Optimization of cellular activity of G9a inhibitors 7-aminoalkoxy- quinazolines

Article abstract of DOI:10.1021/jm200903z

Protein lysine methyltransferase G9a plays key roles in the transcriptional repression of a variety of genes via dimethylation of lysine 9 on histone H3 (H3K9me2) of chromatin as well as dimethylation of nonhistone proteins including tumor suppressor p53. We previously reported the discovery of UNC0321 (3), the most potent G9a inhibitor to date, via structure-based design and structure-activity relationship (SAR) exploration of the quinazoline scaffold represented by BIX01294 (1). Despite its very high in vitro potency, compound 3 lacks sufficient cellular potency. The design and synthesis of several generations of new analogues aimed at improving cell membrane permeability while maintaining high in vitro potency resulted in the discovery of a number of novel G9a inhibitors such as UNC0646 (6) and UNC0631 (7) with excellent potency in a variety of cell lines and excellent separation of functional potency versus cell toxicity. The design, synthesis, and cellular SAR of these potent G9a inhibitors are described.

Full text of DOI:10.1021/jm200903z

ARTICLE
pubs.acs.org/jmc
Optimization of Cellular Activity of G9a Inhibitors
7-Aminoalkoxy-quinazolines
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Feng Liu,†, Dalia Barsyte-Lovejoy,‡, Abdellah Allali-Hassani,‡ Yunlong He,§ J. Martin Herold,†
Xin Chen,† Christopher M. Yates,^ Stephen V. Frye,† Peter J. Brown,‡ Jing Huang,§ Masoud Vedadi,‡
Cheryl H. Arrowsmith,‡ and Jian Jin*,†
†Center for Integrative Chemical Biology and Drug Discovery, Division of Medicinal Chemistry and Natural Products, UNC Eshelman
School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
‡Structural Genomics Consortium, University of Toronto, Toronto, Ontario, M5G 1L7, Ontario, Canada
§Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,
United States
S Supporting Information
b
ABSTRACT: Proteinlysinemethyltransferase G9a plays key roles inthetranscriptional
repression of a variety of genes via dimethylation of lysine 9 on histone H3 (H3K9me2)
of chromatin as well as dimethylation of nonhistone proteins including tumor
suppressor p53. We previously reported the discovery of UNC0321 (3), the most
potent G9a inhibitor to date, via structure-based design and structureÀactivity relation-
ship (SAR) exploration of the quinazoline scaffold represented by BIX01294 (1).
Despite its very high in vitro potency, compound 3 lacks sufficient cellular potency. The
design and synthesis of several generations of new analogues aimed at improving cell
membranepermeabilitywhilemaintaininghighin vitropotencyresulted inthe discovery
of a number of novel G9a inhibitors such as UNC0646 (6) and UNC0631 (7) with
excellent potency in a variety of cell lines and excellent separation of functional potency
versus cell toxicity. The design, synthesis, and cellular SAR of these potent G9a inhibitors are described.
’ INTRODUCTION
regulation, and DNA repair, replication and metabolism.7,8
PKMTs themselves are also often components of large regulatory
complexes and act in concert with other epigenetic factors.9
Selective inhibition of individual PKMT catalytic activity in cellular
systems represents a valuable strategy to decipher the complex
signaling mechanisms of histone and protein lysine methylation. A
“toolkit” of potent, selective, and cell-penetrant chemical probes10
of PKMTs will permit biological and therapeutic hypotheses to be
tested with high confidence in cell-based models of human biology
and disease. However, very few such small-molecule tools are
available to the biomedical research community.11À13
G9a (also known as KMT1C or EHMT2) was initially
identified as a H3K9 methyltransferase.14 It was later found that
G9a could methylate various histone15 and nonhistone proteins
including the tumor suppressor p53.16,17 G9a is overexpressed in
various human cancers,17À20 and knockdown of G9a inhibits
cancer cell growth.18,20À22 The dimethylation of p53 K373 by
G9a and GLP (also known as KMT1D or EHMT1), a closely
related methyltransferase that shares 80% sequence identity with
G9a in their respective SET domainsand forms a heterodimer with
G9a, results in the inactivation of p53, whose loss of function is
implicated in over 50% of cancers.17 In addition, G9a has been
The “writers” (the enzymes that produce post-translational
modifications (PTMs)), the “readers” (the proteins that recog-
nize PTMs), and the “erasers” (the enzymes that remove PTMs)
of the histone code1,2 are critical targets for manipulation to
understand the role of the histone code in human disease. Protein
lysine methyltransferases (PKMTs), the methyl “writers” of the
histone code, catalyze the transfer of a methyl group from
S-adenosyl-L-methionine (SAM) to the ε-amino group of lysine
residues of various proteins including histones and nonhistone
proteins.3,4 With the exception of DOT1L, all PKMTs contain
the evolutionarily conserved SET domain, named after Droso-
phila Su(var.)3À9 (suppressor of variegation 3À9), E(z)
(enhancer of zeste), and trithorax.5 Since the first PKMT was
characterized in 2000,6 more than 50 human PKMTs have been
identified3,4 and the mechanistic, biochemical, and biological
functions of these proteins are still being elucidated. PKMTs
display significant variations in protein substrate selectivity and the
degree of methylation on lysine from mono- and di-, to trimethyla-
tion. In the context of epigenetic gene regulation, these different
states of lysine methylation encode distinct signals and are
recognized by a host of protein modules such as Tudor and
chromodomains, MBT domains and PHD fingers, usually within
larger proteins, and chromatin remodeling complexes that direct
cellular processes such as chromatin packaging and transcriptional
Received: July 10, 2011
Published: July 22, 2011
r
2011 American Chemical Society
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Figure 1. Structures of known G9a/GLP inhibitors.29,31À34
shown to play a role in cocaine addiction,23 mental retardation,24
maintenance of HIV-1 latency,25 and DNA methylation in em-
bryonic stem (ES) cells.26À28
cells and an excellent ratio of toxicity to functional potency (tox/
function ratio, which is determined dividing the EC50 value of the
observed toxicity by the IC50 value of the functional potency).34
Here we report the design and synthesis of novel analogues of
compound 3 aimed at improving cellular potency of this quinazo-
line series and the SAR of these compounds in cell-based assays
that led to the discovery of compound 5 and novel G9a inhibitors
such as UNC0646 (6) and UNC0631 (7), which have excellent
tox/function ratios in a variety of cell lines.
The recent report by Jenuwein and colleagues of BIX01294 (1),
the first selective small molecule inhibitor of G9a and
GLP,29,30 was an important advance in the PKMT probe discovery
area (Figure 1). We have explored the 2,4-diamino-6,7-dimethox-
yquinazoline template represented by1. Ourinitial structure-based
design and synthesis in combination with SAR exploration led to
the discovery of potent and selective G9a inhibitors UNC0224 (2)
and UNC0321 (3, the most potent G9a inhibitor (Morrison
Ki = 63 pM) to date) and valuable SAR for the quinazoline
template.31,32 Cheng and co-workers have also reported SAR of
the quinazoline scaffold that resulted in the discovery of E72 (4) as
a potent and selective GLP inhibitor.33 However, compounds 334
and 433 are less potent in cellular assays than1 even though 3 and 4
are more potent than 1 in in vitrobiochemicalassays. Compound1
is active at reducing the H3K9me2 levels in bulk histones and at
G9a target genes,29 butithaspoorseparation of functional potency
versus cell toxicity.29,34 We have recently reported UNC0638 (5),
a chemical probe of G9a/GLP with robust on-target activities in
’ RESULTS AND DISCUSSION
It was hypothesized that the poor cellular potency of com-
pound 3 was likely due to its poor cell membrane permeability
and low lipophilicity (ALogP = 1.9). To improve cell membrane
permeability, and thus cellular potency of this chemical series, we
exploited the SAR at the 2-amino, 4-amino, and 7-aminoalkoxy
regions of this quinazoline scaffold discovered previously. For the
7-aminoalkoxy region, the four groups (marked in blue in Figure 2)
that yield high in vitro potency32 were selected as affinity anchors.
Because few modifications to the 4-(piperidin-4-yl)amino moiety
Figure 2. Compounds designed to achieve balanced in vitro potency and physicochemical properties aiding cell penetration to improve cellular
potency.
