 from Molecular Forecaster to establish its binding
mode using the crystal structure of the human catalytic
Dnmt3A/3L complex (PDB: 2QRV). The results from the docking
show a linear conformation for inhibitor 49 (green in Fig. 5)
with the amide group being in the expected trans conformation.
Fig. 6 Global methylation level of MDA-MB-231 cells measured by luminometric
assay (LUMA). Data are presented as mean of triplicate. 5-Azacytidine was used as
a reference compound at 1 mM.
Fig. 5 Docking of 49 in DNMT3A (PDB: 2QRV) using Forecasterꢁ (49 in green,
SAH in magenta).
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availability of the drug in the cell or in the nucleus. The second spectra were obtained with complete proton decoupling. Purity
possibility is that in contrast to 5-azaC which is incorporated into of tested compounds was determined by HPLC analysis on a
DNA and trap the DNMTs, the compounds tested are catalytic Beckman Gold HPLC system using a C18 column and acetoni-
inhibitors of DNMTs and it is possible that the level of inhibition trile (containing 0.01% of TFA) and water (containing 0.01% of
is insufficient to block DNA methylation during replication. We TFA) as the mobile phase. The purity of all nal compounds is
have previously shown that inhibition of DNMT results in inhi- greater than 95%. Compounds 41, 42, 43, 49, and 50 were
bition of DNA replication which in turn reduces the level of synthesized following a literature procedure.45
substrate for DNA methylation and thus the impact of DNMT
N-BOC protection: general procedure. In a round bottom
inhibition on methylation of newly synthesized DNA.41 Further ask, 10 mmol of amine 11 or 15b and c (1.0 eq.), 10 mmol of
modications of the compounds are possibly required to BOC2O (1.0 eq.) and 20 mmol of Et3N (2.0 eq.) in 50 mL of
increase potency and permeability in order to achieve effective i-PrOH were stirred at r.t. Progress of the reaction was moni-
inhibition of DNA methylation during DNA synthesis in tored by TLC (hexane–EtOAc, 4 : 1). Aer reaction completion,
living cells.
the solvent was evaporated under vacuum. The residue was
puried by ash column chromatography (hexane–EtOAc,
10 : 1) to afford the N-BOC amines 16a–c.
Conclusion
Heck reaction: general procedure. A mixture of 16a–c
The identication of small-molecules that modulate epigenetic (1.0 mmol, 1.0 eq.), LiCl (2.0 mmol, 2.0 eq.) and PdCl2(PPh3)2
processes can open new avenues for the treatment of cancer,42 (0.05 mmol, 0.05 eq.) in DMF (4 mL) in a microwave vial (5 mL)
mental health diseases, and psychopathology disorders.43 Drugs was degassed by bubbling argon through the solution for 10
that target DNMTs could also increase the options for treatment min. Et3N (2 mmol, 2.0 eq.) and methyl acrylate 17a–c
in the context of combination therapy.44 We performed the (1.25 mmol, 1.25 eq.) were added, and the mixture was degassed
resynthesis of NSC 319745 (a compound discovered by virtual again for 5 min. Then, the sealed vial was stirred in a microwave
screening against DNMT1) and 29 analogues and demonstrated at 130 ꢀC for 3 h. The reaction mixture was diluted with EtOAc
that, contrary to previous reports, NSC 319745 is not a DNMT and washed with aq. sat. solution of NaHCO3. The organic layer
inhibitor. All the analogues of NSC 319745 showed less than was separated, dried over anhydrous Na2SO4, ltered and
20% inhibition against DNMT1 at 100 mM. Seven compounds concentrated under vacuum. The crude mixture was puried by
were found to signicantly inhibit DNMT3A (compounds 24, 26, ash column chromatography (hexane–EtOAc, 8 : 1) to afford
28, 29, 49, 50, and 54). An inhibition curve against DNMT3A was compounds 18a–e.
measured for compound 49, giving an apparent EC50 of 36 mM.
