Selective non-nucleoside inhibitors of human DNA methyltransferases active in cancer including in cancer stem cells

Article abstract of DOI:10.1021/jm4012627

DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.

Full text of DOI:10.1021/jm4012627

Article
pubs.acs.org/jmc
Selective Non-nucleoside Inhibitors of Human DNA
Methyltransferases Active in Cancer Including in Cancer Stem Cells
Sergio Valente,†,□ Yiwei Liu,‡,□ Michael Schnekenburger,§ Clemens Zwergel,†,∥ Sandro Cosconati,⊥
Christina Gros,# Maria Tardugno,† Donatella Labella,† Cristina Florean,§ Steven Minden,§
Hideharu Hashimoto,‡ Yanqi Chang,‡ Xing Zhang,‡ Gilbert Kirsch,∥ Ettore Novellino,∇
Paola B. Arimondo,# Evelina Miele,○ Elisabetta Ferretti,◆ Alberto Gulino,◆ Marc Diederich,§,¶
Xiaodong Cheng,‡ and Antonello Mai*,†
†Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Universita
‡Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, United States
§Laboratoire de Biologie Molec
ulaire et Cellulaire du Cancer (LBMCC), Hopital Kirchberg, 9, rue Edward Steichen,
L-2540 Luxembourg
∥Laboratoire d’Ingen
ierie Molec
France
⊥DiSTABiF, Seconda Universita
#USR3388 CNRS-Pierre Fabre ETaC, CRDPF, 3 Avenue H. Curien, 31035 Toulouse Cedex 01, France
̀
di Roma, P.le Aldo Moro 5, 00185 Roma, Italy
́
̂
́
́ ́
ulaire et Biochimie Pharmacologique, Universite de Lorraine−Metz, 1 Boulevard Arago, 57070 Metz,
̀
di Napoli, Via G. Vivaldi 43, 81100 Caserta, Italy
∇Dipartimento di Farmacia, Universita
̀
di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy
○Istituto Italiano di Tecnologia and Dipartimento of Medicina Molecolare, Sapienza Universita
00161 Roma, Italy
̀
di Roma, Viale Regina Elena 291,
◆Dipartimento di Medicina Sperimentale e Dipartimento di Medicina Molecolare, Sapienza Universita
Viale Regina Elena 291, 00161 Rome, Italy
̀
di Roma,
¶Department of Pharmacy, College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
S
* Supporting Information
ABSTRACT: DNA methyltransferases (DNMTs) are important enzymes involved in epigenetic control of gene expression and
represent valuable targets in cancer chemotherapy. A number of nucleoside DNMT inhibitors (DNMTi) have been studied in
cancer, including in cancer stem cells, and two of them (azacytidine and decitabine) have been approved for treatment of
myelodysplastic syndromes. However, only a few non-nucleoside DNMTi have been identified so far, and even fewer have been
validated in cancer. Through a process of hit-to-lead optimization, we report here the discovery of compound 5 as a potent non-
nucleoside DNMTi that is also selective toward other AdoMet-dependent protein methyltransferases. Compound 5 was potent at
single-digit micromolar concentrations against a panel of cancer cells and was less toxic in peripheral blood mononuclear cells than
two other compounds tested. In mouse medulloblastoma stem cells, 5 inhibited cell growth, whereas related compound 2 showed
high cell differentiation. To the best of our knowledge, 2 and 5 are the first non-nucleoside DNMTi tested in a cancer stem cell line.
including cancer.3−5 In contrast to genetic origins of cancer,
epigenetic aberrations are reversible events that occur at early
stages in tumor genesis, and in the past decade, many interactions
and connections have been reported between genetic and
INTRODUCTION
■
Epigenetic regulation of gene expression is mediated through at
least five series of events involving changes of chromatin at the
molecular level: DNA modifications, histone modifications,
epigenetic changes that highlight the complex, multifactorial
histone variants, noncoding RNAs, and nucleosome remodel-
ing.1,2 Epigenetic control of transcription is essential to drive cells
toward their normal phenotype, and epigenetic deregulation
could lead to initiation and progression of human diseases
Received: August 14, 2013
Published: January 4, 2014
© 2014 American Chemical Society
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Chart 1. Nucleoside and Non-nucleoside DNMT Inhibitors
nature of such disease.4 Among the five epigenetic events,
DNA methylation has been extensively studied. Three DNA
methyltransferases (DNMTs), DNMT1, DNMT3A, and DNMT3B,
catalyze the transfer of a methyl group from S-adenosyl-L-methionine
(AdoMet) to the C5-position of cytosine predominantly in CpG
dinucleotides.6−9 The maintenance methyltransferase DNMT1
is most abundant in somatic cells and has a greater activity
for hemi- rather than unmethylated substrates.8,9 Differently, de
novo methyltransferase DNMT3A is only present in low
amounts in somatic cells and shows no preference for hemi- or
unmethylated DNA.10
In normal cells, DNA methylation plays a key role in many
physiological events (genomic imprinting control, X-chromosome
inactivation, maintenance of chromosomal stability, silencing of
genesand endogenous retroviruses, and embryonicdevelopment),
following a pattern according to which certain genomic sites are
hypermethylated (gene silencing), whereas other sites, such as
CpG islands, are hypomethylated (gene transcription).9,11 In
cancer cells, aberrant DNA methylation leads to hypermethylation
at CpG islands, joined to a global hypomethylation, giving rise to
genomic instability and inactivation of tumor-suppressor genes.9,11
Hypermethylation of CpG islands is so biologically relevant and
specific that for each cancer a typical hypermethylome can be
drawn.12 In addition to cancer, very recently aberrant DNA
methylation has been linked to premature senescence and aging
diseases such as Hutchinson−Gilford Progeria and Werner
syndrome,13,14 to neural plasticity and GABAergic signaling
modulation,15,16 and to heart failure and atrial fibrillation through
methylation of the homeobox gene Pitx2c.17 Moreover, DNMT1
and DNMT3A proteins have been found to be overexpressed in
rheumatoid arthritis and osteoarthritis.18
been approved by the U.S. FDA for clinical use against
hematological malignancies, but despite their high efficacy,
such drugs suffer from poor bioavailability, chemical instability,
and toxic side effects.9,19 Some non-nucleoside compounds have
been reported as DNMTi (Chart 1), but they typically share low
potency and/or low selectivity for DNMTs, and their mechanism
of inhibition is unknown.21−25
Among non-nucleoside inhibitors, SGI-1027 (1) (Figure 1),
identified among a series of quinoline-based compounds
developed as anticancer drugs, has attracted our attention
because of its high potency in both enzyme and cell assays.26
Because the structure of 1 shows four fragments (4-amino-
quinoline + 4-aminobenzoic acid + 1,4-phenylenediamine + 2,4-
diamino-6-methylpyrimidine) linked in sequence with para/para
orientation, we prepared a series of regioisomers of 1 by shifting
each fragment’s linkage from the para to the meta or ortho position
(Figure 1) and obtained compounds 2−9 (Figure 1). In addition,
we prepared two related compounds (10 and 11) showing either a
bis-quinoline or bis-pyrimidine structure and two truncated
compounds (12 and 13) lacking the “right” pyrimidine or the
“left” quinoline portion, respectively (Figure 1).
CHEMISTRY
■
Compounds 1−9 were prepared by coupling the 2-, 3-, or 4-
(quinolin-4-ylamino)benzoic acid, 14a−c, with the N4-(2-, 3-, or
4-aminophenyl)-6-methylpyrimidine-2,4-diamine, 15a−c, in the
presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) and triethylamine in dry N,N-
dimethylformamide as solvent (Scheme 1). Intermediate
compounds 14a−c were prepared by reaction between 4-
chloroquinoline with the appropriate ethyl 2-, 3-, or 4-
aminobenzoate and 37% hydrochloric acid in ethanol at 80 °C.