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Table 1. SAR of the 7-Aminoalkoxy Regiona
a IC50 or EC50 values are the average of at least duplicate assay runs with standard deviation (SD) values that are 3-fold less than the average. b ALogP
values were calculated using Pipeline Pilot v7.5.
Scheme 1. Synthesis of Compounds 17a
a (a) 1-Chloro-3-iodopropane, K2CO3, CH3CN, reflux; (b) HNO3, Ac2O, 0 ꢀC to rt, 75% over two steps; (c) pyrrolidine, K2CO3, NaI, cat.
tetrabutylammonium iodide, CH3CN, reflux, 81%; (d) Fe dust, NH4OAc, AcOEtÀH2O, reflux, 63%; (e) NaOCN, AcOH, H2O, rt; (f) NaOH, H2O,
MeOH, reflux; (g) N,N-diethylaniline, POCl3, reflux, 50% over three steps; (h) 1-isopropylpiperidin-4-amine, DIEA, THF, rt, 94%; (i) various amines,
CF3COOH, i-PrOH, 160 ꢀC, microwave, 74À82%.
except the N-capping group (which does not make interactions with
the protein) on the piperidine nitrogen are tolerated,31,32 the three
N-capping groups (marked in red in Figure 2) were selected to
increase lipophilicity while maintaining high in vitro potency.
Because modifications to the 2-amino moiety are well tolerated,31,32
various homopiperazine, azepine, piperidine, and cyclohexyl groups
(marked in green in Figure 2) were selected to further increase
lipophilicity. These combination compounds were designed to
achieve balanced in vitro potency and physicochemical properties
aiding cell penetration.
In-Cell Western (ICW) assay,34 recently developed by us, was
used to assess the cellular potency of these compounds. This assay
allows rapid processing of multiple compounds for H3K9me2
immunofluorescence signal and normalization to cell number via
the use of the nucleic acid dye DRAQ5. The compounds were first
evaluated in MDA-MB-231 cells because this cell line possesses
robust H3K9me2 levels. Most promising compounds were sub-
sequently evaluated in a number of other cancer and normal cell
lines. Viability of cells after inhibitor treatment was assessed by an
MTT (3-(4,5-dimethylthiazol-2-yl)->2,5-diphenyl tetrazolium
bromide) assay, which measures the conversion of yellow MTT
to purple formazan by cellular mitochondrial reductase.
The inhibitory activity of these compounds on G9a was
evaluated using the fluorescence-based S-adenosyl-L-homocys-
teine hydrolase (SAHH)-coupled assay32 that we previously
reported. The H3K9me2 antibody cell immunofluorescence
For the 7-aminoalkoxy region, compounds 2, 3, 8, and 9, the
four most potent G9a inhibitors (in biochemical assays) that we
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Table 2. SAR of the 2-Amino Regiona
a IC50 or EC50 values are the average of at least duplicate assay runs with standard deviation (SD) values that are 3-fold less than the average. b ALogP
values were calculated using Pipeline Pilot v7.5.
previously reported,32 were evaluated in the ICW and MTT
assays (Table 1). Similar to compound 3, compound 2 showed
poor potency in reducing H3K9me2 levels in MDA-MB-231
cells even though it was highly potent in the G9a enzyme
inhibitory assay. On the other hand, compounds 8 and 9, which
are more lipophilic than compounds 2 and 3 as evident by their
ALogP values, showed higher cellular potency in the ICW assay.
All four compounds had low cell toxicity (EC50 > 10 μM) in the
MTT assay. These initial findings validated our hypothesis that
the poor cellular potency of compound 3 was due to its low
lipophilicity, and high cellular potency could be achieved by
increasing lipophilicity of this series. On the basis of these results,
we selected the pyrrolidin-1-ylpropoxy (8) and piperidin-1-
ylpropoxy (9) groups as the preferred 7-aminoalkoxy moieties
for optimizing other regions of this quinazoline series.
To efficiently explore the 2-amino region, a new synthetic
route (9 steps) was developed (Scheme 1). Unlike our previously
published synthetic route,32 this new synthesis allows us to install
various 2-amino groups at the last step of the synthetic sequence.
Commercially available methyl 4-hydroxy-3-methoxybenzoate
(10) was reacted with 1-chloro-3-iodopropane, followed by
nitration, to afford the chloride 11. Substitution of the chloride
with pyrrolidine and subsequent reduction of the nitro group
yielded the aniline 13. Formation of urea from the aniline 13 and
subsequent ring closure afforded the intermediate 15, which
was then converted to the 2,4-dichloroquinazoline 16. Two
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consecutive chloro displacement reactions by various amines
provided the desired final products 17aÀ17i in good overall
yields. Synthesis of compound 5, the 2-cyclohexyl analogue of
compounds 17aÀ17i, from the intermediate 13 was reported by
us previously.34
toxicity of these highly potent G9a inhibitors was assessed
using the MTT assay. While most of these inhibitors had a
relatively poor tox/function ratio (<10), compounds 17h, 17i,
and 5 had a good separation of functional potency (reducing
H3K9me2 levels) versus cell toxicity (tox/function ratio >45).
In particular, compound 5 had an excellent tox/function ratio
of 140.
Because compound 5 showed high cellular potency and ex-
cellent separation of functional potency versus cell toxicity, we
furtherinvestigatedthis 2-cyclohexyl quinazolinesubseries. Thus, a
new 9-step synthesis (Scheme 2) was developed to efficiently
explore the 7-aminoalkoxy region. Benzyl protection of com-
mercially available 4-hydroxy-3-methoxybenzonitrile (18), fol-
lowed by nitration and subsequent reduction, afforded the
aniline 19. Amide formation from the aniline 19 and subse-
quent cyclization yielded the quinazolinone 20, which was then
converted to the 4-chloro quinazoline 21. Displacement of the
chloro intermediate 21 with 1-isopropylpiperidin-4-amine, fol-
lowed by debenzylation, afforded the phenol 23. Mitsunobu
reactions of the phenol 23 with various amino alcohols yielded
the desired compounds 24a, 24b, and 5.
Compared to compound 5, compound 24a was less potent
in the ICW assay (Table 3). This is likely due to the about 10-
fold lower potency of compound 24a in the G9a biochemical
assay although 24a is slightly more lipophilic. The combina-
tion of lower in vitro potency and lower lipophilicity of
compound 24b compared to compound 5 resulted in lower
cellular potency as expected (Figure 3). Interestingly, both
compounds 24a and 24b had low cell toxicity, similar to
compound 5.
Compounds 17aÀ17i and 5 were highly potent inhibitors
(IC50 < 0.025 μM) in the G9a SAHH-coupled biochemical assay
(Table 2). Various N-capping groups (17aÀ17g) on the outer
nitrogen of the homopiperazine moiety were well tolerated. In
addition, azepane (17i), piperidine (5), and cyclohexyl groups
(5) also showed high in vitro potency. Importantly, in addition to
maintaining high in vitro potency, these new compounds have
improved lipophilicity compared to compounds 2 and 3. We
were pleased to find that the combination of high in vitro potency
and improved lipophilicity indeed resulted in high cellular
potency (IC50 < 0.5 μM in the ICW assay). In particular, more
lipophilic compounds such as 17d (ALogP 4.7), 17e (ALogP
5.0), 17g (ALogP 4.4), 17i (ALogP 4.2), and 5 (ALogP 4.8) had
cellular potency (IC50) equal to or less than 0.1 μM. Plotting
cellular potency of the inhibitors in Tables 1 and 2 and inhibitors
to be discussed below as a function of ALogP values of these
inhibitors reveals that cellular potency strongly correlates with
lipophilicity (Figure 3). These results validated our design
strategy for improving cellular potency. Furthermore, cell
We next synthesized a number of combination compounds
including 6, 7, and 25 À 27 using the synthetic route outlined in
Scheme 1 and evaluated them in the G9a SAHH-coupled, ICW, and
MTT assays. Compounds 6, 7, 25, and 26, which had high in vitro
potency versus G9a and improved lipophilicity, were highly potent
(IC50 < 0.06 μM) in reducing H3K9me2 levels in MDA-MB-231
cells and had low cell toxicity (Table 4). In particular, compounds 6
and 7 were more potent than compound 5 and had a similar tox/
function ratio in MDA-MB-231 cells (Table 5). On the other hand,
many combination compounds failed to improve cellular potency
and reduce cell toxicity. For example, compound 27 was about
10-fold less potent in reducing cellular levels of H3K9me2 than
compounds 6 and 7. The lower cellular potency of compound 27 is
Figure 3. Analysis of cellular potency of G9a inhibitors as a function of
their ALogP values reveals that cellular potency increases as lipophilicity
increases for inhibitors that have similar in vitro potency. The inhibitors
are color-coded with their G9a in vitro potency.