Hydrogenation: general procedure. A solution of the
Molecular docking studies suggest that compound 49 binds in substrate 18a–e in MeOH (0.06 M) was stirred at room
the SAM pocket. For all the compounds prepared, no activity temperature over a 10% Pd/C catalyst (10% w/w) under a
was detected against EZH2 and DOT1L, two lysine histone hydrogen atmosphere for 3 h. Aer reaction completion, the
methyltransferases. A moderate reduction of the methylation mixture was ltered through a pad of celite and washed with
level in MDA-MB-231 cells was observed for compound 54 at CH2Cl2. The collected organic phase was evaporated under
200 mM. Compounds 49 and 54 are two small-molecule inhibi- vacuum. The crude product was used directly in the next step
tors of DNMT3 that could represent a potential new starting without further purication.
point for lead optimization programs. Medicinal chemistry
N-BOC deprotection: general procedure. CF3CO2H (50 eq.)
activities on this series to generate permeable and more potent was added dropwise to a 0 ꢀC 0.25 M anhydrous CH2Cl2
compounds are underway in our laboratories and results will be solution of the N-BOC protected compound (1.0 eq.) obtained
reported in due course.
from the previous step. The resulting mixture was stirred for
3 h at r.t. and the solvent was removed under reduced pres-
sure. The residue was taken up in CH2Cl2 and washed twice
with aq. sat. solution of NaHCO3. The organic layer was
separated, dried over anhydrous Na2SO4, ltered and
Experimental section
Chemistry
General methods. All reactions were run under an argon concentrated under vacuum. The product 19a–e was obtained
atmosphere in non ame dried glassware. Unless otherwise as a free base and was used directly in the next step without
stated, commercial reagents were used without further puri- further purication.
cation. Nuclear magnetic resonance spectra were recorded on a
Amide synthesis: general procedure. A 0.4 M anhydrous
Bruker Avance-III 300 MHz spectrometer. Chemical shis for CH2Cl2 solutionꢀ of the benzoyl chloride (1.1 eq.) was added
1H-NMR spectra are recorded in parts per million from tetra- dropwise to a 0 C anhydrous 0.2 M CH2Cl2 solution of amine
methylsilane with the solvent resonance as the internal stan- 19a–e (1.0 eq.) and Et3N (2.0 eq.). The resulting mixture was
dard. Data are reported as follows: chemical shi, multiplicity stirred at r.t. Aer completion of the reaction, as indicated by
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet), TLC analysis, the mixture was diluted with CH2Cl2 and washed
coupling constant J in Hz, integration, and assignment. with brine. The organic layer was separated, dried over anhy-
Chemical shis for 13C spectra are recorded in parts per million drous Na2SO4, ltered and concentrated under vacuum. The
from tetramethylsilane using the central peak of deutero- residue was puried by ash column chromatography (1%
chloroform (77.00 ppm) as the internal standard. All 13C-NMR MeOH/CH2Cl2), providing the corresponding amide.
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at 37 ꢀC for 2 hours. 8 mL are then transferred into a streptavidin
96-well scintillant coated Flashplateꢂ (PerkinElmer) containing
190 mL of 20 mM SAH in 50 mM Tris–HCl pH 7.4. The Flashplateꢂ
was agitated at room temperature for 1 hour, washed three times
with 200 mL of 0.05% Tweenꢀ-20 in 50 mM Tris–HCl pH 7.4, and
read in 200 mL of 50 mM Tris–HCl pH 7.4 on a TopCount NXT
(PerkinElmer).
Saponication: general procedure. To a 0 ꢀC 0.05 M THF
solution of the methyl ester (1.0 eq.) obtained in the previous
step was added a 1 M aqueous solution of LiOH (5.0 eq.). The
resulting mixture was stirred at r.t. overnight. The reaction
mixture was then diluted with 1 M NaOH and washed twice with
ether. The water phase was separated and carefully acidied
with conc. HCl until pH ꢁ3 was reached. The crude product was
extracted three times with CH2Cl2. The organic fraction was
dried over anhydrous Na2SO4, ltered and concentrated under
vacuum. The residue was puried by ash column chromatog-
raphy (5–10% MeOH/CH2Cl2) providing the corresponding acid
10 and 20–31.
