Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase

Article abstract of DOI:10.1021/acs.jmedchem.6b01645

G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including 4 (MS0124) and 18 (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme.

Full text of DOI:10.1021/acs.jmedchem.6b01645

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Article
Discovery of Potent and Selective Inhibitors for
G9a-Like Protein (GLP) Lysine Methyltransferase
Yan Xiong, Fengling Li, Nicolas Babault, Aiping Dong, Hong Zeng, Hong Wu, Xin
Chen, Cheryl H. Arrowsmith, Peter J. Brown, Jing Liu, Masoud Vedadi, and Jian Jin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01645 • Publication Date (Web): 30 Jan 2017
Downloaded from http://pubs.acs.org on February 4, 2017
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Journal of Medicinal Chemistry
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Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine
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Yan Xiong, Fengling Li, Nicolas Babault, Aiping Dong, Hong Zeng, Hong Wu, Xin Chen, Cheryl
H. Arrowsmith,‡ Peter J. Brown, Jing Liu, Masoud Vedadi,* and Jian Jin*,†
,⊥
‡
†
,‡,§
†Department of Pharmacological Sciences and Department of Oncological Sciences, Icahn School of
Medicine at Mount Sinai, New York, New York 10029, United States
‡Structural Genomics Consortium, University of Toronto, Toronto, Ontario, M5G 1L7, Canada
§Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, M5S 1A8,
Canada
⊥Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto,
Toronto, Ontario, M5G 2M9, Canada
ABSTRACT: G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases
(PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a
heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and non-histone
substrates. Although they are closely related, GLP and G9a possess distinct physiological and
pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to
dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual
inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP
inhibitors including 4 (MS0124) and 18 (MS012), which are > 30-fold and 140-fold selective for GLP
over G9a and other methyltransferases respectively. The cocrystal structures of GLP and G9a in the
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complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the
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challenges in structure-based design of selective inhibitors for either enzyme.
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INTRODUCTION
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Protein lysine methyltransferases (PKMTs) catalyze the transfer of the methyl group from the
cofactor S-5′-adenosyl-L-methionine (SAM) to the ε-amino group of the targeted lysine residue in histone
and non-histone substrates, leading to mono-, di- or trimethylation.1-3 Lysine methylation does not change
the charge state of the residue. Instead, this modification increases the bulkiness of the amino group and
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alters the hydrophobicity and hydrogen bond formation potential. As a result, lysine methylation has
dramatic effects on the interactions of the modified protein with other proteins, thus its physiological
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function. Depending on the methylation state and location, histone lysine methylation can lead to either
activation or repression of gene expression and transcription.3-7 In general, methylation of histone H3
lysine 4, 36, and 79 (H3K4, H3K36, and H3K79) is associated with transcription activation and
methylation of histone H3 lysine 9 and 27 (H3K9 and H3K27) is typically a hallmark of transcription
repression.8-11
GLP (also known as euchromatic histone-lysine N-methyltransferase 1 (EHMT1) or lysine
methyltransferase 1D (KMT1D)) and G9a (also known as EHMT2 or KMT1C) are two closely related
PKMTs with approximately 80% sequence identity in their respective suppressor of variegation 3−9
enhancer-of-zeste trithorax (SET) domains.12, 13 It has been shown that the heterodimeric complex of G9a
and GLP is the main functional methyltransferase in mouse embryonic stem cells, depositing mono- and
dimethylated H3K9 (H3K9me1 and H3K9me2).14, 15 Besides H3K9, other histone substrates, such as
histone H1,16, 17 H3K27,18-20 and H3K56, have been reported. G9a and/or GLP can also methylate a
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variety of non-histone proteins, including the tumor suppressor p53, sirtuin 1 (SIRT1), reptin,
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myogenic differentiation 1 (MyoD), chromodomain Y-like protein (CDYL1), widely interspaced zinc
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finger motifs protein (WIZ), and G9a. Furthermore, G9a and/or GLP have been reported to play roles
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tumor cell growth and metathesis,23,
in germ cell development and meiosis, embryo development,
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immune response,34, 35 cocaine-induced plasticity, cognition and adaptive behavior,4, 37 and provirus
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silencing. Dysregulation of G9a and/or GLP has been implicated in many human diseases such as
cancer, inflammatory diseases, and neurogenerative disorder.4, 6, 32, 37, 39, 40
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Although G9a and GLP share high sequence homology and many biological activities, they do
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possess distinct physiological and pathophysiological functions. For example, the ankyrin repeat domain
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of G9a mainly associates with H3K9me2, whereas the ankyrin domain of GLP preferentially associates
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with H3K9me1. Mutations at the ankyrin domain of G9a and GLP result in drastic phenotype differences
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in mice. In addition, G9a and GLP often display distinct tissue-specific expression profiles. Different
from G9a, GLP is highly expressed in brown adipose tissue and controls brown adipose cell fate and
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energy homeostasis. Moreover, G9a and GLP have opposite roles during early steps of skeletal muscle
terminal differentiation. GLP, but not G9a, stabilizes MyoD, which is the major player in triggering
muscle terminal differentiation, by down-regulating proteasome subunit-encoding gene expression.44
Potent and selective small-molecule inhibitors of G9a/GLP are useful tools for investigating
biological functions of these proteins and testing the therapeutic hypotheses associated with them. Over
the last decade, a number of G9a/GLP inhibitors have been reported.12, 13, 45-56 Compound 1 (BIX01294)
was the first selective dual G9a/GLP inhibitor (Figure 1).12 It was discovered via high throughput
screening and is competitive with the peptide substrate and non-competitive with the cofactor SAM.