The obtained ethyl benzoates, 16a−c, underwent basic
hydrolysis using 2 N potassium hydroxide to afford the
corresponding acids, 14a−c. The same reaction conducted
DNMT inhibitors (DNMTi) have been validated as useful
tools to reactivate tumor-suppressor genes and to reprogram can-
cer cells toward growth arrest and death.9,19,20 Two nucleoside
analogue DNMTi (azacytidine and decitabine, Chart 1) have
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Figure 1. Chemical structures of SGI-1027 (1) and related analogues 2−13 described in this study. Their IC50 values (μM) from nanoscale HTS against
human DNMT1 are reported in the brackets.
with 4-chloro-6-methylpyrimidin-2-amine and the opportune 2-,
3-, or 4-nitroanilines in the presence of 37% hydrochloric acid
and ethanol at 80 °C furnished the nitro-intermediates, 17a−c,
which were in turn reduced with stannous chloride dihydrate and
37% hydrochloric acid in ethanol to the corresponding anilines,
15a−c.
Chemical and physical data of final compounds 1−13
(Table S1) as well as those of intermediate compounds 14−22
(Table S2) are listed in the Supporting Information.
methylpyrimidin-2-amine with ethyl 4-aminobenzoate afforded
intermediate ester 20, which was hydrolyzed to acid 21 and was
then coupled with 17c to furnish bispyrimidine 11 (Scheme 2B).
Truncated compounds 12 and 13 were prepared by reaction
of 16c with 4-phenylendiamine (12) or by reaction of 17c
with 4-nitrobenzoic acid and subsequent reduction of the
nitro group of intermediate 22 to the corresponding amine (13)
(Scheme 3).
Bisquinoline 10 was obtained by treating 4-chloroquinoline
with 4-nitroaniline and, after reduction of the nitro group of the
resulting intermediate 18 to the corresponding aniline 19, by
coupling 19 with 16c (Scheme 2A). Treatment of 4-chloro-6-
RESULTS AND DISCUSSION
■
DNMT1, DNMT3A2/3L, PRMT1, and GLP Inhibition Assays.
From a nanoscale prescreen performed on 1−13 against human
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a
Scheme 1. Synthesis of Compounds 1−9
a
Reagents and conditions: (a) 37% HCl, CH3CH2OH, 80 °C, 2 h; (b) 2 N KOH, CH3CH2OH; (c) stannous chloride dihydrate, 37% HCl,
CH3CH2OH, 1 h, 80 °C; (d) (C2H5)3N, PyBOP, anhydrous DMF, N2 atmosphere, room temperature, 1 h.
a
Scheme 2. Synthesis of Compounds 10 and 11
a
Reagents and conditions: (a) 37% HCl, CH3CH2OH, 80 °C, 2 h; (b) stannous chloride dihydrate, 37% HCl, CH3CH2OH, 80 °C, 1 h; (c)
(C2H5)3N, PyBOP, anhydrous DMF, N2 atmosphere, room temperature, 1 h; (d) 2 N KOH, CH3CH2OH.
a
Scheme 3. Synthesis of Compounds 12 and 13
a
Reagents and conditions: (a) (C2H5)3N, PyBOP, anhydrous DMF, N2 atmosphere, room temperature, 1 h; (b) stannous chloride dihydrate, 37%
HCl, CH3CH2OH, 80 °C, 1 h.
DNMT1 using poly(dI−dC) as substrate, compounds 2, 4, and
5, obtained by replacing either or both the para with the meta
linkages in the structure of 1, emerged as highly efficient
DNMT1 inhibitors, whereas the ortho regioisomers were less
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Figure 2. Inhibitory activities of 1, 2, 4, 5, 10, and 11 against human DNMT1 (hemimethylated substrate) and the DNMT3A2/DNMT3L complex
(unmethylated substrate).
Figure 3. Competition experiments performed with compound 5 on DNMT1 by varying AdoMet concentrations at a near-saturating DNA
concentration (A, B) or by varying DNA concentrations at a near-saturating AdoMet concentration (C, D). (A) IC50 of compound 5 plotted against
[AdoMet]/KmAdoMet. (B) Velocity plot against [AdoMet] for different concentrations of compound 5. The lines represent the nonlinear regressions by
the Michaelis−Menten equation. (C) IC50 of 5 plotted against [DNA]/KmDNA. The line represents the linear regression. (D) Velocity plot against
[DNA] for different concentrations of 5. The lines represent the nonlinear regressions with sigmoidal dose−response. On each graph, the mean of two
independent experiments is represented SEM.
potent (3 and 6−8) or totally inactive (9). Bisquinoline 10
displayed a similar potency as 1 against DNMT1, whereas bis-
pyrimidine 11 was slightly less efficient, and truncated compounds
12 and 13 showed a severe drop in inhibition (Figure 1).
To perform a more wide and accurate inhibition assay against
DNMTs, we tested the most potent compounds, 2, 4, 5, 10,
and 11, in comparison with 1 against human DNMT1 using a
hemimethylated substrate and against the human DNMT3A2/
DNMT3L complex using an unmethylated substrate (Figure 2).
Under these assay conditions, meta/meta analogue 5 (IC50 = 9 μM)
displayed a 4-fold higher activity than 1 against DNMT1, whereas
the para/meta (2) and meta/para (4) analogues were 1.5- and 7.6-
fold less potent, respectively. Bisquinoline 10 showed the same
DNMT1 inhibiting activity as 1, whereas bispyrimidine 11 was
2-fold less potent.
Against DNMT3A2/DNMT3L, all of the inhibitors were
more efficient than against DNMT1, and 5 was again the most
potent, with IC50 = 2.8 μM [IC50 (1) = 10 μM]. Among the
remaining compounds, 11 and 2 were 2-fold, 10 was 3-fold, and 4
was 4-fold less potent than 1 (Figure 2).
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To determine the mechanism of DNMT1 inhibition by
compound 5, we carried out competition experiments by varying
the concentration of either AdoMet or DNA, and we used both
the IC50 and the velocity plot methods to analyze the data.
As shown in Figure 3, the IC50 analysis of 5 against the AdoMet
concentration shows nearly constant IC50 values as the AdoMet
concentration increases (Figure 3A,B), suggesting a non-
competitive inhibition.27 Differently, the IC50 of 5 increases
linearly with DNA concentration, suggesting a DNA-competitive
behavior (Figure 3C,D).27 The velocity plot against DNA
concentration displays sigmoidal deformations. These deforma-
tions have been described by Copeland and Horiuchi28 and are
explained by potential nonspecific substrate−inhibitor inter-
actions (DNA interactions), tight-binding, or a time-dependent
inhibitor.28 Competition studies performed on structurally
different compound 11 gave similar results (Figure S1 in the
Supporting Information).
sinefungin (PDB 3SWR, Hashimoto and Cheng, unpublished
data) and DNMT1 (residues 646−1600) in complex with both
AdoHcy and a 19 bp DNA duplex (PDB 3PTA).32 These two
DNMT1 structures share a similar overall 3D arrangement,
with some conformational differences mainly in the DNA-
binding CXXC domain region (residues 646−692, Figure S2 in
the Supporting Information). The latter is adjacent to the
methyltransferase domain, which is the putative inhibitor binding
site, and in principle its conformation could influence the inhibitor
binding. Therefore, we decided to dock 5 in both of the afore-
mentioned structures by employing the Glide program of the
Schrodinger package.33
̈
Docking results achieved on the DNMT1 structure alone
(unbound to DNA) revealed that in the lowest-energy binding
conformation (docking score −6.95) 5 should be able to span
between the AdoMet binding site and the CXXC region (Figure 4).
To determine the specificity of 1, 2, 4, 5, 10, and 11 for
DNMTs among other AdoMet-dependent enzymes, we tested
them against PRMT1, a protein arginine methyltransferase,29
and G9a-like protein (GLP), a histone H3 lysine 9 methyl-
transferase.30,31 Under the tested conditions, the new derivatives
showed very low (if any) PRMT1- and GLP-inhibiting activities,
resulting in the tested compounds being more DNMT-selective
than 1, which displayed only 4- and 1.9-fold lower activities
against PRMT1 and GLP, respectively, when compared with
DNMT1 inhibition. In contrast, 5 was 33- and 11-fold less potent
against PRMT1 and GLP than against DNMT1 (Table 1).