DNMT3A inhibition assay. The procedure described in ref.
36 was used on catalytic human DNMT3A. Briey, a 50-labelled
biotin oligonucleotide is hybridized to its complementary
strand labelled with 6-carboxyuorescein at the 30-end into a
384 well microplate (black Optiplates; Perkin Elmer) pre-coated
with avidin. The duplex contains a unique CpG site overlapping
with a restriction site of a methylation sensitive restriction
enzyme. The human C-terminal DNMT3A (a.a. 623–912),
produced as described in the literature,46 was added in each well
(100 nM) and mixed with the chemical compounds at desired
concentrations and freshly prepared SAM (20 mM nal
concentration) to start the reaction in a total volume of 50 mL.
Aer 1 hour incubation at 37 ꢀC each well was washed three
times with PBS, Tween-20 0.05%, NaCl (500 mM) and three
more times with PBST. Specic uorescent signals were detec-
ted with the methylation-sensitive restriction enzyme Hpy-
CH4IV (NEB) as described and measured on a Perkin Elmer
Envision detector.
3-(4-(2-(5-Chloro-2-methoxybenzamido)ethyl)phenyl)-prop-
1
anoic acid 10. Rf 0.3 (5% MeOH/CH2Cl2). H-NMR (300 MHz,
CDCl3): d 10.94 (s(br), 1H, CO2H), 8.15 (d, J ¼ 2.7 Hz, 1H, Ph),
7.86 (s (br), 1H, NH), 7.33 (dd, J ¼ 8.7, 2.7 Hz, 1H, Ph), 7.17 (s
(br), 4H, Ph), 6.82 (d, J ¼ 8.7 Hz, 1H, Ph), 3.74–3.68 (m, 5H, Me,
CH2), 2.97–2.84 (m, 4H, 2 ꢂ CH2), 2.67 (t, J ¼ 7.6 Hz, 2H, CH2).
13C-NMR (75 MHz, CDCl3): d 177.9, 164.1, 156.0, 138.5, 137.1,
132.3, 131.9, 129.1, 128.5, 126.7, 122.8, 112.8, 56.1, 40.9, 35.6,
35.1, 30.2. HPLC: Rt ¼ 12.9 min, 99.9%.
5-Chloro-N-(4-hydroxyphenethyl)-2-methoxybenzamide 49.
Rf 0.4 (5% MeOH/CH2Cl2). 1H-NMR (300 MHz, CDCl3): d 8.23 (d,
J ¼ 2.7 Hz, 1H, Ph), 7.94 (s (br), 1H, NH), 7.42 (dd, J ¼ 8.7, 2.7 Hz,
1H, Ph), 7.16 (d, J ¼ 8.4 Hz, 2H, Ph), 6.89 (t, J ¼ 8.7 Hz, 3H, Ph),
3.83 (s, 3H, Me), 3.80–3.74 (m, 2H, CH2), 2.89 (t, J ¼ 6.6 Hz, 2H,
CH2). 13C-NMR (75 MHz, CDCl3): d 164.1, 156.0, 154.6, 132.4,
132.0, 130.9, 130.0, 126.8, 122.8, 115.5, 112.8, 56.2, 41.2, 34.6.