Scheme 2. Synthesis of Compounds 24 and 5a
a (a) BnBr, K2CO3, DMF, rt; (b) HNO3, Ac2O, 0 ꢀC to rt; (c) Fe dust, NH4Cl, i-PrOH-H2O, reflux, 67% over 3 steps; d) cyclohexane carbonyl chloride,
DIEA, DMF-DCM, 0 ꢀC to rt; (e) NaOH, H2O2, EtOH, reflux, 48% over 2 steps; (f) N,N-diethylaniline, POCl3, reflux, 79%; (g) 1-isopropylpiperidin-4-
amine, DIEA, i-PrOH, 160 ꢀC, microwave, 94%; (h) Pd/C, 1,4-cyclohexadiene, EtOH, reflux; (i) various amino alcohols, PPh3, DIAD, THF, 0 ꢀC to rt,
36À65% over 2 steps.
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Table 3. SAR of 2-Cyclohexyl Quinazoline Analoguesa
a IC50 or EC50 values are the average of at least duplicate assay runs with standard deviation (SD) values that are 3-fold less than the average. b ALogP
values were calculated using Pipeline Pilot v7.5.
Table 4. Cellular Potency and Cell Toxicity of Combination Compoundsa
a IC50 or EC50 values are the average of at least duplicate assay runs with standard deviation (SD) values that are 3-fold less than the average. b ALogP
values were calculated using Pipeline Pilot v7.5.
likely due to the combination of its lower G9a in vitro potency and
lower lipophilicity compared to compounds 6 and 7 (Figure 3).
Because compounds 6 and 7 showed excellent separation of
functional potency versus cell toxicity in MDA-MB-231 cells, these
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Table 5. Potency and Toxicity of Compounds 5, 6, and 7 in a Variety of Cell Linesa
ICW IC50 (μM)
MTT EC50 (μM)
tox/function ratio (MTT EC50/ICW IC50)
5
6
7
5
6
7
5
6
7
breast carcinoma
prostate carcinoma
colon carcinoma
human fibroblast
MDA-MB-231
MCF7
0.081
0.070
0.026
0.010
0.025
0.018
11
3.3
4.7
2.8
1.7
140
110
130
470
110
94
7.6
PC3
0.059
0.048
0.012
0.014
0.026
0.024
14
2.8
7.2
3.8
2.5
240
94
230
510
150
100
22RV1
4.5
HCT116 wt
0.21
0.24
0.068
0.086
0.051
0.072
11
11
8.7
12
5.8
6.4
52
46
130
140
110
89
HCT116 p53À/À
IMR90
0.12
0.010
0.046
2.3
3.6
1.8
19
360
39
a IC50 or EC50 values are the average of at least duplicate assay runs with standard deviation (SD) values that are 3-fold less than the average.
reported the robust effects of compound 5 on reducing
H3K9me2 levels in various cellular systems.34 However, the
effects of the quinazoline series on cellular levels of p53
K373me2 have not been reported previously. Thus, we
evaluated the effects of compound 5 on reducing p53
K373me2 levels in H1299 cells (p53 negative), which were
cotransfected with p53 and G9a or GLP. As shown in Figure 4,
compound 5 at 0.25 μM effectively inhibited the dimethyla-
tion of p53 K373 without significant changes to levels of total
p53 and G9a or GLP in H1299 cells. These results together
with our previous H3K9me2 findings34 strongly suggest that
compound 5 is an effective inhibitor that blocks the methyl-
transferase activity of G9a and GLP in cells.
Figure 4. Compound 5 at 0.25 μM effectively inhibited the dimethyla-
tion of p53 K373 in H1299 cells. p53 expression vector was cotrans-
fected with or without expression vectors for G9a or GLP into H1299
cells. Immediately after transfection, cells were treated with compound 5
or DMSO for 24 h. Cellular levels of p53 K373me2, total p53 and G9a
or GLP were subject to Western blot analysis using antibodies.
’ CONCLUSIONS
To improve cellular potency of the quinazoline series, we
designed several generations of novel analogues aimed at achiev-
ing balanced G9a in vitro potency and physicochemical properties
aiding cell penetration. New synthetic routes to efficiently
synthesize these novel compounds were developed. SAR studies
of these newly synthesized compounds in the G9a biochemical
(SAHH-coupled), cell-based functional (anti-H3K9me2 ICW),
and cell toxicity (MTT) assays validated our inhibitor design
strategy and resulted in the discovery of compounds 6 and 7,
which had high cellular potency and excellent separation of
functional potency versus cell toxicity in a variety of cell lines.
Compounds 6 and 7 together with compound 5, the previously
reported G9a chemical probe, are valuable small molecule tools
for the biomedical research community to further investigate the
biology of G9a and its role in chromatin remodeling as well as
PTMs of other proteins.
two compounds were further evaluated in a variety of cell lines to
characterize their cellular potency and cell toxicity. As shown in
Table 5, compounds 6 and 7 were highly potent in reducing
H3K9me2 levels in these cell lines and had low cell toxicity. Similar
to compound 5, compounds 6 and 7 had excellent tox/function
ratios in these cell lines. In particular, compound 6 had an
outstanding tox/function ratio in MCF7 (470), 22RV1 (510),
and IMR90 (360) cells, making this compound potentially more
useful for investigating G9a biology in these specific cell lines. In
general, compounds 6 and 7 had higher cellular potency but also
higher cell toxicity compared to compound 5. While compound 5
has been selected as a chemical probe of G9a/GLP34 because of its
low cell toxicity and lower molecular weight, compounds 6 and 7
are valuable alternative tool compounds that may be more suitable
for investigating specific cellular systems (vide supra).
The excellent selectivity of compound 5 for G9a/GLP over a wide
range of epigenetic and nonepigenetic targets has been reported
previously.34 Consistent with these results, we found that compound
6was also a potent GLP inhibitor (IC50 < 0.015 μMintheGLPSAHH-
coupled assay) and highly selective for G9a/GLP over several other
PKMTs and PRMTs including SETD7 (a H3K4 PKMT), SUV39H2
(a H3K9 PKMT), SETD8 (a H4K20 PKMT), and PRMT3 (IC50 >
10 μM in the SAHH-coupled assays against these four targets).
In addition to methylating histone substrates,14,15,35 G9a and
GLP dimethylate K373 of the tumor suppressor p53.17 We
’ EXPERIMENTAL SECTION
Chemistry General Procedures. HPLC spectra for all com-
pounds were acquired using an Agilent 6110 series system with UV
detector set to 254 nm. Samples were injected (5 μL) onto an Agilent
Eclipse Plus 4.6 mm  50 mm, 1.8 μM, C18 column at room
temperature. A linear gradient from 10% to 100% B (MeOH + 0.1%
acetic acid) in 5.0 min was followed by pumping 100% B for another
2 min with A being H2O + 0.1% acetic acid. The flow rate was 1.0 mL/min.
Mass spectra (MS) data were acquired in positive ion mode using an
Agilent 6110 single quadrupole mass spectrometer with an electrospray
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ionization (ESI) source. High-resolution mass spectra (HRMS) were
acquired using a Shimadzu LCMS-IT-TOF time-of-flight mass spectro-
meter. Nuclear magnetic resonance (NMR) spectra were recorded at
Varian Mercury spectrometer with 400 MHz for proton (1H NMR) and
100 MHz for carbon (13C NMR); chemical shifts are reported in
ppm (δ).36 Preparative HPLC was performed on Agilent Prep 1200
series with UV detector set to 220 nm. Samples were injected onto a
Phenomenex Luna 75 mm  30 mm, 5 μM, C18 column at room
temperature. The flow rate was 30 mL/min. A linear gradient was used
with 10% of MeOH (A) in 0.1% TFA in H2O (B) to 100% of MeOH (A).