13
Based on the crystal structure of the GLP–1 complex, our group and others have discovered more potent
G9a/GLP inhibitors including UNC0224,45 UNC0321,46 and 2 (E72).47 Further optimization of this
chemical series led to the discovery of potent, selective and cell-active G9a/GLP dual inhibitors 3
(UNC0638)48, 49, 57 and UNC0642,50 which has sufficient in vivo pharmacokinetic properties and is
suitable for animal studies (Figure 1). Among all of these inhibitors, compound 1 is the only one that has
some selectivity for GLP over G9a (about 3–5 fold).13, 49 Since G9a and GLP are not functionally
redundant and have been shown to have distinct biological functions, G9a or GLP selective inhibitors will
be valuable tools to study distinct functions of these two closely related PKMTs. Here, we describe the
design, synthesis, and biochemical evaluation of new compounds based on the quinazoline scaffold shared
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by compounds 1 – 3. Our structure–activity relationship (SAR) studies resulted in the discovery of potent
and selective GLP inhibitors including 18 (MS012), which is remarkably selective for GLP over G9a (>
140-fold) in biochemical assays.
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RESULTS AND DISCUSSION
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Discovery of 4 (MS0124) as a GLP Selective Inhibitor. To identify selective inhibitors for GLP
or G9a, we screened our quinazoline compound collection against these two enzymes using radioactivity
based scintillation proximity biochemical assays, which measure the potency of test compounds at
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inhibiting the transfer of the methyl group from H-SAM to the peptide substrates. We were pleased to
find compound 4 was 34-fold more selective for GLP (IC50 = 13 ± 4 nM) over G9a (IC50 = 440 ± 63 nM)
(Table 1). We were also able to obtain cocrystal structures of both GLP and G9a in complex with 4 in the
presence of SAM at resolutions of 2.6 Å and 1.95 Å, respectively (Figure 2 and Supporting Table 1).
These two cocrystal structures are extremely similar, with a root-mean-square deviation (RMSD) of 0.31
Å. Compound 4 occupies the peptide substrate binding site with the same binding mode for GLP and G9a.
The interactions between compound 4 and these two enzymes are conserved. For example, the secondary
amino group at the C4 position forms a direct hydrogen bond with the side chain of GLP Asp1171 and
the equivalent G9a Asp1083. In addition, the N1 nitrogen on the pyrimidine ring forms another direct
hydrogen with the side chain of GLP Asp1176 and G9a Asp1088. Moreover, the oxygen atoms of both
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- and 7-methoxy groups interact with a water molecule in both structures.
To improve potency and selectivity of 4, we conducted structure-activity relationship (SAR)
studies at the following four regions: (1) N-capping group of the piperidine moiety, (2) 6-methoxy moiety,
(3) 7-methoxy moiety, and (4) 2-morpholine moiety (Tables 1−3).
Synthesis. We previously developed a two-step synthetic sequence to explore the 2-amino and 4-
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amino regions of the 6,7-dimethoxyquinazoline scaffolds. Using this efficient synthesis, we converted
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the commercially available 2,4-dichloro-6,7-dimethoxyquinazoline 5 to compounds 4 and 7−22 by two
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consecutive chloro displacement reactions (Scheme 1 and Tables 1 and 3).
To investigate the SAR of 6- and 7-methoxy moieties, we synthesized analogs with ethoxy,
isopropoxy, fluoro and methyl groups at C6 and C7 positions (Scheme 2 and Table 2). We developed four
synthetic routes to prepare 4,5-disubtituted-2-cyanoaniline intermediates 29, 30, 35, and 36. Briefly,
etherification of commercially available phenols, 23 and 24, followed by nitration and subsequent nitro
reduction produced 2-cyanoanilines (29 and 30) bearing different alkoxy groups (Scheme 2A). The fluoro
and methyl substituted intermediates, 35 and 36, were synthesized from anilines 31 and 32 through
bromination and subsequent cyanation reactions (Scheme 2B). The 4,5-disubstituted-2-cyanoaniline
intermediates 29, 30, 35, and 36 were then reacted with methyl chloroformate to yield carbamates 37.
After saponification of the cyano group, cyclization reaction afforded quinazolinediones 38. Chlorination
and subsequent two consecutive chloro substitutions provided quinazolines 41−48 (Scheme 2C and Table
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Quinazoline analogs with alkyl groups at the C2 position (53 and 54) were synthesized from
aniline 49. Amide coupling of 49 with acid chlorides provided intermediates 50, which were then
converted to 51 through the saponification and subsequent cyclization reaction. Chlorination followed by
chloro displacement provided 2-alkyl analogs 53 and 54 (Scheme 3 and Table 3).