Table 1. Inhibitory Activities of 1, 2, 4, 5, 10, and 11 against
a
PRMT1 and GLP
Figure 4. Predicted binding mode of 5 in the DNMT1 structure
unbound to DNA (PDB 3SWR). Compound 5 is depicted as orange
sticks, and the enzyme catalytic and CXXC domains are depicted as cyan
(right side) and brown (left side) ribbons and sticks, respectively. H-
bonds are depicted as dashed red lines.
Indeed, changing one or both the para/meta linkages (1, 2, 4, and
5) with ortho ones (3 and 6−9) by modifying the ligand shape
from a stretched to a more condensed one should prevent the
ligand from spanning between the two sites, explaining why the
presence of ortho linkages is not productive in terms of inhibitory
potency. As depicted in Figure 4, the 4-aminoquinoline fragment
of 5 is embedded in a lipophilic region normally occupied by
the AdoMet adenine ring, establishing hydrophobic contacts
with I1167, M1169, P1225, and I1247 as well as a T-shaped
interaction with F1145. In this position, substitution of the
quinoline ring with a smaller pyrimidine one (11) or with a
hydrogen (13) would cause a partial loss of the interactions
within the aforementioned cleft, thus explaining the drop in
inhibitory potency. Also, the attached central fragment (3-
aminobenzoic acid + 1,3-phenylenediamine) of 5 is embedded in
a region in which cation−π and π−π interactions are established
with R1574 and W1170, respectively. The intrinsic rigidity of this
fragment allows to project the 2,4-diamino-6-methylpyrimidine
terminal at the crevice between the CXXC and methyltransferase
domains. In this position, the terminal hydrophilic head of 5
forms three H-bonds with the M694 and P692 backbone CO and
with the D1571 side chain. Given the critical role of the CXXC
region for the DNMT1 enzymatic activity,34 it might be
speculated that favorable ligand interactions with this region
PRMT1
GLP
compound IC50 (μM)
DNMT1
selectivity
IC50 (μM)
DNMT1
selectivity
1
2
3
4
5
6
139
4.0
65
1.9
300
5.8
600
400
100
100
500
11.5
1.5
300
1.1
300
33.3
3.0
11.1
3.0
100
>1000
>14.7
7.3
a
The DNMT1 selectivity (as the PRMT1 or GLP/DNMT1 IC50
ratio) for each compound is reported. Inhibition assays were per-
formed in duplicate. The errors in the determinations of IC50’s are
within 20% of their values.
Docking Studies of Compound 5 in DNMT1 Structures.
To delineate better the mechanism(s) for the inhibitory potency
of 5 against DNMT1, molecular modeling studies were per-
formed. In particular, compound 5 was docked in two different
DNMT1 structures available in the Protein Data Bank (PDB):
DNMT1 (residues 600−1600) crystallized in complex with
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Figure 5. Cellular studies on quinoline-based DNMTi. (a−e) Antiproliferative effects (left) and cell-death induction (right) of 1, 2, and 5 on U-937 (a),
RAJI (b), MDA-MB-231 (c), PC-3 (d), and PBM (e) cells. (f) IC50 values of cell viability relative to the above cells treated with 1, 2, and 5 for 48 h. (g)
Nuclear-morphology analysis after Hoechst and PI staining in U-937 and RAJI cells treated with increasing doses of 5 for 72 h. Data represent the mean
( SD) of at least three independent experiments. Pictures are representative of three independent experiments.
should also be critical for inhibitory potency, thus explaining why
replacement of the 2,4-diamino-6-methylpyrimidine (5) frag-
ment with a quinoline (10) one or with an hydrogen (12) does
not enhance DNMT1 inhibition.
Interestingly, the same calculations did not succeed in sug-
gesting a well-defined binding pose for compound 5 in the enzyme/
DNA structure. Indeed, in the DNMT1/DNA complex, when
merging the coordinates of 5 achieved in the absence of DNA, it
seems clear that the subtle rearrangements of the CXXC domain
induced by the presence of DNA would sterically hamper 5
from spanning from the AdoMet interaction site to the crevice
between the CXXC and methyltransferase domains (Figure S4b
in the Supporting Information). Results of analogous docking
studiesperformed on1 are reportedinthe Supporting Information
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(Figures S3 and S4a). Almost the same binding orientations were
obtained either in the free DNMT1 or in the DNMT1/DNA
structure, suggesting that, whereas 5 can in principle be
recognized by the DNA-unbound enzyme conformation (i.e.,
in a DNA-competitive binding mode), 1 should be able to bind
DNMT1 independently from the presence of DNA.
Azacytidine has been shown to reactivate SALL4 expression and
transcription in acute promyelocytic leukemia NB4 cells36 as
well as in colorectal cancers37 through DNMT inhibition. In
IDH1 mutant glioma cells, decitabine induced a dramatic loss
of stemlike properties and efficient adoption of markers of
differentiation as well as decreased replicative potential and
tumor growth in vivo.38 To date, no non-nucleoside DNMTi has
been tested in a cancer stem cell context. We tested compounds 2
and 5 at different dosages in mouse MbSCs, a cancer stem
cell line expressing high levels of DNMTs (Figure S7 in the
Supporting Information), to determine their effects on cell
proliferation and differentiation. In these assays, compound 5
arrested the MbSC clonogenic activity, induced cell adhesion and
differentiation, and impaired significantly the MbSC growth rate,
evaluated by both quantifying PCNA levels and MTT assay
(Figure 6a,b), whereas 2 was less effective. In MbSCs differentiation
Competition experiments also outline that the inhibitory
potency of 5 is not affected by increasing concentrations of the
AdoMet cofactor. These data might suggest that either the ligand
is also able to adapt to binding sites that are different from that of
the cofactor or the same compound can also bind the enzyme/
cofactor complex. In this respect, the DNMT1 structures (PDB
3PTA and 3SWR) were also searched for different binding sites
with the SiteMap software available within the Maestro suite.33
Results of these predictions demonstrate that the most probable
site is the one occupied by the cofactor, whereas four additional
less probable sites could exist (Figure S5a). Nevertheless, when
Glide was employed to predict a possible binding position of 5 in
these enzyme regions, weak binding affinities were predicted
(docking scores below 5.0), mainly because the selected clefts,
being rather shallow and solvent-exposed, are less prone to
provide efficient ligand interactions. This would suggest that the
highest affinity DNMT1 binding site is the cofactor one. We then
attempted to dock 5 in the DNMT1/AdoHcy (mimicking
AdoMet substrate analogue) complex to probe whether the
ligand is still able to bind this complex. Indeed, these studies
suggest that in the presence of the cofactor the ligand would still
positively interact with the enzyme complex, adopting a confor-
mation (docking score −6.76, Figure S5b) in which the ligand is
sandwiched between the substrate and part of the enzyme CXXC
domain region (residues 646−650). This might rationalize why
increasing AdoMet concentrations do not abrogate the ligand
inhibition efficiency.
Effects of Quinoline-Based DNMTi in a Panel of Cancer
Cell Lines. To study the effects of these DNMTi in a cellular
context, we tested 1, 2, 4, 5, 10, and 11 in a panel of cancer cells
(histiocytic lymphoma, U-937; breast cancer, MDA-MB-231;
Burkitt’s lymphoma, RAJI; and prostate cancer, PC-3). Trypan
blue exclusion assays were carried out to determine their effects
on cell proliferation and viability. The effect of the tested com-
pounds in peripheral blood mononuclear cells (PBMCs) was
also determined to assess for differential toxicity. In full agree-
ment with their DNMT-inhibition potency, compounds 1, 2, and
5 displayed the highest antiproliferative effects and the strongest
cell-death induction in all of the cancer cells tested (Figure 5a−d).
Compounds 4, 10, and 11 showed both lower activity and cyto-
toxicity in these assays (Figure S6 in the Supporting Information).