All compounds that were evaluated in biological assays had >95% purity
using the HPLC methods described above.
2.2 Hz, 2H), 2.19À2.11 (m, 2H), 2.11À2.02 (m, 2H), 2.01À1.94 (m, 2H),
1.79À1.70 (m, 4H), 1.54 (ddd, J = 14.9, 11.8, 3.7 Hz, 2H), 1.04 (d,
J = 6.6 Hz, 6H). 13C NMR (100 MHz, CDCl3, two overlapping peaks)
δ 158.70, 158.10, 154.05, 149.76, 145.22, 107.08, 102.79, 101.67, 67.40,
59.07, 57.46, 56.76, 54.58, 54.25, 53.01, 48.77, 47.88, 46.83, 45.98, 32.75,
28.64, 27.98, 23.56, 18.60. HPLC: 97%; tR 1.18 min. MS (ESI): 540
[M + H]+.
2-(4-Ethyl-1,4-diazepan-1-yl)-N-(1-isopropylpiperidin-4-yl)-
6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine
(17b). Compound 17b was prepared as a white solid by the same
procedures as the preparation of compound 17a (80% yield). 1H NMR
(400 MHz, CDCl3) δ 6.89 (s, 1H), 6.71 (s, 1H), 4.95 (d, J = 7.1 Hz, 1H),
4.15 (t, J = 6.8 Hz, 2H), 4.10À4.01 (m, 1H), 3.98À3.92 (m, 2H),
3.90À3.82 (m, 5H), 2.94À2.85(m, 2H), 2.80À2.71 (m, 3H), 2.64À2.46
(m, 10H), 2.36À2.26 (m, 2H), 2.20À2.13 (m, 2H), 2.12À2.04 (m, 2H),
2.02À1.93 (m, 2H), 1.82À1.70 (m, 4H), 1.56 (ddd, J = 14.6, 12.0, 3.5 Hz,
2H), 1.13À0.99 (m, 9H). 13C NMR (100 MHz, CDCl3) δ 158.75, 158.15,
154.10, 149.80, 145.26, 107.12, 102.81, 101.69, 67.43, 56.84, 55.94, 54.64,
54.47, 54.30, 53.08, 51.71, 48.76, 47.92, 46.28, 45.88, 32.77, 28.68,
27.97, 23.60, 18.63, 12.51. HPLC: 97%; tR 1.14 min. MS (ESI): 554
[M + H]+.
2-(4-Isopropyl-1,4-diazepan-1-yl)-N-(1-isopropylpiperidin-4-
yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine
(17c). Compound 17c was prepared as an off-white solid by the same
procedures as the preparation of compound 17a (82% yield). 1H NMR
(400 MHz, CDCl3) δ 6.87 (s, 1H), 6.72 (s, 1H), 4.98 (d, J = 7.1 Hz, 1H),
4.13 (t, J = 6.8 Hz, 2H), 4.09À3.99 (m, 1H), 3.93À3.87 (m, 2H),
3.88À3.80 (m, 5H), 2.95À2.83 (m, 3H), 2.78À2.69 (m, 3H), 2.62À2.53
(m, 4H), 2.53À2.43 (m, 4H), 2.33À2.23 (m, 2H), 2.20À2.11 (m, 2H),
2.10À2.02 (m, 2H), 1.94À1.85 (m, 2H), 1.81À1.68 (m, 4H), 1.54 (ddd,
J = 14.6, 12.0, 3.6 Hz, 2H), 1.04 (d, J = 6.6 Hz, 6H), 0.98 (d, J = 6.5 Hz,
6H). 13C NMR (100 MHz, CDCl3, two overlapping peaks) δ 158.74,
158.14, 154.00, 149.85, 145.12, 107.08, 102.78, 101.72, 67.39, 56.77, 55.20,
54.57, 54.25, 53.02, 52.26, 50.68, 48.76, 47.89, 47.80, 45.77, 32.76, 28.78,
28.65, 23.56, 18.61. HPLC: 99%; tR 1.25 min. MS (ESI): 568 [M + H].
2-(4-Cyclohexyl-1,4-diazepan-1-yl)-N-(1-isopropylpiperidin-
4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-
amine (17d). Compound 17d was prepared as a white solid by the
same procedures as the preparation of compound 17a (78% yield). 1H
NMR (400 MHz, CDCl3) δ 6.87 (s, 1H), 6.72 (s, 1H), 4.98 (d, J = 7.1 Hz,
1H), 4.13 (t, J = 6.8 Hz, 2H), 4.09À3.97 (m, 1H), 3.92À3.78 (m, 7H),
2.91À2.84 (m, 2H), 2.83À2.77 (m, 2H), 2.77À2.68 (m, 1H), 2.65À2.60
(m, 2H), 2.58 (t, J = 7.4 Hz, 2H), 2.53À2.39 (m, 5H), 2.33À2.24 (m, 2H),
2.18À2.10 (m, 2H), 2.10À2.00 (m, 2H), 1.92À1.83 (m, 2H), 1.82À1.68
(m, 8H), 1.62À1.47 (m, 3H), 1.25À1.06 (m, 5H), 1.04 (d, J = 6.5 Hz, 6H).
13C NMR (100 MHz, CDCl3) δ 158.73, 158.13, 153.98, 149.86, 145.09,
107.07, 102.76, 101.72, 67.37, 64.43, 56.75, 54.56, 54.24, 53.01, 52.64, 51.26,
48.75, 47.99, 47.89, 45.78, 32.75, 29.34, 28.85, 28.64, 26.49, 26.19, 23.55,
18.60. HPLC: 99%; tR 2.16 min. MS (ESI): 608 [M + H]+.
2-(4-(Cyclohexylmethyl)-1,4-diazepan-1-yl)-N-(1-isopropyl
piperidin-4-yl)-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quina-
zolin-4-amine (17e). Compound 17e was prepared as a white solid
by the same procedures as the preparation of compound 17a (81% yield).
1H NMR (400 MHz, CDCl3) δ 6.87 (s, 1H), 6.73 (s, 1H), 4.99 (d, J = 7.1
Hz, 1H), 4.13 (t, J = 6.8 Hz, 2H), 4.09À3.98 (m, 1H), 3.94À3.87 (m, 2H),
3.87À3.76 (m, 5H), 2.91À2.83 (m, 2H), 2.78À2.67 (m, 3H), 2.62À2.52
(m, 4H), 2.52À2.43 (m, 4H), 2.29 (t, J = 10.7 Hz, 2H), 2.22 (d, J = 6.9 Hz,
2H), 2.15 (d, J = 10.8 Hz, 2H), 2.11À2.01 (m, 2H), 1.96À1.86 (m, 2H),
1.81À1.48 (m, 11H), 1.47À1.34 (m, 1H), 1.26À1.10 (m, 3H), 1.04 (d, J =
6.5 Hz, 6H), 0.88À0.74 (m, 2H). 13C NMR (100 MHz, CDCl3, two
overlapping peaks) δ 158.75, 158.10, 153.99, 149.86, 145.10, 107.07,
102.76, 101.72, 67.38, 64.33, 56.75, 55.34, 54.56, 54.24, 53.01, 48.77,
47.89, 46.62, 45.96, 36.32, 32.74, 32.00, 28.64, 27.93, 26.96, 26.30, 23.56,
18.60. HPLC: 99%; tR 2.39 min. MS (ESI): 622 [M + H]+.
2,4-Dichloro-6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quin-
azoline (16). A solution ofcompound 13 (3.6 g, 11.67 mmol) (prepared
from commercially available methyl 3-methoxy-4-hydroxy benzoate (10)
according to the procedures described previously34) in acetic acid
(20 mL) and water (10 mL) was treated with sodium cyanate (1.14 g,
17.5 mmol) while stirring, and then the mixture was stirred overnight at rt.
Afterthe resulting mixture wasconcentrated, the desired crude product14
was obtained as a brown slurry oil. MS (ESI): 352 [M + H]+. A mixture of
crude compound 14 in MeOH (30 mL) was neutralized with 10% NaOH
aqueous solution, and then an additional 10% NaOH aqueous solution
(10 mL) was added. The mixture was refluxed for 3 h, cooled to room
temperature, and neutralized with concentrated HCl to give a precipitate.