SAR in GLP and G9a Biochemical Assays. All of the synthesized compounds were evaluated
in the GLP and G9a radioactivity based scintillation proximity biochemical assays. IC50 values of the
compounds are summarized in Tables 1−3.
For the N-capping group of the piperidine moiety, we found that a variety of alkyl groups were
well tolerated for both GLP and G9 (Table 1). Replacing the methyl group (4) with the ethyl (7), n-propyl
(8), i-propyl (9), or cyclopropyl (10) group did not significantly change the potency for either GLP or
G9a. These SAR results are consistent with the crystal structures of GLP and G9a in complex with 4,
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which show the N-capping group is solvent exposed. Since the methyl group in 4 provides slightly better
selectivity and is the smallest substituent, we chose the methyl group as the N-capping group for the
following SAR studies.
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In contrast to the N-capping groups, the 6-methoxy moiety was much more sensitive to structural
changes (Table 2). Displacing the methoxy group with the ethoxy (41) or isopropoxy (42) group totally
diminished their activities against both GLP and G9a (IC50 > 5,000 nM). From the cocrystal structures of
G9a and GLP in complex with 4, the 6-methoxy group occupies a shallow binding groove. A larger group
such as ethoxy or isopropoxy likely clashes with the binding groove, thus decreasing the potency. The
removal of the oxygen atom from the methoxy group and installation of a smaller group also led to less
potent analogs. In addition, GLP was more sensitive to these structural changes than G9a. For example,
6-fluoro group (43) decreased the potency for GLP (IC50 = 1,230 ± 340 nM) by 95-fold and for G9a (IC50
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3,690 ± 1,780 nM) by 8-fold. Similarly, 6-methyl group (44) led to a 62-fold potency drop for GLP
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cocrystal structures.
The SAR of the C7 substituents followed the similar trend as the 6-substituents (Table 2). The
ethoxy (45) or isopropoxy (46) group abolished the activities against both GLP and G9a. From the
cocrystal structures, the 7-methoxy group is located at the neck of the lysine binding channel and also
interacts with the water molecule. In our previous report, the pyrrolidin-1-yl-propoxy group at this
position greatly increased G9a potency by forming an addition hydrogen bond with Leu1086 and cation-
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π interaction with Tyr1154. The drastic potency loss of the ethoxy and isopropoxy analogs highlights
the importance of the interactions between the basic amino group and Leu1086 and Tyr1154. We also
found that, while the fluoro group (47) completely abolished the activities against both GLP and G9a, the
methyl group (48) was tolerated with only about 2−3 fold potency loss for GLP (IC50 = 29 ± 12 nM) and
G9a (IC50 = 1,150 ± 47 nM).
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Having established initial SAR for the N-capping group, and 6- and 7-methoxy regions, we next
explored the 2-morpholine moiety using cyclic amines (11−15), cyclic alkyl groups (53 and 54), and
acyclic amines (16−22). We first compared the pyrrolidinyl (11), piperidinyl (12) and azepanyl (14)
analogs and found the effect of the ring size on the potency for GLP was minimal with IC50 values of 12
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1 nM, 12 ± 1 nM, and 16 ± 3 nM, respectively. However, the ring size was more important for
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modulating G9a potency. Among the three groups, the 5-membered pyrrolidinyl group (11) led to the
weakest G9a inhibition (IC50 = 313 ± 133 nM) and the six-membered piperidinyl group (12) resulted in
the most potent G9a inhibitor (IC50 = 31 ± 9 nM). Substitutions on the cyclic amino groups also exhibited
a drastic impact on the potency for both GLP and G9a. Compared with the piperidinyl analog 12, the 4,4-
difluoro substituted piperidinyl group (13) decreased potency for GLP by 8-fold and for G9a by 34-fold.
Compared with the azepanyl analog 14, the methylhomopiperazinyl analog 15 increased potency for GLP
by 4-fold and G9a by 21-fold. Replacing the 2-cycloamino group with a 2-cycloalkyl group led to much
less potent compounds for both GLP and G9a. For example, compound 53 was 3-fold less potent for GLP
and 14-fold less potent for G9a, compared with 12. In addition, compound 54 was 26-fold potent for GLP
and 4-fold potent for G9a, compared with 4. These SAR results indicate the important role of the 2-amino
group for maintaining high potency for both GLP and G9a. We next explored a number of acyclic alkyl
amines (16−22) at the 2-position. For the secondary amine analogs 16−19, the length of the alkyl chain
had the most profound effect on the potency for GLP over G9a. We were delighted to find that compound
18, which contains a 2-hexylamino group, was > 140-fold selective for GLP over G9a, representing the
most selective GLP inhibitor to date. The tertiary amine analogs 20−22, which possess an extra N-methyl
substitute, did not improve potency or selectivity for GLP over G9a.