Compounds 1 and 5 showed comparable potency against tumor
cells, but 5 was the less toxic in PBMCs (Figure 5e,f). Nuclear
morphological changes were further observed by fluorescence
microscopy after Hoechst and propidium iodide (PI) staining in
both U-937 and RAJI cells treated with increasing doses of 5 to
assess the levels of necrosis and apoptosis induced by the
compound (Figure 5g). In U-937 cells, 5 displayed massive
apoptosis at 10 μM followed by necrosis at 25 μM, whereas in
RAJI cells, 5 mainly led to necrosis at 25 μM without triggering
any apoptotic response at lower concentrations.
Figure 6. Effects of 2 and 5 in MbSCs. (a) PCNA mRNA levels and (b)
MTT assay of MbSCs after 48 h of 2 and 5 treatment or DMSO as
control (Ctr). *P < 0.05 versus untreated cells (ctr). (c) mRNA levels of
βIII-tubulin (βIIItub) in 2- and 5-treated MbSCs for 48 h. DMSO was
used as control.*P < 0.05 versus untreated cells (ctr). (d) Representative
bright-field images of MbSCs after 2 or 5 treatment (48 h, 10 μM) or
DMSO as control.
assays, evaluated by both βIII-tubulin RT-PCR and phase-contrast
images (Figure 6c,d), 2 showed the highest differentiation effect
after treatment with lower doses (10 μM), whereas 5 required
higher concentrations (50 μM) to reach significance. To the best of
our knowledge, 2 and 5 are the first examples of non-nucleoside
DNMTi tested in cancer stem cells (CSCs).
CONCLUSIONS
■
Through chemical manipulation applied on the structure of 1, we
identified compound 5, a novel non-nucleoside DNMTi more
potent than 1 and more selective toward other AdoMet-
dependent protein methyltransferases (PRMT1 and GLP).
Tested on a panel of cancer cells (leukemia, U937; breast
cancer, MDA-MB-231; Burkitt’s lymphoma, RAJI; and prostate
cancer, PC-3) as well as on PBMCs, compound 5 displayed
Effects of 2 and 5 in Medulloblastoma Stem Cells
(MbSCs). DNA methylation also plays a key role in cancer stem
cells: A comparison of the epigenomes of normal and cancer
stem cells and pluripotent and differentiated states revealed a
clear link between DNMTs and transcribed loci.35 In cancer stem
cells, the effect of DNMT inhibitors has been poorly investigated.
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The reaction was then kept at 80 °C for 1 h. Afterward, the reaction was
quenched at room temperature by a 2 N sodium carbonate solution
(20 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL),
washed with brine (3 × 30 mL), dried with anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The crude solid was
recrystallized from cyclohexane to give the pure product 15b. 1H NMR
(DMSO-d6, 400 MHz) δ 2.06 (s, 3H, −CH3), 4.92 (bs, 2H, −NH2-
aniline), 5.87 (s, 1H, pyrimidine proton), 5.97 (bs, 2H, -NH2-
pyrimidine), 6.19 (d, 1H, J = 8.0 Hz, aniline proton), 6.80 (d, 1H, J =
8.0 Hz, aniline proton), 6.87−6.91 (m, 2H, aniline protons), 8.64 (bs,
1H, −NH). 13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 102.7, 105.0,
107.8, 130.3, 143.2, 147.8, 163.1, 164.5, 170.2. MS (EI) m/z [M]+ calcd
for C11H13N5, 215.1171; found, 215.1167.
comparable activity as 1 and with less toxicity. In MbSCs at
10 μM, 5 significantly blocked proliferation but required higher
doses (50 μM) to induce differentiation, whereas related com-
pound 2, which was less potent as an antiproliferative agent,
showed high differentiating activity. The anticancer activity
displayed by 2 and 5 in the tested cancer cells, including in cancer
stem cells, suggests their use as potent and selective non-
nucleoside DNMTi for cancer therapy.
EXPERIMENTAL SECTION
■
Chemistry. Melting points were determined on a Buchi 530 melting-
point apparatus and are uncorrected. H NMR and 13C NMR spectra
1
General Procedure for the Synthesis of Compounds 1−12
and 22. Example: Synthesis of N-(3-(2-Amino-6-methylpyrimidin-4-
ylamino)phenyl)-3-(quinolin-4-ylamino)benzamide (5). Triethyl-
amine (3.04 mmol, 0.42 mL) and benzotriazol-1-yl-oxytripyrrolidino-
phosphonium hexafluorophosphate (PyBOP) (0.91 mmol, 0.47 g)
were added to a solution of 3-(quinolin-4-ylamino)benzoic acid 14b
(0.76 mmol, 0.2 g) in anhydrous N,N-dimethylformamide (5 mL) under
a nitrogen atmosphere. The resulting mixture was stirred for 30 min at
room temperature followed by the addition of N4-(3-aminophenyl)-6-
methylpyrimidine-2,4-diamine 15b (0.76 mmol, 0.16 g) under a nitrogen
atmosphere, and the reaction was stirred overnight. The reaction was
quenched with water (50 mL) and extracted with ethyl acetate (3 ×
30 mL). The combined organic extracts were dried, and the residue
obtained upon evaporation of solvent was purified by column
chromatography (SiO2 eluting with ethyl acetate/methanol 10:1) to
provide pure 5. 1H NMR (DMSO-d6, 400 MHz) δ 2.10 (s, 3H, −CH3),
5.94 (s, 1H, pyrimidine proton), 6.07 (bs, 2H, −NH2-pyrimidine), 7.06
(d, 1H, J = 4.8 Hz, quinoline proton), 7.26 (t, 1H, J = 8.0 Hz, benzene
proton), 7.35 (d, 1H, J = 7.6 Hz, quinoline proton), 7.55−7.61 (m, 4H,
benzene and quinoline protons), 7.72−7.75 (m, 2H, benzene protons),
7.91 (d, 1H, J = 8.0 Hz, quinoline proton), 7.95 (s, 1H, benzene proton),
8.03 (s, 1H, benzene proton), 8.42 (d, 1H, J = 8.4 Hz, quinoline proton),
8.52 (d, 1H, J = 5.6 Hz, quinoline proton), 9.04 (bs, 1H, −NH-
pyrimidine), 9.16 (bs, 1H, −NH-quinoline), 10.18 (s, 1H, −CONH−).
13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 108.0, 111.6, 111.8, 112.8,
113.4, 117.5, 121.2, 121.6, 124.2, 125.7, 129.2, 129.6, 129.7, 133.9, 135.0,
136.7, 138.7, 142.5, 142.6, 149.7, 151.6, 163.1, 164.5, 164.7, 170.2. MS
(EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found, 461.1969.
N-(4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-
4-ylamino)benzamide (1, SGI-1027). 1H NMR (DMSO-d6, 400 MHz)
δ 2.10 (s, 3H, −CH3), 5.88 (s, 1H, pyrimidine proton), 6.14 (bs, 2H,
−NH2-pyrimidine), 7.21 (d, 1H, J = 5.4 Hz, quinoline proton), 7.48−
7.81 (m, 8H, benzene and quinoline protons), 7.93−8.03 (m, 3H,
benzene and quinoline protons), 8.42 (d, 1H, J = 8.4 Hz, quinoline
proton), 8.52 (d, 1H, J = 5.6 Hz, quinoline proton), 8.97 (bs, 1H, −NH-
pyrimidine), 9.25 (bs, 1H, −NH-quinoline), 10.06 (bs, 1H,
−CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 111.4
(2C), 112.8, 117.7 (2C), 121.6, 122.4 (2C), 124.2 (2C), 125.7, 127.9, 129.2,
129.6, 130.2 (2C), 136.5, 138.7, 149.3, 149.7, 151.6, 163.1, 164.5, 164.7,
170.2. MS (EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found, 461.1969.
N-(3-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-
4-ylamino)benzamide (2). 1H NMR (DMSO-d6, 400 MHz) δ 2.10 (s,
3H, −CH3), 5.95 (s, 1H, pyrimidine proton), 6.07 (bs, 2H, −NH2-
pyrimidine), 7.21−7.25 (m, 2H, benzene and quinoline protons), 7.37
(d, 1H, J = 8.2 Hz, benzene proton), 7.50−7.61 (m, 4H, benzene and
quinoline protons), 7.75 (t, 1H, J = 7.4 Hz, quinoline proton), 7.93−
8.03 (m, 4H, benzene and quinoline protons), 8.42 (d, 1H, J = 8.4 Hz,
quinoline proton), 8.52 (d, 1H, J = 5.6 Hz, quinoline proton), 9.03 (bs,
1H, −NH-pyrimidine), 9.25 (bs, 1H, −NH-quinoline), 10.05 (bs, 1H,
−CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 108.0, 111.4
(2C), 111.6, 112.8, 113.4, 121.6, 124.2 (2C), 125.7, 129.2, 129.6, 129.7,
130.2 (2C), 136.7, 138.7, 142.6, 149.3, 149.7, 151.6, 163.1, 164.5, 164.7,
170.2. MS (EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found,
461.1969.