The resulting precipitate was collected and dried to provide the desired
crude product 15 (2.60 g). MS (ESI): 320 [M + H]+. A mixture of the
crude compound 15 (2.60 g, 8.14 mmol) and N,N-diethylaniline (1.3 mL,
8.14 mmol) in POCl3 (45 mL) was heated at reflux for 7 h. The reaction
mixture was concentrated in vacuo and saturated aq NaHCO3 (50 mL)
was added. The resulting mixture was extracted with chloroform (30 mL Â 3).
The combined organic layers were dried, concentrated, and purified by
flash column chromatography on silica gel (0À20% MeOH/CH2Cl2) to
afford the title compound 16 as a yellow solid (2.06 g, 50% over three
steps). 1H NMR (400 MHz, CDCl3) δ 7.30 (s, 1H), 7.25 (s, 1H), 4.26
(t, J = 6.5 Hz, 2H), 4.02 (s, 3H), 2.74 (t, J = 7.3 Hz, 2H), 2.64 (m, 4H),
2.23À2.15 (m, 2H), 1.87À1.79 (m, 4H). MS (ESI): 356 [M + H]+.
N-(1-Isopropylpiperidin-4-yl)-6-methoxy-2-(4-methyl-1,4-dia-
zepan-1-yl)-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine
(17a). A mixture of compound 16 (0.72 g, 2.02 mmol), 1-isopropylpi-
peridin-4-amine (0.87 g, 6.06 mmol) (this amine and other noncommer-
cially available amines were prepared according to the literature
procedures37), and DIEA (0.67 mL, 4.04 mmol) in THF (10 mL) was
stirred overnight at rt. After concentration in vacuo, the crude product was
purified by silica gel chromatography (0À20% MeOH (1% NH3)/CH2Cl2)
to afford the desired product 2-chloro-N-(1-isopropylpiperidin-4-yl)-6-
methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine as a yellow
solid (0.88 g, 94% yield). 1H NMR (400 MHz, CDCl3) δ 7.12 (s, 1H),
6.78 (s, 1H), 5.36 (d, J = 7.7 Hz, 1H), 4.31À4.20 (m, 1H), 4.17 (t, J = 6.7
Hz, 2H), 3.96 (s, 3H), 2.90 (d, J = 11.9 Hz, 2H), 2.84À2.73 (m, 1H),
2.67À2.59 (m, 2H), 2.57À2.46 (m, 4H), 2.45À2.34 (m, 2H), 2.22À2.05
(m, 4H), 1.84À1.71 (m, 4H), 1.65À1.55 (m, 2H), 1.07 (d, J = 6.6 Hz, 6H).
MS (ESI): 462 [M + H]+. Then, a mixture of the intermediate (71 mg,
0.149 mmol), 1-methylhomopiperazine (34 mg, 0.30 mmol), and TFA
(68 mg, 0.60 mmol) in i-PrOH (0.25 mL) was heated by microwave
irradiation to 160 ꢀC for 15 min in a sealed tube. After concentration in
vacuo, the crude product was purified by preparative HPLC with a
gradient from 10% of MeOH (A) in 0.1% TFA in H2O (B) to 100% of
MeOH (A). The resulting product was basified with saturated aq
NaHCO3 and extracted with CH2Cl2 to afford the title compound 17a
as a white solid (64 mg, 79% yield). 1H NMR (400 MHz, CDCl3) δ 6.87
(s, 1H), 6.71 (s, 1H), 4.98 (d, J = 7.1 Hz, 1H), 4.13 (t, J = 6.8 Hz, 2H),
4.08À3.98 (m, 1H), 3.97À3.92 (m, 2H), 3.89À3.82 (m, 5H), 2.91À2.84
(m, 2H), 2.78À2.69 (m, 1H), 2.69À2.63 (m, 2H), 2.61À2.56 (m, 2H),
2.55À2.50 (m, 2H), 2.51À2.44 (m, 4H), 2.34 (s, 3H), 2.29 (td, J = 11.5,
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Journal of Medicinal Chemistry
ARTICLE
N-(1-Isopropylpiperidin-4-yl)-6-methoxy-2-(4-phenyl-1,4-
diazepan-1-yl)-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine
(17f). Compound 17f was prepared as a yellow solid by the same
procedures as the preparation of compound 17a (74% yield). 1H NMR
(400 MHz, CDCl3) δ 7.22 (t, J = 7.9 Hz, 2H), 6.90 (s, 1H), 6.80À6.72
(m, 3H), 6.65 (t, J = 7.2 Hz, 1H), 4.99 (d, J = 7.1 Hz, 1H), 4.18 (t, J = 6.8
Hz, 2H), 4.14À3.99 (m, 3H), 3.90 (s, 3H), 3.71À3.61 (m, 4H), 3.51 (t, J =
6.2 Hz, 2H), 2.96À2.87 (m, 2H), 2.83À2.70 (m, 1H), 2.62 (t, J = 7.3 Hz,
2H), 2.58À 2.45 (m, 4H), 2.32 (t, J = 10.7 Hz, 2H), 2.22À2.05 (m, 6H),
1.85À1.72 (m, 4H), 1.58 (ddd, J = 14.7, 11.9, 3.5 Hz, 2H), 1.08 (d, J =
6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 158.40, 158.31, 154.12,
149.87, 147.69, 145.37, 129.52, 115.76, 111.66, 107.14, 102.93, 101.63,
67.42, 56.81, 54.60, 54.29, 53.06, 50.56, 48.83, 47.93, 47.78, 47.10, 46.12,
32.86, 28.67, 24.89, 23.59, 18.61. HPLC: 98%; tR 3.29 min. MS (ESI): 602
[M + H]+.
(20 mL) and NaOH (400 mg, 10.0 mmol) in ethanol (100 mL) was stirred
for 2 h under reflux. After adding 100 mL of water, the reaction mixture was
cooled to rt and the resulting precipitate was collected, washed with water,
and dried to afford the title compound 20 as a white solid (1.35 g, 48% yield
over 2 steps). 1H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H), 7.60 (s, 1H),
7.50À7.30 (m, 5H), 7.15 (s, 1H), 5.26 (s, 2H), 3.99 (s, 3H), 2.66À2.53 (m,
1H), 2.03 (d, J = 12.2 Hz, 2H), 1.96À1.86 (m, 2H), 1.83À1.74 (m, 1H),
1.68À1.52 (m, 2H), 1.49À1.24 (m, 3H).
7-(Benzyloxy)-4-chloro-2-cyclohexyl-6-methoxyquinazo-
line (21). A mixture of the compound 20 (1.32 g, 3.61 mmol) and N,N-
diethylaniline (0.30 mL, 1.80 mmol) in POCl3 (20 mL) was heated at reflux
for 4 h. The reaction mixture was concentrated in vacuo, and saturated aq
NaHCO3 (20 mL) was added. The resulting mixture was extracted with
chloroform (20 mL Â 3). The combined organic layers were dried,
concentrated, and purified by flash column chromatography on silica gel
(0À10% ethyl acetate/hexanes) to afford the title compound 21 as a yellow
solid (1.10 g, 79% yield). 1H NMR (400 MHz, CDCl3) δ 7.51À7.30 (m,
7H), 5.30 (s, 2H), 4.04 (s, 3H), 2.92 (tt, J = 11.7, 3.4 Hz, 1H), 2.10À1.99
(m, 2H), 1.93À1.84 (m, 2H), 1.79À1.64 (m, 3H), 1.49À1.27 (m, 3H).