Characterization of 4 and 18 in Selectivity, Biophysical and Mechanism of Action Studies
(MOA). To further assess the selectivity of 4 and 18, we tested them against 29 protein lysine
methyltransferases (PKMTs), protein arginine methyltransferases (PRMTs), DNA methyltransferases
(DNMTs), and RNA methyltransferases (RNMTs). We found that 4 did not inhibit any of these
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methyltransferases at 10 µM (Figure 3A). While 18 did not inhibit any of these methyltransferase at 1
µM, it displayed modest inhibition (57%) of the N6-adenosine-methyltransferase subunit METTL3 and
14 complex (METTL3-14) at 10 µM (Figure 3B).
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
We next confirmed bindings of 4 and 18 to GLP and G9a using isothermal titration calorimetry
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
ITC) in the presence of SAM (Figure 4). The binding of 4 and 18 to GLP and G9a is enthalpy driven.
Compound 4 displayed higher binding affinity to GLP (K = 40 ± 5 nM) than G9a (K = 445 ± 40 nM)
Figure 4A and 4B). Similarly, compound 18 also exhibited stronger binding affinity to GLP (K = 46 ±
= 610 ± 68 nM) (Figure 4C and 4D). Thus, in ITC biophysical assays, 4 and 18 were
d
d
(
d
1
1
5 nM) than G9a (K
d
1-fold and 13-fold selective for GLP over G9a respectively.
To assess MOA of 4 and 18, we evaluated the effect of peptide and SAM concentrations on IC50
values of 4 and 18 against GLP. As illustrated in Figures 5A and 5C, increasing the peptide substrate
concentration significantly increased the IC50 values of 4 and 18, indicating that both compounds are
peptide competitive. As illustrated in Figures 5B and 5D, increasing the SAM concentration decreased
the IC50 values of 4 and 18, indicating that both compounds are SAM uncompetitive.
Cocrystal Structures of 18. We solved the ternary crystal structures of GLP and G9a in complex
with 18 in the presence of the cofactor SAM (Figure 6A-C, and Supporting Table 2). Similar to 4,
compound 18 conserved three key hydrogen bonds with GLP and G9a: (1) the secondary amino group at
the 4-position interacts with GLP Asp1171 and G9a Asp1083; (2) the N1 nitrogen on the quinazoline ring
interacts with GLP Asp1176 and G9a Asp1088; (3) the oxygen atoms of both 6- and 7-methoxy groups
interact weakly with a water molecule in both GLP and G9a structures. Interestingly, the 2-hexylamino
group of 18 points to a different direction compared to the 2-(3’-dimethylamino)propylamino group of
4
7
47
compound 2 in the cocrystal structure of GLP-SAH-2 (Figure 6D). The salt bridge formed between
the 3’-dimethylamino group of 2 and Asp1131 likely leads to the 2-(3’-dimethylamino)propylamino side
chain adapting to the different geometry. Notably, the crystal structures of GLP and G9a in the complex
with 4 or 18 revealed the virtually identical inhibitor binding modes and ligand–protein interactions, and
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Journal of Medicinal Chemistry
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did not provide insight for the structural basis of the high selectivity for GLP over G9a. We are currently
conducting additional studies to understand the structural basis of the observed high selectivity and will
report our progress in future publications.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Lastly, we assessed membrane permeability of compound 18 in a Caco-2 cell permeability assay
and found that 18 had poor membrane permeability (5.0 nm/s) and high efflux ratio (80). Thus, 18 is not
suitable for cellular studies. Future work will attempt to improve membrane permeability and reduce
efflux while maintaining the high selectivity for GLP over G9a.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSIONS
By testing our quinazoline compound collection, we identified compound 4 as a GLP selective
inhibitor. We next conducted SAR studies and explored four regions of 4, which led to the discovery of
compound 18, which is > 140-fold selective for GLP over G9a. We confirmed the bindings of 4 and 18
to GLP and G9a by ITC. Both 4 and 18 are also selective for GLP over a broad range of other PKMTs,
PRMTs, DNMTs, and RNMTs. We also obtained high-resolution X-ray cocrystal structures of GLP and
G9a in complex with either 4 or 18, which further confirmed the bindings of these inhibitors to GLP and
G9a and revealed that the inhibitors bind to GLP and G9a in virtually identical binding modes. This work
will help develop high-quality chemical probes of GLP.
EXPERIMENTAL SECTION
Chemistry General Procedures. HPLC spectra for all compounds were acquired using an Agilent 1200
Series system with DAD detector. Chromatography was performed on a 2.1×150 mm Zorbax 300SB-C18
5
μm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic
acid as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0−1 min),
−99% B (1−4 min), and 99% B (4−8 min). High-resolution mass spectra (HRMS) data were acquired in
1
positive ion mode using an Agilent G1969A API-TOF with an electrospray ionization (ESI) source.