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 (M+) and base peaks
1
are given. All compounds were routinely checked by TLC, H NMR,
and 13C NMR spectra. TLC was performed on aluminum-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 reduced pressure of ca. 20 Torr. Organic solutions were
dried over anhydrous sodium sulfate. Elemental analysis was used to
determine the purity of the described compounds, which was found to
be >95%. Analytical results are within 0.40% of the theoretical values.
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 Ester Intermediates
16a−c and 20 and Nitro Intermediates 17a−c and 18. Example:
Synthesis of Ethyl 3-(Quinolin-4-ylamino)benzoate (16b). 4-Chlor-
oquinoline (6.05 mmol, 0.99 g), ethyl 3-aminobenzoate (6.05 mmol, 1
g), and a catalytic amount (4 drops) of 37% hydrochloric acid were
refluxed for 2 h. The reaction was allowed to cool to room temperature,
and the precipitated solid was filtered off, washed with water (3 × 5 mL),
and recrystallized by methanol to afford pure 16b as a hydrochloride
salt. 1H NMR (DMSO-d6, 400 MHz) δ 1.34 (t, 3H, J = 7.2 Hz,
−COOCH2CH3), 4.36 (q, 2H, J = 7.2 Hz, −COOCH2CH3), 6.88 (d,
1H, J = 5.2 Hz, quinoline proton), 7.74 (m, 2H, benzene protons), 7.84
(t, 1H, J = 7.4 Hz, benzene proton), 8.00 (d, 1H, J = 8.2 Hz, quinoline
proton), 8.06−8.12 (m, 3H, quinoline and benzene protons), 8.56
(d, 1H, J = 8.2 Hz, quinoline proton), 8.81 (d, 1H, J = 5.2 Hz, quinoline
proton), 11.08 (bs, 1H, −NH), 14.68 (bs, 1H, H+). 13C NMR (DMSO-
d6, 100 MHz) δ 14.1, 60.9, 112.8, 114.2, 119.9, 121.6, 122.1, 124.2,
125.7, 129.2, 129.6, 130.9, 133.7, 138.7, 142.3, 149.7, 151.6, 165.9. MS
(EI) m/z [M]+ calcd for C18H16N2O2, 292.1212; found, 292.1218.
General Procedure for the Synthesis of Acid Intermediates
14a−c and 21. Example: Synthesis of 3-(Quinolin-4-ylamino)-
benzoic Acid (14b). A solution of ethyl 3-(quinolin-4-ylamino)benzoate
16b (1.71 mmol, 0.5 g) and 2 N KOH (6.84 mmol, 0.38 g) in ethanol
(15 mL) was stirred overnight at room temperature. Then, the solvent
was evaporated, and 2 N HCl was slowly added until pH 5.0 was
obtained. The colorless solid was filtered, washed with water (3 × 5 mL),
and recrystallized from methanol to obtain pure 14b. 1H NMR (DMSO-
d6, 400 MHz) δ 7.00 (d, 1H, J = 5.2 Hz, quinoline proton), 7.51−7.58
(m, 2H, quinoline and benzene protons), 7.63 (d, 1H, benzene proton),
7.68−7.75 (m, 2H, quinoline and benzene protons), 7.90−7.93 (m, 2H,
quinoline and benzene protons), 8.39 (d, 1H, J = 8.0 Hz, quinoline
proton), 8.51 (d, 1H, J = 5.2 Hz, quinoline proton), 9.17 (bs, 1H, NH),
12.0 (bs, 1H, COOH). 13C NMR (DMSO-d6, 100 MHz) δ 112.8, 119.5
(2C), 120.2, 121.6, 124.2, 125.7, 129.2, 129.6, 131.1 (2C), 138.7, 149.7,
151.1, 151.6, 169.3. MS (EI) m/z [M]+ calcd for C16H12N2O2, 264.0899;
found, 264.0902.
General Procedure for the Synthesis of Anilines 15a−c, 19,
and 13. Example: Synthesis of N4-(3-Aminophenyl)-6-methylpyr-
imidine-2,4-diamine (15b). To a cooled solution of 6-methyl-N4-(3-
nitrophenyl)pyrimidine-2,4-diamine 17b (1.63 mmol, 0.5 g) and
stannous chloride dihydrate (8.15 mmol, 1.84 g) in ethanol (10 mL)
was slowly added a 37% hydrochloric acid solution (0.3 mL) at 0 °C.
N-(2-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-4-(quinolin-
4-ylamino)benzamide (3). 1H NMR (DMSO-d6, 400 MHz) δ 2.09 (s, 3H,
−CH3), 5.88 (s, 1H, pyrimidine proton), 6.31 (bs, 2H, −NH2-pyrimidine),
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7.18−7.22 (m, 2H, benzene and quinoline protons), 7.37−7.50 (m,
4H), 7.58−7.65 (m, 2H benzene and quinoline protons), 7.72−7.77 (m,
2H, benzene protons), 7.94−7.96 (m, 2H, quinoline protons), 8.37 (d,
1H, J = 7.6 Hz, quinoline proton), 8.55 (bs, 1H, −NH-pyrimidine), 8.58
(d, 1H, J = 4.8 Hz, quinoline proton), 9.25 (bs, 1H, −NH-quinoline),
10.15 (bs, 1H, −CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9,
93.8, 111.4 (2C), 112.8, 118.9, 121.6, 122.8, 124.2 (2C), 125.5, 125.7,
126.1, 129.2, 129.6, 130.2 (2C), 137.9, 138.7, 147.6, 149.3, 149.7, 151.6,
163.1, 164.5, 170.2, 172.5. MS (EI) m/z [M]+ calcd for C27H23N7O,
461.1964; found, 461.1969.
13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 112.8, 116.4, 117.9, 118.8,
118.9, 121.6, 122.8, 124.2, 125.5, 125.7, 126.1, 128.3, 129.2, 129.6, 132.9,
137.9, 138.7, 147.6, 149.7, 151.6, 151.9, 163.1, 164.5, 167.5, 170.2. MS
(EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found, 461.1969.
4-(Quinolin-4-ylamino)-N-(4-(quinolin-4-ylamino)phenyl)-
1
benzamide (10). H NMR (DMSO-d6, 400 MHz) δ 6.83 (d, 1H, J =
6.0 Hz, quinoline proton), 7.20 (d, 1H, J = 4.8 Hz, quinoline proton),
7.42 (d, 2H, J = 8.8 Hz, benzene protons), 7.53 (d, 2H, J = 8.4 Hz,
benzene protons), 7.60−7.68 (m, 2H, quinoline protons), 7.79 (t, 1H,
J = 7.2 Hz, quinoline proton), 7.83 (t, 1H, J = 7.2 Hz, quinoline proton),
7.94−7.97 (m, 4H, benzene and quinoline protons), 8.07 (d, 2H, J = 7.6
Hz, benzene protons), 8.45−8.49 (m, 2H, quinoline protons), 8.57−
8.61 (m, 2H, quinoline protons), 9.49 (bs, 1H, −NH-quinoline), 9.82
(bs, 1H, −NH-quinoline), 10.35 (bs, 1H, −CONH−). 13C NMR
(DMSO-d6, 100 MHz) δ 111.4 (2C), 112.8 (2C), 117.7 (2C), 121.6
(2C), 122.4 (2C), 124.2 (3C), 125.7 (2C), 127.9, 129.2 (2C), 129.6
(2C), 130.2 (2C), 138.7 (2C), 141.5, 149.3, 149.7 (2C), 151.6 (2C),
164.7. MS (EI) m/z [M]+ calcd for C31H23N5O, 481.1903; found,
481.1908.