HPLC: Rt ¼ 12.2 min, 95.3%
EZH2 inhibition assay. PRC2, biotinylated nucleosome (both
prepared in-house), H3K27Me3 activator peptide (RKQLAT-
KAARK(Me3)SAPATGGVKKP-NH2, New England Peptide, Gard-
ner, MA) and compound (in DMSO) were incubated in 50 mM
Tris, pH 8.5, 5 mM MgCl2, 1 mM DTT, 70 mM Brij-35, and 0.1 mg
mLꢃ1 BSA for 30 minutes. Reaction was initiated by addition of
3H-SAM to nal conditions of 5 nM PRC2, 200 nM nucleosome
(concentration expressed as H3), activator peptide (3.6 mM) and
(S)-3-(4-(Carboxymethoxy)phenyl)-2-(5-chloro-2-methoxy-
benzamido)propanoic acid 54. Rf 0.3 (5% MeOH/CH2Cl2).
1H-NMR (300 MHz, CD3OD): d 8.60 (d, J ¼ 6.9 Hz, 1H, NH), 7.95
(d, J ¼ 2.7 Hz, 1H, Ph), 7.51 (dd, J ¼ 9.0, 2.7 Hz, 1H, Ph), 7.21–
7.16 (m, 3H, Ph), 6.93 (d, J ¼ 8.7 Hz, 2H, Ph), 4.89–4.85 (m, 1H),
4.68 (s, 2H, CH2), 3.88 (s, 3H, Me), 3.26–3.21 (m, 2H, CH2). 13C-
NMR (75 MHz, CD3OD): d 174.4, 165.8, 158.7, 158.1, 134.0,
131.8, 131.7, 130.6, 127.3, 123.6, 115.7, 115.1, 65.9, 57.2, 55.4,
37.3. HPLC: Rt ¼ 11.1 min, 97.9%.
3
200 nM H-SAM in a total volume of 25 mL in 384 well Greiner
plates. For compound analysis, assays were ten point dose-
responses with a nal total DMSO of 0.8% (v/v). Typically assays
were run for 60 minutes with <35% substrate turnover. Aer
that, reaction assays were quenched by addition of 20 mL of
2 mM SAH and 200 mM EDTA in 50 mM Tris, pH 8.5. Reactions
were transferred to streptavidin coated FlashPlates, incubated
for 2 hours, aspirated, washed, and read on a TopCount.
DOT1L inhibition assay. Run in a manner similar to the
Biology
DNMT1 inhibition assay. His-DNMT1 (182 kDa, human) was
expressed and puried as described in ref. 29a. DNMT1 activity EZH2 assay except that GST-DOT1L1-416 (prepared in-house47)
was measured by following the transfer of radioactively labeled was used at a nal concentration of 12 nM enzyme, 30 nM
methyl from SAM to a biotinylated oligonucleotide containing biotinylated oligonucleosome, 500 nM 3H-SAM and 30 minute
one hemimethylated CpG site as described in ref. 29b with some reaction time course in a buffer consisting of 50 mM Tris, pH
changes
8.5, 5 mM MgCl2, 1 mM DTT, and 80 mM Tween-20. Subsequent
In the Flashplateꢂ assay, the reaction was performed in a 10 quench and work-up were as for EZH2.
mL total reaction volume in low volume NBSꢂ 384-well micro-
Cell line and culture conditions. Breast MDA-MB-231 cells
plates (Corning), containing the tested compound (up to 1% (#HTB-231) were purchased from the American Type Culture
DMSO), 1 mM of a SAM/[methyl-3H] SAM (3 TBq per mmol, Per- Collection (ATCC, Manassas, Virginia). MDA-MB-231 cells were
kinElmer) mix in a ratio of 3-to-1 (isotopic dilution 1 : 3), 0.3 mM of cultured in Dulbecco's modied Eagle's medium (Invitrogen).
biotinylated hemimethylated DNA duplex (50-GATmCGCmC 2 ꢂ 106 MDA-MB-231 cells were plated the day before treatment
GATGmCGmCGAATmCGmCGATmCGATGmCGAT-30 and BIOT-50- on 10 cell culture plates. The following day, the cells were
ATCGCATCGATCGCGATTCGCGCATCGGCGATC-30), and 90 nM treated with compounds at concentrations of 100 and 200 mM
of DNMT1 in methylation buffer (20 mM HEPES pH 7.2, 1 mM and with 1 mM of 5-Azadc. The treatment was repeated for six
EDTA, 50 mM KCl, 25 mg mLꢃ1 BSA). The reaction was incubated days, every second day. On day six, cells were harvested.