Nuclear Magnetic Resonance (NMR) spectra were acquired on a Bruker DRX-600 spectrometer (600
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Journal of Medicinal Chemistry
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13
1
13
MHz H, 150 MHz C) or a Varian Mercury spectrometer (400 MHz H, 100 MHz C). Chemical shifts
are reported in ppm (δ). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector
set to 254 nm. Samples were injected into a Phenomenex Luna 75 x 30 mm, 5 μm, C18 column at room
temperature. The flow rate was 40 mL/min. A linear gradient was used with 10% (or 50%) of MeOH (A)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in H
2
O (with 0.1 % TFA) (B) to 100% of MeOH (A). HPLC was used to establish the purity of target
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compounds. All final compounds had > 95% purity using the HPLC methods described above.
6
2
0
,7-Dimethoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (4) To a solution of
,4-dichloro-6,7-dimethoxyquinazoline (50 mg, 0.19 mmol) in DMF (2 mL) were added K CO (55 mg,
.40 mmol) and 4-methylpiperidin-1-amine (0.05 mL, 0.40 mmol). After being stirred for 2 h, the reaction
2
3
was quenched with water and extracted with DCM (3 x 5 mL). The combined organic layers were dried
over anhydrous Na SO , filtered, and concentrated under reduced pressure. The residue was dissolved in
2
4
i-PrOH (1 mL) and treated with HCl (0.1 mL, 4 M in dioxane, 0.4 mmol) and morpholine (0.03 mL, 0.34
mmol). The solution was heated in a microwave reactor at 160 °C for 15 min before being concentrated
under reduced pressure. The resulting residue was purified by preparative HPLC to yield the title
1
compound (60 mg, 82% over 2 steps). H NMR (600 MHz, CDCl
3
) δ 6.92 (s, 1H), 6.79 (s, 1H), 5.21 (d,
J = 7.2 Hz, 1H), 4.17-4.12 (m, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 3.83-3.79 (m, 8H), 2.90 (d, J = 10.8 Hz,
1
3
2H), 2.33 (s, 3H), 2.21-2.15 (m, 4H), 1.71-1.61 (m, 2H); C NMR (151 MHz, CDCl
3
) δ 158.95, 158.32,
1
54.45, 149.05, 145.68, 106.11, 103.39, 100.69, 67.10, 56.32, 55.98, 54.80, 47.75, 46.20, 44.67, 32.13.
+
HRMS(ESI-TOF) m/z: [M+H] calcd for C20
H N
30 5
3
O , 388.2343, found 388.2382.
N-(1-Ethylpiperidin-4-yl)-6,7-dimethoxy-2-morpholinoquinazolin-4-amine (7). The title compound
1
(
75% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz, CDCl
3
) δ
6
.87 (s, 1H), 6.73 (s, 1H), 5.13 (d, J = 7.6 Hz, 1H), 4.08-4.05 (m, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.78-
3.73 (m, 8H), 2.93 (d, J = 12.0 Hz, 2H), 2.41 (q, J = 6.8 Hz, 2H), 2.14-2.08 (m, 4H), 1.63-1.54 (m, 2H),
.07 (q, J = 7.2 Hz, 3H); MS (ESI) m/z 402.3 [M+H]+.
1
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6
,7-Dimethoxy-2-morpholino-N-(1-propylpiperidin-4-yl)quinazolin-4-amine (8). The title compound
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
(76% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz, CDCl
3
) δ
6.89 (s, 1H), 6.74 (s, 1H), 5.12 (d, J = 7.2 Hz, 1H), 4.13-4.10 (m, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.81-
3
0
.74 (m, 8H), 2.92 (d, J = 11.6 Hz, 2H), 2.30 (q, J = 6.8 Hz, 2H), 2.15-2.11 (m, 4H), 1.61-1.47 (m, 4H),
.90 (q, J = 7.2 Hz, 3H); MS (ESI) m/z 416.1 [M+H]+.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N-(1-Isopropylpiperidin-4-yl)-6,7-dimethoxy-2-morpholinoquinazolin-4-amine (9). The title
1
compound (85% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl ) δ 6.88 (s, 1H), 6.78 (s, 1H), 5.24 (d, J = 7.2 Hz, 1H), 4.12-4.03 (m, 1H), 3.91 (s, 3H), 3.89 (s,
3
3
H), 3.81-3.74 (m, 8H), 2.91 (d, J = 12.0 Hz, 2H), 2.81-2.75 (m, 1H), 2.33 (t, J = 10.8 Hz, 2H), 2.16-2.14
(
m, 2H), 1.67-1.58 (m, 2H), 1.06 (d, J = 6.8 Hz, 6H); MS (ESI) m/z 416.3 [M+H]+.
N-(1-Cyclopropylpiperidin-4-yl)-6,7-dimethoxy-2-morpholinoquinazolin-4-amine (10). The title
1
compound (77% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 6.91 (s, 1H), 6.70 (s, 1H), 5.01 (d, J = 6.8 Hz, 1H), 4.18-4.08 (m, 1H), 3.95 (s, 3H), 3.92 (s,
3
1
4
H), 3.83-3.77 (m, 8H), 3.04 (d, J = 12.4 Hz, 2H), 2.39 (td, J = 11.6, 3.6 Hz, 2H), 2.15-2.11 (m, 2H),
.66-1.60 (m, 1H), 1.54 (qd, J = 11.2, 4.0 Hz, 2H), 0.49-0.45 (m, 2H), 0.44-0.40 (m, 2H); MS (ESI) m/z
14.3 [M+H]+.