N-(4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-3-(quinolin-
1
4-ylamino)benzamide (4). H NMR (DMSO-d6, 400 MHz) δ 2.08
(s, 3H, −CH3), 5.87 (s, 1H, pyrimidine proton), 6.10 (bs, 2H, −NH2-
pyrimidine), 7.06 (d, 1H, J = 5.6 Hz, quinoline proton), 7.54−7.74 (m,
9H, benzene and quinoline protons), 7.90 (d, 1H, J = 8.4 Hz quinoline
proton), 8.01 (s, 1H, benzene proton), 8.46 (d, 1H, J = 8.4 Hz, quinoline
proton), 8.51 (d, 1H, J = 4.0 Hz, quinoline proton), 9.01 (bs, 1H, −NH-
pyrimidine), 9.28 (bs, 1H, −NH-quinoline), 10.30 (s, 1H, −CONH−).
13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 111.8, 112.8, 117.5, 117.7
4-(2-Amino-6-methylpyrimidin-4-ylamino)-N-(4-(2-amino-6-
methylpyrimidin-4-ylamino)phenyl)benzamide (11). 1H NMR
(DMSO-d6, 400 MHz) δ 2.09 (s, 3H, −CH3), 2.13 (s, 3H, −CH3),
5.86 (s, 1H, pyrimidine proton), 5.95 (s, 1H, pyrimidine proton), 6.10
(bs, 2H, −NH2-pyrimidine), 6.26 (bs, 2H, −NH2-pyrimidine), 7.65
(m, 4H, benzene protons), 7.89 (m, 4H, benzene protons), 8.91 (bs, 1H,
−NH-pyrimidine), 9.32 (bs, 1H, −NH-pyrimidine), 9.96 (bs, 1H,
−CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9 (2C), 93.8 (2C),
111.4 (2C), 117.7 (2C), 122.4 (2C), 124.2, 127.9, 130.2 (2C), 136.5,
144.3, 163.1 (2C), 164.5, 164.7, 167.0, 167.7, 170.2. MS (EI) m/z [M]+
calcd for C23H23N9O, 441.2026; found, 441.2023.
(2C), 121.2, 121.6, 122.4 (2C), 124.2, 125.7, 127.9, 129.2, 129.6, 133.9,
135.0, 136.5, 138.7, 142.5, 149.7, 151.6, 163.1, 164.5, 164.7, 170.2. MS
(EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found, 461.1969.
N-(2-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-3-(quinolin-
4-ylamino)benzamide (6). 1H NMR (DMSO-d6, 400 MHz) δ 2.05 (s,
3H, −CH3), 5.86 (s, 1H, pyrimidine proton), 6.23 (bs, 2H, −NH2-
pyrimidine), 7.04 (d, 1H, J = 5.6 Hz, quinoline proton), 7.20 (m, 2H,
benzene and quinoline protons), 7.51−7.74 (m, 7H, benzene and
quinoline protons), 7.90−7.92 (m, 2H, benzene and quinoline protons),
8.40 (d, 1H, J = 8.4 Hz, quinoline proton), 8.47 (bs, 1H, −NH-
pyrimidine), 8.51 (d, 1H, J = 5.4 quinoline proton), 9.11 (bs, 1H, −NH-
quinoline), 10.19 (bs, 1H, −CONH−). 13C NMR (DMSO-d6, 100
MHz) δ 23.9, 93.8, 111.8, 112.8, 117.5, 118.9, 121.2, 121.6, 122.8, 124.2,
125.5, 125.7, 126.1, 129.2, 129.6, 133.9, 135.0, 137.9, 138.7, 142.5, 147.6,
149.7, 151.6, 163.1, 164.5, 164.7, 170.2. MS (EI) m/z [M]+ calcd for
C27H23N7O, 461.1964; found, 461.1969.
N-(4-Aminophenyl)-4-(quinolin-4-ylamino)benzamide (12). 1H
NMR (DMSO-d6, 400 MHz) δ 4.91 (bs, 2H, −NH2-benzene), 6.56
(d, 2H, J = 7.6 Hz, benzene protons), 7.18 (d, 1H, J = 4.8 Hz, quinoline
proton), 7.38 (d, 2H, J = 8.4 Hz, benzene protons), 7.46 (d, 2H, J = 8.4
Hz, benzene protons), 7.60 (t, 1H, J = 7.2 Hz, quinoline proton), 7.74
(t, 1H, J = 7.2 Hz, quinoline proton), 7.93 (d, 1H, J = 8.0 Hz, quinoline
proton), 7.97 (d, 2H, J = 8.4 Hz, benzene protons), 8.38 (d, 1H, J = 8.4
Hz, quinoline proton), 8.57 (d, 1H, J = 4.4 Hz, quinoline proton),
9.20 (bs, 1H, −NH-quinoline), 9.79 (bs, 1H, −CONH−). 13C NMR
(DMSO-d6, 100 MHz) δ 111.4 (2C), 112.8, 116.5 (2C), 121.6, 122.4
(2C), 124.2 (2C), 125.7, 127.9, 129.2, 129.6, 130.2 (2C), 138.7, 144.0,
149.3, 149.7, 151.6, 164.7. MS (EI) m/z [M]+ calcd for C22H18N4O,
354.1481; found, 354.1486.
N-(4-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-2-(quinolin-
1
4-ylamino)benzamide (7). H NMR (DMSO-d6, 400 MHz) δ 2.07
(s, 3H, −CH3), 5.85 (s, 1H, pyrimidine proton), 6.08 (bs, 2H, −NH2-
pyrimidine), 7.15 (d, 1H, J = 5.4 Hz, quinoline proton), 7.26 (t, 1H, J =
6.8 Hz, quinoline proton), 7.48−7.75 (m, 8H, benzene and quinoline
protons), 7.92 (m, 2H, quinoline protons), 8.16 (d, 1H, J = 8.0 Hz,
quinoline proton), 8.55 (d, 1H, J = 4.0 Hz, quinoline proton), 8.95
(bs,1H, −NH-pyrimidine), 10.26 (bs, 1H, −NH- quinoline), 10.41 (bs,
1H, −CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 112.8,
116.4, 117.7 (2C), 117.9, 118.8, 121.6, 122.4 (2C), 124.2, 125.7, 127.9,
128.3, 129.2, 129.6, 132.9, 136.5, 138.7, 149.7, 151.6, 151.9, 163.1, 164.5,
167.5, 170.2. MS (EI) m/z [M]+ calcd for C27H23N7O, 461.1964; found,
461.1969.
N-(3-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-2-(quinolin-
4-ylamino)benzamide (8). 1H NMR (DMSO-d6, 400 MHz) δ 2.08 (s,
3H, −CH3), 5.91 (s, 1H, pyrimidine proton), 6.05 (bs, 2H, −NH2-
pyrimidine), 7.12−7.28 (m, 3H, benzene and quinoline protons), 7.61−
7.73 (m, 3H, benzene and quinoline protons), 7.71−7.76 (m, 3H,
benzene protons), 7.91−7.96 (m, 2H, benzene and quinoline protons),
8.16 (d, 1H, J = 8.0 Hz, quinoline proton), 8.55 (d, 1H, J = 4.8 Hz,
quinoline proton), 9.02 (bs, 1H, −NH-pyrimidine), 10.10 (bs, 1H,
−NH-quinoline), 10.40 (bs, 1H, −CONH−). 13C NMR (DMSO-d6,
100 MHz) δ 23.9, 93.8, 108.0, 111.6, 112.8, 113.4, 116.4, 117.9, 118.8,
121.6, 124.2, 125.7, 128.3, 129.2, 129.6, 129.7, 132.9, 136.7, 138.7, 142.6,
149.7, 151.6, 151.9, 163.1, 164.5, 167.5, 170.2. MS (EI) m/z [M]+ calcd
for C27H23N7O, 461.1964; found, 461.1969.