1568 | Med. Chem. Commun., 2013, 4, 1562–1570
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DNA extraction and luminometric methylation assay. DNA
was extracted by phenol–chloroform as follows: cells were
resuspended and incubated in DNA extraction buffer (500 mL)
containing proteinase K (20 mL; 20 mg mlꢃ1; Roche, Basel,
Switzerland) at 50 ꢀC for 12 h. Samples were treated with
RNAase A (50 U mgꢃ1; 30 min; Roche) and phenol–chloroform
(1 : 1). To precipitate DNA, ethanol [95% (v/v)] was added. The
pellet was washed in ethanol 70% and redissolved in DNAse free
water. DNA was then digested with methylation sensitive
(HpaII) or insensitive (MspI) restriction enzymes in combina-
tion with an internal control restriction enzyme (EcoRI) to
normalize for the DNA input. Both HpaII and MspI restriction
enzymes recognize and cleave 50-CCGG-30 sequences producing
50-CG overhangs, whereas EcoRI recognizes and cleaves
50-GAATTC-30 sequences and produces 50-AATT overhangs. The
extent of cleavage is determined by a bioluminometric poly-
merase extension assay based on a four-step pyrosequencing
reaction. Samples were incubated (37 ꢀC, 4 h) and then heat
inactivated (80 ꢀC, 20 min). Digested genomic DNA (15 mL) was
mixed with pyrosequencing annealing buffer (15 mL; Qiagen,
Toronto, ON, Canada). Samples were transferred to 24-well
pyrosequencing plates for sequencing (PyroMark 24; Biotage,
Uppsala, Sweden). The nucleotide dispensation order used was
based on the study by Pilsner et al.48 Peak heights for C and A
represent the HpaII and MspI cuts (methylation) and EcoRI
(input DNA), respectively. The formula for calculating %
genomic methylation is: 1-[(HpaII (C)/EcoRI (A)/MspI (C)/EcoRI
(A))] ꢂ 100. All samples were run in triplicate.
17 Many other epigenetic enzymes are also currently being
explored as potential target for the development of
therapeutic agents: (a) P. Jones, Med. Chem. Commun.,
2012, 3, 135–161; (b) R. A. Copeland, E. J. Olhava and
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Acknowledgements
This work was supported by Pharmaqam, the Natural Sciences
and Engineering Research Council of Canada (NSERC), the
´
´
`
´
Universite du Quebec a Montreal (UQAM), and a grant from the
Canadian Institute of Health Research to MS. PBA was funded
´
´
by Region Midi-Pyrenees (Equipe d'Excellence) et FEDER
18 (a) J. Fahy, A. Jeltsch and P. B. Arimondo, Expert Opin. Ther.
Pat., 2012, 22, 1427–1442; (b) M. Szyf, Biochim. Biophys. Acta,
2010, 1799, 750–759; (c) C. Gros, J. Fahy, L. Halby, I. Dufau,
´
´
(CNRS-Region Midi-Pyrenees). We thank Constellation Phar-
maceuticals for generously testing our compounds against
EZH2 and DOT1L. AG would like to thank Dr Brian Albrecht, Dr
Richard Cummings, and Pr. Steve Bourgault for useful discus-
sion. IMA thanks UQAM for an undergraduate scholarship, DC
thanks the Israel Cancer Research Fund for a post-doctoral
fellowship.
´
A. Erdmann, J.-M. Gregoire, F. Ausseil, S. Vispe and
P. B. Arimondo, Biochimie, 2012, 94, 2280–2296; (d) Y. Itoh,
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K.-K. Ho, M. V. McCullar and S. B. Kanner, J. Biomol.
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