6
,7-Dimethoxy-N-(1-methylpiperidin-4-yl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (11). The title
1
compound (80% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 6.92 (s, 1H), 6.78 (s, 1H), 5.12 (d, J = 6.8 Hz, 1H), 4.19-4.09 (m, 1H), 3.91 (s, 3H), 3.89 (s,
3H), 3.64-3.60 (m, 4H), 2.85 (d, J = 12.0 Hz, 2H), 2.30 (s, 3H), 2.18-2.13 (m, 4H), 1.96-1.93 (m, 4H),
.64-1.55 (m, 2H); MS (ESI) m/z 372.3 [M+H]+.
1
6
,7-Dimethoxy-N-(1-methylpiperidin-4-yl)-2-(piperidin-1-yl)quinazolin-4-amine (12). The title
1
compound (80% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
MeOH-d4) δ 7.64 (s, 1H), 7.11 (s, 1H), 4.58-4.46 (m, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 3.87-3.81 (m, 4H),
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.63 (d, J = 12.8 Hz, 2H), 3.26-3.19 (m, 1H), 2.89 (s, 3H), 2.34 (d, J = 12.9 Hz, 2H), 2.10-1.96 (m, 2H),
.82-1.66 (m, 7H); MS (ESI) m/z 386.3 [M+H]+.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2-(4,4-Difluoropiperidin-1-yl)-6,7-dimethoxy-N-(1-methylpiperidin-4-yl)quinazolin-4-amine (13).
1
The title compound (84% over 2 steps) was prepared according to synthetic procedure for 4. H NMR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(400 MHz, CDCl
3
) δ 6.89 (s, 1H), 6.71 (s, 1H), 5.01 (d, J = 7.2 Hz, 1H), 4.15-4.05 (m, 1H), 3.99 (t, J =
6.0 Hz, 4H), 3.96 (s, 3H), 3.95 (s, 3H), 2.89-2.86 (m, 2H), 2.33 (s, 3H), 2.22-2.13 (m, 4H), 2.06-1.96 (m,
H), 1.69-1.59 (m, 2H); MS (ESI) m/z 422.3 [M+H]+.
4
2
-(Azepan-1-yl)-6,7-dimethoxy-N-(1-methylpiperidin-4-yl)quinazolin-4-amine (14). The title
1
compound (79% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl ) δ 6.88 (s, 1H), 6.72 (s, 1H), 4.97 (d, J = 6.4 Hz, 1H), 4.12-4.05 (m, 1H), 3.93 (s, 3H), 3.91 (s,
3
3
H), 3.78 (t, J = 6.0 Hz, 4H), 2.88-2.85 (m, 2H), 2.33 (s, 3H), 2.17-2.13 (m, 4H), 1.82-1.74 (m, 4H), 1.64-
1.53 (m, 6H); MS (ESI) m/z 400.3 [M+H]+.
6
,7-Dimethoxy-2-(4-methyl-1,4-diazepan-1-yl)-N-(1-methylpiperidin-4-yl)quinazolin-4-amine (15).
1
The title compound (78% over 2 steps) was prepared according to synthetic procedure for 4. H NMR
(400 MHz, MeOH-d4) δ 7.38 (s, 1H), 6.89 (s, 1H), 4.15 – 4.07 (m, 1H), 3.93 – 3.89 (m, 2H), 3.88 (s, 3H),
3
2
.87 (s, 3H), 3.82 (t, J = 6.4 Hz, 2H), 2.94 (d, J = 12.1 Hz, 2H), 2.76-2.70 (m, 2H), 2.60-2.57 (m, 2H),
.34 (s, 3H), 2.30 (s, 3H), 2.17 (td, J = 12.1, 2.2 Hz, 2H), 2.12-2.04 (m, 2H), 2.04-1.96 (m, 2H); MS (ESI)
m/z 415.3 [M+H]+.
2
4
N -Hexyl-6,7-dimethoxy-N -(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (16). The title
1
compound (85% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 6.84 (s, 1H), 6.82 (s, 1H), 5.32 (d, J = 6.8 Hz, 1H), 4.79 (br, 1H), 4.13-4.11 (m, 1H), 3.87 (s,
3
1
H), 3.85 (s, 3H), 3.45- 3.40 (m, 2H), 2.83 (d, J = 11.6 Hz, 2H), 2.28 (s, 3H), 2.15- 2.10 (m, 4H), 1.62-
.57 (m, 4H), 1.45-1.37 (m, 2H). 0.93 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 374.3 [M+H]+.