4-Amino-N-(4-(2-amino-6-methylpyrimidin-4-ylamino)phenyl)-
1
benzamide (13). H NMR (DMSO-d6, 400 MHz) δ 2.06 (s, 3H,
−CH3), 5.71 (bs, 2H, −NH2-benzene), 5.85 (s, 1H, pyrimidine proton),
6.08 (bs, 2H, −NH2-pyrimidine), 6.60 (d, 2H, J = 8.4 Hz, benzene
protons), 7.59−7.64 (m, 4H, benzene protons), 7.71 (d, 2H, J = 8.4 Hz,
benzene protons), 8.88 (bs, 1H, −NH-pyrimidine), 9.66 (bs, 1H,
−CONH−). 13C NMR (DMSO-d6, 100 MHz) δ 23.9, 93.8, 114.3 (2C),
117.7 (2C), 122.4 (2C), 124.2, 127.9, 130.2 (2C), 136.5, 151.8, 163.1,
164.5, 164.7, 170.2. MS (EI) m/z [M]+ calcd for C18H18N6O, 334.1542;
found, 334.1545.
Nanoscale DNMT1 Prescreen and HotSpot DNMT Assay.
Compounds 1−13 were tested in 10-dose IC50 mode with 2-fold serial
dilutions at a starting concentration of 500 μM against human DNMT1
using poly(dI−dC) (0.001 mg/mL) as a substrate in the presence of
AdoMet (1 μM) as a cofactor. Control compounds, AdoHcy (S-(5′-
adenosyl)-L-homocysteine) and sinefungin, were tested in 10-dose IC50
mode with 3-fold serial dilutions starting at 100 μM.
Protein Purification. The expression and purification of human
DNMT1 with an N-terminal deletion of 600 residues (residues 601−
1600) and the human DNMT3A2/DNMT3L complex have been
described.7 Recombinant rat PRMT127 and the human G9a-like protein
(GLP) C-terminal fragment containing both the ankyrin repeats and
catalytic SET domain (residues 734−1235; pXC758)29 were purified as
described.
DNMT1 Inhibition Assay. For DNMT1, methyl transfer activity
inhibition assays were performed in 20 μL reactions containing 4.6 mM
[methyl-3H]-AdoMet (10.0 Ci/mmol; PerkinElmer), 1.0 mM DNA
N-(2-(2-Amino-6-methylpyrimidin-4-ylamino)phenyl)-2-(quinolin-
1
4-ylamino)benzamide (9). H NMR (DMSO-d6, 400 MHz) δ 2.01
(s, 3H, −CH3), 5.81 (s, 1H, pyrimidine proton), 6.27 (s, 2H, −NH2-
pyrimidine), 7.12−7.24 (m, 4H, benzene and quinoline protons), 7.48
(d, 1H, J = 7.6 Hz, benzene proton), 7.54−7.61 (m, 3H, benzene
protons), 7.71−7.76 (m, 2H, benzene and quinoline protons), 7.92−
7.97 (m, 2H, benzene and quinoline protons), 8.08 (d, 1H, J = 8.8 Hz,
quinoline protons), 8.57 (m, 2H, quinoline and −NH-pyrimidine
protons), 10.44 (bs, 1H, −NH-quinoline), 10.57 (bs, 1H, −CONH−).
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oligonucleotides, 0.2 μM DNMT1, 1 mM EDTA, and 50 mM Tris-HCl,
pH 7.5. The DNA substrates were 36 bp hemimethylated (GAC)12.
Enzymes were preincubated with AdoMet and various concentrations of
inhibitors for 5 min at 37 °C before the addition of substrate DNA. After
a 15 min incubation, the reactions were terminated by the addition of 1%
SDS and 1 mg/mL of protease K and heating at 50 °C for 15 min. The
reaction mixtures were spotted on DE81 paper circles (Whatman),
washed with 5 mL of cold 0.2 M NH4HCO3 (twice), 5 mL of deionized
water (twice), and 5 mL of ethanol (once). The dried circles were
subjected to liquid scintillation counting with Cytoscint scintillant. All
curves were fit individually using Origin 7.5 software (OriginLab).
DNMT3A Inhibition Assay. For the DNMT3a2/3L complex, the
inhibition assays were performed 20 μL reactions containing 4.6 mM
[methyl-3H]-AdoMet, 1.0 mM DNA oligonucleotides, 0.3 μM enzyme,
0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 2.5% (v/v) glycerol,
and 50 mM Tris-HCl, pH 7.5. The DNA substrates were 28 bp and
contained two CpG sites: 5′-ACA GTA CGT CAA GAT CTT GAC
GTA CTG T-3′ and the complementary strand.
PRMT1 and GLP Inhibition Assays. For histone methylation
inhibitions, the assays were performed in 20 μL reactions containing
4.6 mM [methyl-3H]-AdoMet, 50 μg/mL histone from calf thymus
(Sigma), 12 μg/mL (0.3 μM) PRMT1 or 10 μg/mL (0.17 μM) GLP,
100 mM KCl, 5 mM dithiothreitol (DTT), and 50 mM Tris-HCl, pH
8.5. Enzymes were preincubated with AdoMet and various concen-
trations of inhibitors for 5 min at 37 °C (for PRMT1) or 30 °C
(for GLP) before the addition of histone substrates. After incubation
(6.5 min for PRMT1 or 5 min for GLP), the reactions were terminated
by the addition of 20% trichloroacetic acid (TCA, Fisher Scientific). The
reaction mixtures were spotted on GF/A paper circles (Whatman) and
washed three times with 3 mL of 10% TCA and once with 3 mL of
ethanol. The dried circles were subjected to liquid scintillation counting
with Cytoscint scintillant. All curves were fit individually using Origin
7.5 software (OriginLab).
of 0.01 kJ mol−1 Å−1. All complex pictures were rendered employing the
UCSF Chimera software.41
U-937, RAJI, PC-3, MDA-MB-231, and PBM Cellular Assays.
U-937 (histiocytic lymphoma), RAJI (Burkitt’s lymphoma), PC-3
(prostate cancer), MDA-MB-231 (breast cancer) cell lines were
purchased from Deutsche Sammlung fur Mikroorganismen and
̈
Zellkulturen (DSZM). Cells were cultured in RPMI 1640 (Lonza)
supplemented with 10% fetal calf serum (Lonza) and 1% antibiotic−
antimycotic (Lonza). Peripheral blood mononuclear cells (PBMCs)
were isolated and cultured as previously described.42 Cells in expo-
nential growth phase were treated with compounds at the indicated
concentrations. Proliferation and viability were assessed by trypan blue
exclusion analysis at the indicated time points. Morphological
determination of apoptosis and necrosis was performed as described
previously.43
Medulloblastoma Cancer Stem Cell (MbSC) Assays and Stem
Cell Cultures and Treatments. MbSCs were isolated and cultivated
as described.44 In detail, MbSCs were isolated from fresh tumor
specimens from Ptch1+/−. Cells were obtained after mechanical and
enzymatic dissociation and cultured in serum-free DMEM-F12
supplemented with glucose (0.6%), insulin (25 mg/mL), N-acetyl-L-
cystein (60 mg/mL), heparin (2 mg/mL), B27 (1×), EGF (20 ng/mL),
and bFGF (20 ng/mL). MbSCs were also treated to differentiate in vitro
after withdrawal of EGF/bFGF and addition of differentiating factors
(platelet derived growth factor, PDGF) for 48 h. Compounds 2 and 5
were resuspended in DMSO at 1 mM. Cells were treated with increasing
concentration of 2 or 5 (1, 10, and 50 μM) for 48 h, and DMSO was used
as control. Unless otherwise indicated, media and supplements were
purchased from Gibco-Invitrogen (Life Science), and chemicals were
from Sigma-Aldrich (St. Louis, MO).