7-(Benzyloxy)-2-cyclohexyl-N-(1-isopropylpiperidin-4-yl)-
6-methoxyquinazolin-4-amine (22). A mixture of compound 21
(0.68 g, 1.77 mmol), 1-isopropylpiperidin-4-amine (0.77 g, 5.41 mmol),
and DIEA (0.60 mL, 3.60 mmol) in i-PrOH (3.0 mL) was heated by
microwave irradiation to 160 ꢀC for 15 min in a sealed tube. After
concentration in vacuo, the crude product was purified by silica gel chrom-
atography (0À10% MeOH/CH2Cl2) to afford the title compound 22 as a
white solid (0.81 g, 94% yield). 1H NMR (400 MHz, MeOH-d4) δ 7.52 (s,
1H), 7.45 (d, J = 7.2 Hz, 2H), 7.34 (t, J = 7.3 Hz, 2H), 7.31À7.24 (m, 1H),
7.11 (s, 1H), 5.18 (s, 2H), 4.27À4.19 (m, 1H), 3.94 (s, 3H), 2.98 (d, J= 11.7
Hz, 2H), 2.81À2.71(m, 1H), 2.69À2.57 (m, 1H), 2.35 (t, J=11.4Hz, 2H),
2.13 (d, J = 11.4 Hz, 2H), 1.91À1.82 (m, 4H), 1.79À1.61 (m, 5H),
1.49À1.23 (m, 3H), 1.10 (d, J = 6.5 Hz, 6H). MS (ESI): 489 [M + H]+.
2-Cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-
(pyrrolidin-1-yl)propoxy)quinazolin-4-amine (5). A mixture of
compound 22 (0.81 g, 1.65 mmol), 1,4-cyclohexadiene (1.6 mL, 16.5
mmol), and 10 wt % Pd/C (250 mg) in ethanol (16 mL) was heated to
reflux 2 h. The reaction mixture was filtered and concentrated to provide the
debenzylated product 23 as a yellow solid which was used without
purification. MS (ESI): 399 [M + H]+. Under nitrogen gas, DIAD (270 μL,
1.38 mmol) was added into an ice-cooled mixture of compound 21
(110 mg, 0.276 mmol), 3-(pyrrolidin-1-yl)propan-1-ol (142 mg, 1.10
mmol), and PPh3 (399 mg, 1.52 mmol) in THF (4.0 mL). The reaction
mixture was stirred overnight at rt. After concentration in vacuo, the
crude product was purified by preparative HPLC with a gradient from
10% of MeOH (A) in 0.1% TFA in H2O (B) to 100% of MeOH (A). The
resulting product was basified with a saturated solution of NaHCO3 and
extracted with CH2Cl2 to afford 56 mg of the desired product 5 as a white
solid (91 mg, 65% yield over 2 steps). The characterization data (1H and
13C NMR, HPLC, HRMS) of the title compound match the reported.34
2-Cyclohexyl-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-(3-
(piperidin-1-yl)propoxy)quinazolin-4-amine (24a). Compound
24a was prepared as a white solid from compound 23 and 3-(piperidin-1-
yl)propan-1-ol via Mitsunobu reaction (52% over two steps). 1H NMR
(400 MHz, CDCl3) δ 7.13 (s, 1H), 6.82 (s, 1H), 5.22 (d, J = 7.3 Hz, 1H),
4.25À4.07 (m, 3H), 3.88 (s, 3H), 2.93À2.84(m, 2H), 2.81À2.63 (m,
2H), 2.48À2.25 (m, 8H), 2.19 (d, J = 11.3 Hz, 2H), 2.08À1.90 (m, 4H),
1.81 (d, J = 12.6 Hz, 2H), 1.75À1.47 (m, 9H), 1.47À1.17 (m, 5H), 1.05
(d, J = 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 169.44, 157.93,
153.68, 148.40, 147.38, 108.51, 106.73, 100.07, 67.73, 56.43, 55.78, 54.64,
54.62, 48.59, 47.97, 47.85, 32.67, 32.01, 26.49, 26.48, 26.32, 26.08, 24.53,
18.56. HPLC: 99%; tR 3.10 min. MS (ESI): 524 [M + H]+.
2-(4-Benzyl-1,4-diazepan-1-yl)-N-(1-isopropylpiperidin-4-yl)-
6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine
(17g). Compound 17g was prepared as an off-white solid by the same
1
procedures as the preparation of compound 17a (78% yield). H NMR
(400 MHz, CDCl3) δ 7.25À7.12 (m, 5H), 6.82 (s, 1H), 6.67 (s, 1H), 4.95
(d, J = 7.0 Hz, 1H), 4.08 (t, J = 6.8 Hz, 2H), 4.02À3.91 (m, 1H), 3.90À3.84
(m, 2H), 3.84À3.75 (m, 5H), 3.55 (s, 2H), 2.87À2.77 (m, 2H), 2.74À2.61
(m, 3H), 2.59À2.50 (m, 4H), 2.47À2.38 (m, 4H), 2.21 (t, J = 11.3 Hz,
2H), 2.15À1.95 (m, 4H), 1.94À1.83 (m, 2H), 1.73À1.63 (m, 4H), 1.48
(ddd, J = 14.6, 11.9, 3.5 Hz, 2H), 0.98 (d, J = 6.5 Hz, 6H). 13C NMR (100
MHz, CDCl3) δ 158.71, 158.12, 154.04, 149.76, 145.19, 139.61, 128.91,
128.20, 126.87, 107.06, 102.79, 101.72, 67.39, 62.30, 56.76, 56.28, 54.98,
54.58, 54.25, 53.02, 48.76, 47.87, 46.58, 46.01, 32.72, 28.63, 28.15, 23.56,
18.58. HPLC: 98%; tR 2.27 min. MS (ESI): 616 [M + H]+.
N-(1-Isopropylpiperidin-4-yl)-6-methoxy-2-(piperidin-1-yl)-
7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine (17h). Com-
pound 17h was prepared as a white solid by the same procedures as
1
the preparation of compound 17a (76% yield). H NMR (400 MHz,
CDCl3) δ 6.90 (s, 1H), 6.75 (s, 1H), 5.10 (d, J = 7.2 Hz, 1H), 4.21À4.01
(m, 3H), 3.87 (s, 3H), 3.80À3.73 (m, 4H), 2.98À2.86 (m, 2H), 2.82À2.72
(m, 1H), 2.65À2.57 (m, 2H), 2.58À2.45 (m, 4H), 2.40À2.28 (m, 2H),
2.20À2.12 (m, 2H), 2.12À2.02 (m, 2H), 1.82À1.69 (m, 4H), 1.69À1.53
(m, 8H), 1.07 (d, J = 6.6 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 158.95,
158.27, 154.01, 149.36, 145.46, 106.97, 102.93, 101.65, 67.35, 56.71, 54.74,
54.24, 53.02, 48.48, 47.89, 45.18, 32.47, 28.54, 26.07, 25.19, 23.56, 18.51.
HPLC: 97%; tR 2.92 min. MS (ESI): 511 [M + H]+.
2-(Azepan-1-yl)-N-(1-isopropylpiperidin-4-yl)-6-methoxy-
7-(3-(pyrrolidin-1-yl)propoxy)quinazolin-4-amine (17i). Com-
pound 17i was prepared as a white solid by the same procedures as the
preparation of compound 17a (78% yield). 1H NMR (400 MHz, CDCl3)
δ 6.89 (s, 1H), 6.72 (s, 1H), 4.96 (d, J = 6.9 Hz, 1H), 4.15 (t, J = 6.8 Hz,
2H), 4.11À4.00 (m, 1H), 3.87 (s, 3H), 3.77 (t, J = 5.9 Hz, 4H), 2.93À2.85
(m, 2H), 2.80À2.68 (m, 1H), 2.60 (t, J = 7.4 Hz, 2H), 2.55À2.44 (m, 4H),
2.35À2.24 (m, 2H), 2.17 (d, J = 11.3 Hz, 2H), 2.13À2.02 (m, 2H),
1.85À1.67 (m, 8H), 1.63À1.47 (m, 6H), 1.05 (d, J = 6.5 Hz, 6H). 13C
NMR (100 MHz, CDCl3) δ158.76, 158.15, 154.03, 149.91, 145.04, 107.06,
102.71, 101.81, 67.41, 56.84, 54.61, 54.29, 53.06, 48.83, 47.94, 46.99, 32.82,
28.69, 28.66, 27.53, 23.59, 18.62. HPLC: 98%; tR 3.09 min. MS (ESI): 525
[M + H]+.