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4
2
6
,7-Dimethoxy-N -(1-methylpiperidin-4-yl)-N -pentylquinazoline-2,4-diamine (17). The title
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
compound (76% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl ) δ 6.83 (s, 1H), 6.82 (s, 1H), 5.40 (d, J = 5.2 Hz, 1H), 4.96 (br, 1H), 4.16-4.07 (m, 1H), 3.87 (s,
3
3
H), 3.84 (s, 3H), 3.39 (q, J = 6.8 Hz, 2H), 2.84-2.81 (m, 2H), 2.26 (s, 3H), 2.14-2.08 (m, 4H), 1.63-1.54
(
m, 4H), 1.34-1.31 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 388.2 [M+H]+.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4
N -Hexyl-6,7-dimethoxy-N -(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (18). The title
1
compound (81% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 6.86 (s, 1H), 6.76 (s, 1H), 5.14 (d, J = 7.2 Hz, 1H), 4.77 (br, 1H), 4.15-4.12 (m, 1H), 3.92 (s,
3
4
H), 3.90 (s, 3H), 3.46-3.40 (q, J = 6.8 Hz, 2H), 2.50 (d, J = 12.0 Hz, 2H), 2.30 (s, 3H), 2.18-2.12 (m,
+
13
H), 1.60-1.56 (m, 4H), 1.32-1.28 (m, 6H), 0.88 (t, J = 7.2 Hz, 3H); MS (ESI): 402 [M+H] ; C NMR
(151 MHz, CDCl
3
) δ 158.68, 158.39, 154.77, 145.62, 104.35, 103.15, 101.36 (2C), 56.48, 56.12, 54.81,
+
4
7.97, 46.27, 41.58, 32.19, 31.68, 30.01, 26.79, 22.66, 14.10. HRMS(ESI-TOF) m/z: [M+H] calcd for
C
22
H
36
N
5
O
2
, 402.2864, found 402.2896.
2
4
N -Heptyl-6,7-dimethoxy-N -(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (19). The title
1
compound (75% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400 MHz,
CDCl
3.85 (s, 3H), 3.82 (s, 3H), 3.39 (dd, J = 12.9, 6.9 Hz, 2H), 2.81 (d, J = 11.9 Hz, 2H), 2.25 (s, 3H), 2.17-
.98 (m, 4H), 1.67-1.48 (m, 4H), 1.43-1.14 (m, 8H), 0.83 (t, J = 6.9 Hz, 3H); MS (ESI) m/z 416.2
3
) δ 6.84 (s, 1H), 6.82 (s, 1H), 5.46 (d, J = 6.7 Hz, 1H), 4.95-4.80 (m, 1H), 4.21- 4.03 (m, 1H),
1
[
M+H]+.
2
2
N -Butyl-6,7-dimethoxy-N -methyl-N4-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (20). The
1
title compound (83% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400
MHz, CDCl
3
) δ 6.90 (s, 1H), 6.74 (s, 1H), 5.02 (d, J = 7.0 Hz, 1H), 4.10 (dt, J = 11.0, 5.3 Hz, 1H), 3.93
(s, 3H), 3.91 (s, 3H), 3.79-3.55 (t, J = 7.2 Hz, 2H), 3.18 (s, 3H), 2.86 (t, J = 10.2 Hz, 2H), 2.31 (s, 3H),
2
.16-2.11 (m, 4H), 1.72-1.48 (m, 4H), 1.44-1.27 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 388.3
[
M+H]+.
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6
,7-Dimethoxy-N -methyl-N -(1-methylpiperidin-4-yl)-N2-pentylquinazoline-2,4-diamine (21). The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
title compound (79% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400
MHz, CDCl
3
) δ 6.88 (s, 1H), 6.77 (s, 1H), 5.10 (d, J = 6.8 Hz, 1H), 4.13-4.03 (m, 1H), 3.90 (s, 3H), 3.87
(
s, 3H), 3.62 (t, J = 7.2 Hz, 2H), 3.17 (s, 3H), 2.85 (d, J = 12.0 Hz, 2H), 2.29 (s, 3H), 2.15-2.09 (m, 4H),
1
.64-1.54 (m, 4H), 1.37-1.23 (m, 4H), 0.88 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 402.3 [M+H]+.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
N -Hexyl-6,7-dimethoxy-N -methyl-N4-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (22). The
1
title compound (82% over 2 steps) was prepared according to synthetic procedure for 4. H NMR (400
MHz, CDCl
3
) δ 6.88 (s, 1H), 6.78 (s, 1H), 5.13 (d, J = 6.8 Hz, 1H), 4.13-4.03 (m, 1H), 3.90 (s, 3H), 3.87
(
s, 3H), 3.62 (t, J = 7.2 Hz, 2H), 3.17 (s, 3H), 2.85 (d, J = 12.0 Hz, 2H), 2.29 (s, 3H), 2.15-2.09 (m, 4H),
1
.64-1.54 (m, 4H), 1.37-1.23 (m, 6H), 0.88 (t, J = 7.2 Hz, 3H); MS (ESI) m/z 416.3 [M+H]+.