RNA Isolation and Real-Time qPCR. Total RNA was isolated with
Tri-Reagent (Ambion) according to the manufacturer’s procedure, and
sample quantification was done using a Nanodrop spectophotometer
(Thermo Scientific). The reverse transcription was performed using
a high-capacity cDNA reverse-transcription kit (Applied Biosystem),
and quantitative RT-PCR analysis of βIII-tubulin, PCNA, DNMT1,
DNMT3A, and DNMT3B was performed using a TaqMan assay from
Lifetech. mRNA expression was analyzed using the ABI Prism 7900HT
sequence detection system (Applied Biosystem) with a TaqMan gene-
expression assay according to the manufacturer’s protocol (Applied
Biosystem). Each amplification reaction was performed in triplicate, and
the average of the three threshold-cycle values was used to calculate the
relative amount of transcripts in the sample (SDS 2.3 software, Applied
Biosystem). mRNA quantification was expressed, in arbitrary units, as
the ratio of the sample quantity to the calibrator or to the mean values of
control samples. All values were normalized to three endogenous
controls: β-actin, β2-microglobulin, and HPRT.
Competition Studies. Competition studies were performed on
human DNMT1 according to Gros et al.39 Briefly, the reaction was
started by the addition of 94.5 nM DNMT1 to a mix containing the
tested compound (up to 1% DMSO), an AdoMet/[methyl-3H]-
AdoMet mix in a ratio of 3:1 (isotopic dilution 1*:3), and biotinylated
DNA duplex in a 10 μL final volume. In AdoMet competition studies,
AdoMet was varied between 0.5 and 15 μM while DNA was kept at a
near-saturating concentration. In DNA competition studies, DNA was
varied between 0.1 and 0.6 μM while AdoMet was kept at a near-
saturating concentration. The tested compounds were spanned from
IC10 to IC80. For each substrate concentration, the IC50 of the tested
compound was calculated by nonlinear regression fitting with sigmoidal
dose response (variable slope). The linear regressions of IC50 against
DNA
AdoMet
[DNA]/Km
or [AdoMet]/Km
were displayed only if their
Western Blot Assay. Cells were lysed using RIPA buffer (Tris-HCl,
pH 7.6, 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM,
NP40 1%, EDTA 5 mM, NaF 100 mM, sodium pyrophosphate 2 mM)
and protease inhibitors. Lysates were separated on an 8% acrylamide gel
and immunoblotted using standard procedures. The following antibod-
ies were used: anti-DNMT1 (sc-20701; Santa Cruz Biotechnology),
anti-DNMT3a (sc-365769; Santa Cruz Biotechnology), and anti-
DNMT3b (sc-10236; Santa Cruz Biotechnology).
slopes were significantly different from 0. Velocity plots were fitted
by Michaelis−Menten regression or nonlinear regression fitting with
sigmoidal dose response (variable slope). All regressions were
performed with GraphPad Prism 4.03.
Molecular Modeling. Prior to docking calculations, Epik software40
was used to calculate the most relevant ionization and tautomeric state
of compounds 5 and 1. Then, the Glide program of the Schrodinger
̈
package33 was used to dock these compounds into the two selected
DNMT1 X-ray structures (PDB 3SWR, Hashimoto and Cheng,
unpublished data, and PDB 3PTA32). The receptor grid generation
was performed for the box with a center in the putative binding sites of
the two structures. The size of the box was determined automatically.
The extra precision mode (XP) of Glide was used for docking. The
ligand scaling factor was set to 1.0. The geometry of the ligand binding
site of the complex between 5 (or 1) and the DNMT1 structures was
then optimized. The binding site was defined as 5 (or 1) and all amino
acid residues located within 8 Å from the ligands. All of the receptor
residues located within 2 Å from the binding site were used as a shell.
The following parameters of energy minimization were used. The
OPLS2005 force field was used. Water was used as an implicit solvent,
and a maximum of 5000 iterations of the Polak−Ribier conjugate
gradient minimization method was used with a convergence threshold
MTT Assay. MbSC were treated with 1, 10, and 50 μM of compound
2 or 5 for 48 h. The growth of drug-treated cells relative to untreated
cells was measured by MTT assay. Each sample was measured in
triplicates and repeated at least three times.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical and physical data for compounds 1−22; elemental
analyses for compounds 1−13; molecular modeling studies on 1;
cellular studies (RAJI, MDA-MB-231, PC-3, and PMB cells) for
compounds 4, 10, and 11; and expression levels of DNMTs in
medulloblastoma stem cells. This material is available free of
charge via the Internet at http://pubs.acs.org.
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dx.doi.org/10.1021/jm4012627 | J. Med. Chem. 2014, 57, 701−713

Journal of Medicinal Chemistry
Article
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Curr. Opin. Genet. Dev. 1999, 9, 158−163.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +39 06 49913392; Fax: +39 06 491491; E-mail: antonello.
mai@uniroma1.it.
(7) Hashimoto, H.; Liu, Y.; Upadhyay, A. K.; Chang, Y.; Howerton, S.
B.; Vertino, P. M.; Zhang, X.; Cheng, X. Recognition and potential
mechanisms for replication and erasure of cytosine hydroxymethylation.
Nucleic Acids Res. 2012, 40, 4841−4849.
Author Contributions
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function of mammalian DNA methyltransferases. ChemBioChem 2011,
12, 206−222.
□These authors contributed equally to this work.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by FIRB RBFR10ZJQT, FP7
BLUEPRINT/282510, FP7 COST/TD0905, FP7Marie Curie
REDCAT/215009, the Sapienza-IIT Project, Tel
bourg, the Recherche Cancer et Sang Foundation, the Recherches
Scientifiques Luxembourg and Een Haerz fir Kriibskrank Kanner
associations, and the U.S. National Institutes of Health
(GM049245-20 and DK094346-01). X.C. is a Georgia Research
Alliance Eminent Scholar. M.S. is supported by the Action Lions
“Vaincre le Cancer”. C.F. is a recipient of a postdoctoral grant from
́ ́
evie Luxem-
̈
̀ ́
the Ministere de la Culture, de l’Enseignement superieur et de la
Recherche du Luxembourg. M.D. is supported by a NRF from
MEST (Korea for Tumor Microenvironment grant GCRC 2012-
0001184), by a Seoul National University research grant, and by
theResearch Settlement Fund for the new faculty of SNU. P.B.A. is
supported by French Reg
et FEDER).
́ ́ ́
ion Midi-Pyrenees (Equipe d’Excellence
ABBREVIATIONS USED
■
AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-homo-
cysteine; bFGF, basic fibroblast growth factor; CSCs, cancer
stem cells; DMSO, dimethyl sulfoxide; DNMTs, DNA
methyltransferases; DNMTi, DNA methyltransferase inhibitors;
DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
EGF, epidermal growth factor; GABA, gamma-aminobutyric
acid; GLP, G9a-like protein; HPRT, hypoxanthine guanine
phosphoribosyl transferase; IDH1, isocitrate dehydrogenase 1;
MbSCs, medulloblastoma stem cells; MTT, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMCs, peripheral
blood mononuclear cells; PCNA, proliferating cell nuclear
antigen; PDB, Protein Data Bank; PDGF, platelet-derived
growth factor; Pitx2c, pituitary homeobox 2c; PRMT1, protein
arginine methyltransferase 1; PyBOP, benzotriazol-1-yl-oxy-
tripyrrolidinophosphonium hexafluorophosphate; RT-PCR,
real-time polymerase chain reaction; SALL4, Sal-like protein 4;
SET, Su(var.)3-9-enhancer of zeste-trithorax; TCA, trichloro-
acetic acid; TCEP, tris(2-carboxyethyl)phosphine
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Hernandez, E.; Arias-Bofill, D.; Vidal, S.; Cervera, E.; Duenas-Gonzalez,
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