7-(Benzyloxy)-2-cyclohexyl-6-methoxyquinazolin-4(3H)-one
(20). Cyclohexanecarbonyl chloride (1.25 mL, 9.2 mmol) was added into
a solution of compound 19 (1.95 g, 7.64 mmol) (prepared from commer-
cially available 4-hydroxy-3-methoxybenzonitrile (18) according to the
procedures described previously31,32) and DIEA (1.90 mL, 11.5 mmol)
in cosolvent DMF:DCM (10 mL:5 mL) at 0 ꢀC. The reaction mixture was
stirred overnight at rt, 25 mL of water was added, and the resulting mixture
was extracted with CH2Cl2 (15 mL Â 3). The combined organic solvent
was dried with sodium sulfate, concentrated, and used in the next step
without further purification. A mixture of the product, 30 wt % H2O2
2-Cyclohexyl-7-(2-(2-(dimethylamino)ethoxy)ethoxy)-N-(1-
isopropylpiperidin-4-yl)-6-methoxyquinazolin-4-amine (24b).
Compound 24b was prepared as a white solid from compound 23 and
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Journal of Medicinal Chemistry
ARTICLE
2-(2-(dimethylamino)ethoxy)ethanol via Mitsunobu reaction (36% over
two steps). 1H NMR (400 MHz, CDCl3) δ 7.11 (s, 1H), 6.83 (s, 1H),
5.22 (d, J = 7.2 Hz, 1H), 4.27À4.13 (m, 3H), 3.92À3.83 (m, 5H), 3.61 (t,
J = 5.8 Hz, 2H), 2.93À2.85 (m, 2H), 2.80À2.64 (m, 2H), 2.48 (t, J = 5.8
Hz, 2H), 2.40À2.31 (m, 2H), 2.22 (s, 6H), 2.19 (d, J = 10.5 Hz, 2H), 1.95
(d, J = 11.9 Hz, 2H), 1.86À1.76 (m, 2H), 1.75À1.50 (m, 5H), 1.46À1.21
(m, 3H), 1.05 (d, J = 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
169.49, 157.94, 153.43, 148.39, 147.26, 108.52, 107.00, 100.20, 69.62,
69.01, 68.19, 58.84, 56.39, 54.61, 48.62, 47.98, 47.86, 45.91, 32.67, 32.01,
26.49, 26.32, 18.58. HPLC: 99%; tR 2.98 min. MS (ESI): 514 [M + H]+.
N-(1-Cyclohexylpiperidin-4-yl)-2-(4-isopropyl-1,4-diazepan-1-
yl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-amine
(6). Compound 6 was prepared as a white solid by the same procedures as
(p, J = 12.0 Hz, 4H), 1.13À1.00 (m, 1H). HPLC: 99%; tR 2.26 min. MS
(ESI): 594 [M + H]+.
2-(Azepan-1-yl)-N-(1-isopropylpiperidin-4-yl)-6-methoxy-7-
(3-(piperidin-1-yl)propoxy)quinazolin-4-amine (27). Compound
27 was prepared as a white solid by the same procedures as the preparation
of compound 17a (75% yield). 1H NMR (400 MHz, CDCl3) δ 6.88 (s,
1H), 6.72 (s, 1H), 4.95 (d, J = 7.0 Hz, 1H), 4.13 (t, J = 6.8 Hz, 2H),
4.10À4.00 (m, 1H), 3.87 (s, 3H), 3.78 (t, J = 6.0 Hz, 4H), 2.93À2.85 (m,
2H), 2.80À2.68 (m, 1H), 2.50À2.42 (m, 2H), 2.41À2.24 (m, 6H), 2.17
(d, J = 10.8 Hz, 2H), 2.0À1.98 (m, 2H), 1.83À1.73 (m, 4H), 1.62À1.49
(m, 10H), 1.45À1.36 (m, 2H), 1.05 (d, J = 6.5 Hz, 6H). 13C NMR (100
MHz, CDCl3) δ 158.81, 158.15, 154.03, 149.98, 145.03, 107.06, 102.70,
101.80, 67.52, 56.82, 55.90, 54.68, 54.60, 48.82, 47.94, 46.97, 32.83, 28.67,
27.54, 26.63, 26.13, 24.59, 18.62. HPLC: 100%; tR 3.06 min. MS (ESI): 539
[M + H]+.
1
the preparation of compound 17a (81% yield). H NMR (400 MHz,
CDCl3) δ 6.87 (s, 1H), 6.72 (s, 1H), 4.99 (d, J = 7.1 Hz, 1H), 4.11 (t, J =
6.8 Hz, 2H), 4.08À3.98 (m, 1H), 3.93À3.88 (m, 2H), 3.87À3.78 (m,
5H), 3.17À3.00 (m, 1H), 2.96À2.85 (m, 3H), 2.78À2.70 (m, 2H),
2.60À2.52 (m, 2H), 2.49À2.23 (m, 8H), 2.14 (d, J = 11.1 Hz, 2H),
2.07À1.98 (m, 2H), 1.94À1.71 (m, 6H), 1.66À1.47 (m, 7H), 1.45À1.35
(m, 2H), 1.22 (p, J = 11.7 Hz, 4H), 1.15À1.03 (m, 1H), 0.98 (d, J = 6.6
Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 158.75, 158.12, 154.00, 149.85,
145.12, 107.05, 102.78, 101.72, 67.50, 63.86, 56.75, 55.86, 55.20, 54.66,
52.27, 50.69, 48.82, 48.27, 47.78, 45.77, 32.90, 29.05, 28.79, 26.59, 26.47,
26.14, 26.10, 24.56, 18.61. HPLC: 100%;tR 2.25 min. MS (ESI): 622[M +
H]+. HRMS (ESI) calcd for C36H60N7O2 [M + H]+, 622.4808; found,
622.4810.
ALogP Calculation. ALogP values were calculated using Pipeline
Pilot v7.5 (Accelrys Software Inc., San Diego, 2009).
Experimental Procedures for SAHH-Coupled Assays. The
previously described enzyme-coupled assay38 was optimized and em-
ployed to assay the activity of histone methyltransferases. This assay
utilizes SAHH to hydrolyze the methyltransfer product SAH to homo-
cysteine and adenosine in the presence of adenosine deaminase, which
converts adenosine to inosine. The homocysteine concentration is then
determined through conjugation of its free sulfhydryl moiety to a thiol-
sensitive fluorophore, ThioGlo (Calbiochem). For IC50 determinations,
assay mixtures were prepared in 25 mM potassium phosphate buffer pH
7.5, 1 mM EDTA, 2 mM MgCl2, 0.01% Triton X-100 with 5 μM SAHH,
0.3 U/mL of adenosine deaminase from Sigma, 25 μM SAM, and 15 μM
ThioGlo. G9a, GLP, SETD7, SETD8, PRMT3, and SUV39H2 were
assayed at 25, 100, 200, 250, 500, and 100 nM, respectively. Inhibitors
were added at concentrations ranging from 4 to 16 μM. After 2 min
incubation, reactions were initiated by addition of the histone peptides:
10 μM H3(1À25) for G9a, 20 μM H3(1À25) for GLP, 100 μM
H3(1À25) for SETD7, 500 μM H4(1À24) for SETD8, 10 μM
H4(1À24) for PRMT3, and 200 μM H3K9Me1 (1À15) for SUV39H2.
The methylation reaction was followed by monitoring the increase in
fluorescence using Biotek Synergy2 plate reader with 360/40 nm
excitation filter and 528/20 nm emission filter for 20 min in 384-well
plate format. Activity values were corrected by subtracting background
caused by the peptide or the protein. IC50 values were calculated using
Sigmaplot. Standard deviations were calculated from two independent
experiments.
Experimental Procedures for ICW and MTT Assays. MDA-
MB-231, PC3, and HCT116 cells were cultured in RPMI with 10% FBS,
22RV1 cells in alphaMEM and 10% FBS, MCF7 and IMR90 cells in
DMEM with 10% FBS. Cells were treated with inhibitors for 48 h.
In-Cell Western (ICW) Assay. Cells were seeded at 5000À10000
cells in black-walled 96-well plates (Greiner) and exposed to various
inhibitor concentrations for 48 h. The media was removed and 2%
formaldehyde in PBS for fixation was added for 15 min. After five washes
with 0.1% Triton X100 in PBS, cells were blocked for 1 h with 1% BSA in
PBS. Three out of four replicates were exposed to the primary H3K9me2
antibody, Abcam no. 1220 at 1/800 dilution in 1% BSA, PBS for 2 h. One
replicate was reserved for the background control. The wells were
washed five times with 0.1% Tween 20 in PBS, then secondary IR800
conjugated antibody (LiCor) and nucleic acid-intercalating dye, DRAQ5
(LiCor) added for 1 h. After 5 washes with 0.1% Tween 20 PBS, the plates
were read on an Odyssey (LiCor) scanner at 800 nm (H3K9me2 signal)
and 700 nm (DRAQ5 signal). Fluorescence intensity was quantified,
normalized to the background, then to the DRAQ5 signal, and expressed
as a percentage of control. IC50s were calculated using GraphPad Prizm
statistical package with sigmoidal variable slope dose response curve fit.
MTT Assay. The media was removed and replaced with DMEM
10% FBS without phenol red supplemented with 1 mg/mL of MTT and
N-(1-(Cyclohexylmethyl)piperidin-4-yl)-2-(4-isopropyl-1,4-
diazepan-1-yl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quin-
azolin-4-amine (7). Compound 7 was prepared as a white solid by the
1
same procedures as the preparation of compound 17a (81% yield). H
NMR (400 MHz, CDCl3) δ 6.88 (s, 1H), 6.72 (s, 1H), 4.97 (d, J = 6.9 Hz,
1H), 4.13 (t, J = 6.8 Hz, 2H), 4.10À4.00 (m, 1H), 3.94À3.88 (m, 2H),
3.88À3.79 (m, 5H), 2.97À2.88 (m, 1H), 2.88À2.81 (m, 2H), 2.80À2.71
(m, 2H), 2.62À2.54 (m, 2H), 2.50À2.42 (m, 2H), 2.41À2.32 (m, 4H),
2.19À1.98 (m, 8H), 1.96À1.85 (m, 2H), 1.82À1.36 (m, 14H), 1.30À1.09
(m, 3H), 0.99 (d, J = 6.6 Hz, 6H), 0.93À0.80 (m, 2H). 13C NMR (100
MHz, CDCl3) δ 158.79, 158.16, 154.05, 149.87, 145.17, 107.11, 102.81,
101.69, 67.55, 65.89, 56.78, 55.90, 55.23, 54.70, 53.30, 52.28, 50.73, 48.59,
47.80, 45.79, 35.57, 32.45, 32.10, 28.82, 26.95, 26.64, 26.31, 26.15, 24.61,
18.65. HPLC: 99%; tR 2.56 min. MS (ESI): 636 [M + H]+. HRMS (ESI)
calcd for C37H62N7O2 [M + H]+, 636.4965; found, 636.4962.
2-(4-Isopropyl-1,4-diazepan-1-yl)-N-(1-isopropylpiperidin-
4-yl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-amine
(25). Compound 25 was prepared as a white solid by the same procedures
as the preparation of compound 17a (78% yield). 1H NMR (400 MHz,
CDCl3) δ6.87 (s, 1H), 6.72 (s, 1H), 4.98 (d, J= 7.1 Hz, 1H), 4.12 (t, J=6.8
Hz, 2H), 4.09À3.99 (m, 1H), 3.95À3.88 (m, 2H), 3.88À3.78 (m, 5H),
2.97À2.83 (m, 3H), 2.78À2.68 (m, 3H), 2.60À2.53 (m, 2H), 2.49À2.40
(m, 2H), 2.40À2.24 (m, 6H), 2.15 (d, J = 11.1 Hz, 2H), 2.08À1.98 (m,
2H), 1.96À1.84 (m, 2H), 1.61À1.48 (m, 6H), 1.45À1.36 (m, 2H), 1.05
(d, J = 6.5 Hz, 6H), 0.98 (d, J = 6.5 Hz, 6H). 13C NMR (100 MHz, CDCl3,
two overlapping peaks) δ 158.76, 158.14, 154.03, 149.86, 145.14, 107.07,
102.77, 101.74, 67.51, 56.77, 55.87, 55.21, 54.67, 54.58, 52.28, 50.70, 48.76,
47.90, 47.80, 45.78, 32.77, 28.80, 26.60, 26.11, 24.57, 18.61. HPLC: 98%; tR
1.40 min. MS (ESI): 582 [M + H]+.
N-(1-Cyclohexylpiperidin-4-yl)-6-methoxy-2-(4-methyl-1,4-
diazepan-1-yl)-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-amine
(26). Compound 26 was prepared as a white solid by the same procedures
as the preparation of compound 17a (83% yield). 1H NMR (400 MHz,
CDCl3) δ6.87 (s, 1H), 6.72 (s, 1H), 5.03 (d, J= 7.1 Hz, 1H), 4.11 (t, J=6.8
Hz, 2H), 4.07À3.98 (m, 1H), 3.97À3.90 (m, 2H), 3.88À3.79 (m, 5H),
3.27À3.08 (m, 1H), 2.96À2.86 (m, 2H), 2.70À2.62 (m, 2H), 2.58À2.50
(m, 2H), 2.48À2.24 (m, 11H), 2.13 (d, J = 11.0 Hz, 2H), 2.06À1.93 (m,
4H), 1.90À1.71 (m, 4H), 1.66À1.48 (m, 7H), 1.46À1.33 (m, 2H), 1.22
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Journal of Medicinal Chemistry
ARTICLE
incubated for 1À2 h. Live cells reduce yellow MTT to purple formazan.
The resulting formazan was solubilized in acidified isopropyl alcohol and
1% Triton. Formazan signal absorbance was measured at 570 nm and
corrected for the 650 nm background. The data was analyzed as above.
Experimental Procedures for the p53 K373me2 Assay.
H1299 cells were transfected with 1 μg p53 expressing vector and 11 μg
G9a or GLP expressing vector. Immediately after transfection, cells were
treated with 0.25 μM of compound 5 and incubated for 24 h. Cells were
then lysed in NET buffer (50 mM Tris-HCl, pH 8.0, 0.1% NP40, 5 mM
EDTA, 150 mM NaCl with protease inhibitors) and followed by immu-
noprecipitation with DO1 antibody, which recognizing the total p53.
Protein A agarose beads were added to collect the immunoprecipitated
complex. The immunoprecipitated complex was subject to the Western blot
analysis using p53 K373me2 antibody. For p53 and G9a/GLP detection,
whole cell lysate was used.
methyltransferase 1; ES cells, embryonic stem cells; tox/function
ratio, ratio of toxicity to functional potency; SAHH, S-adenosyl-L-
homocysteine hydrolase; ICW, in-cell western; MTT, 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PRMT,
protein arginine methyltransferase; p53 K373me2, dimethylation
of p53 lysine 373; SAH, S-adenosyl-L-homocysteine
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’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
compounds 6 and 7. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 919-843-8459. Fax: 919-843-8465. E-mail: jianjin@
unc.edu.
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Present Addresses
^Viamet Pharmaceuticals, Inc., 2250 Perimeter Park Drive, Suite
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Author Contributions
These authors contributed equally to this work.
’ ACKNOWLEDGMENT
We thank Dr. Ryan Trump for helpful discussions, Dr. Cen Gao
for ALogP calculation, and Dr. R. Bristow for 22RV1 and PC3 cells.
The research described here was supported by the grant
RC1GM090732 from the NIH, the Carolina Partnership and
University Cancer Research Fund from the University of North
Carolina at Chapel Hill, and the Structural Genomics Consortium, a
registered charity (no. 1097737) that receives funds from the
Canadian Institutes of Health Research, the Canada Foundation
for Innovation, Genome Canada through the Ontario Genomics
Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice
Wallenberg Foundation, the Ontario Innovation Trust, the Ontario
Ministry for Research and Innovation, Merck & Co., Inc., the
Novartis Research Foundation, the Swedish Agency for Innovation
Systems, the Swedish Foundation for Strategic Research and the
Wellcome Trust.
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’ ABBREVIATIONS USED
H3K9me2, dimethylation of lysine 9 on histone H3; SAR, structureÀ
activity relationships; PTMs, post-translational modifications;
PKMTs, protein lysine methyltransferases; SAM, S-adenosyl-L-
methionine; SET, suppressor of variegation 3À9, enhancer of zeste,
and trithorax; MBT domains, malignant brain tumor domains;
PHD, plant homeo domain; KMT1C, lysine methyltransferase 1C;
EHMT2, euchromatic histone lysine methyltransferase 2; H3K9,
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