2
-Amino-5-ethoxy-4-methoxybenzonitrile (29a). The title compound (50% over 3 steps) was prepared
1
according to synthetic procedure for 30a. H NMR (600 MHz, CDCl
3
) δ 6.78 (s, 1H), 6.23 (s, 1H), 4.30-
4
.25 (br, 2H), 3.96 (q, J = 7.1 Hz, 2H), 3.81 (s, 3H), 1.40 (t, J = 7.0 Hz, 3H).
2
-Amino-5-isopropoxy-4-methoxybenzonitrile (29b). The title compound (56% over 3 steps) was
1
prepared according to synthetic procedure for 30a. H NMR (600 MHz, CDCl
3
) δ 6.88 (s, 1H), 6.88 (s,
1
H), 6.24 (s, 1H), 4.33-4.29 (m, 1H), 4.26-4.22 (br, 2H), 3.83 (s, 3H), 1.32 (d, J = 6.1 Hz, 6H).
2
-Amino-4-ethoxy-5-methoxybenzonitrile (30a). To a solution of 4-hydroxy-3-methoxybenzonitrile
CO
2.77 g, 20 mmol). The mixture was refluxed overnight. After removal of the solvent, the residue was
suspended in water (50 mL) and extracted with ethyl acetate (3 x 50 mL). The combined organic phase
was dried over anhydrous Na SO , filtered, and concentrated under reduced pressure to provide
intermediate 26a. To a solution of intermediate 26a in Ac O (50 mL) was added HNO (3.6 mL, 39 mmol)
(1.49 g, 10 mmol) in acetone (50 mL) were added ethyl bromide (1.4 mL, 19 mmol), followed by K
2
3
(
2
4
2
3
slowly at 0 °C. The solution was stirred at rt overnight, during which time solid was precipitated out. The
reaction mixture was poured into ice water and filtered. The solid was collected and dried to yield the
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Journal of Medicinal Chemistry
Page 16 of 50
intermediate 28a (2.09 g, 94%) as yellow solid. To a suspension of 28a (2.09 g, 9.4 mmol) in a mixture
of water (50 mL) and ethyl acetate (50 mL) were added iron powder (2.10 g, 37.6 mmol) and NH OAc
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
(4.43 g, 57.4 mmol). The resulting mixture was heated under reflux for 2 h. After cooling to rt, the mixture
was filtered and the filtrate was extracted with ethyl acetate. The organic layer was dried with anhydrous
Na
2
SO
4
, filtered, and concentrated under reduced pressure to yield the title compound (1.06 g, 60%) as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
brown solid. H NMR (400 MHz, CDCl
3
) δ 6.84 (s, 1H), 6.17 (s, 1H), 5.80-5.50 (br, 2H), 4.06 (q, J = 7.0
Hz, 2H), 3.80 (s, 3H), 1.46 (t, J = 7.0 Hz, 3H).
2
-Amino-4-isopropoxy-5-methoxybenzonitrile (30b). The title compound (55% over 3 steps) was
1
prepared according to synthetic procedure for 30a. H NMR (400 MHz, CDCl
3
) δ 6.86 (s, 1H), 6.19 (s,
1
H), 5.72-5.55 (br, 2H), 4.58-4.52 (m, 1H), 3.79 (s, 3H), 1.39 (d, J = 6.1 Hz, 6H).
2
-Bromo-5-methoxy-4-methylaniline (33a). To a solution of 3-methoxy-4-methylaniline (420 mg, 3.1
NBr (1.56 g, 3.2 mmol) at 0 °C. After being stirred at rt
overnight, the reaction was quenched with saturated aqueous NaHCO solution (10 mL). The organic
mmol) in ethyl acetate (15 mL) was added Bu
3
3
3
layer was separated and dried with anhydrous Na
2
SO
4
. After removal of the solvent, the resulting residue
1
was purified by silica gel chromatograph to yield the desire product (585 mg, 90%). H NMR (600 MHz,
CDCl ) δ 7.15 (s, 1H), 6.31 (s, 1H), 4.00-3.90 (br, 2H), 3.79 (s, 3H), 2.11 (s, 3H).
3
2
-Bromo-4-methoxy-5-methylaniline (34a) To a solution of 3-methyl-4-methoxyaniline (600 mg, 4.3
mmol) in acetic acid (10 mL) was added acetic anhydride (0.42 mL, 4.4 mmol) dropwise at rt. The reaction
was heated at 50 °C, at which temperature bromine (0.23 mL, 4.4 mmol) was added. After being stirred
for 2 h at 50 °C, the reaction was cooled to rt and poured into ice-water. The resulting precipitate was
filtered, washed with water and dissolved in ethyl acetate. The solution was washed with saturated sodium
chloride aqueous solution, dried with sodium sulfated and evaporated to dryness to give 2-bromo-4-
methoxy-5-methylacetanilide as a crude product. This intermediate was dissolved in ethanol (10 mL) and
treated with concentrated hydrochloric acid (2.6 mL). The mixture was refluxed for 2 h, before being
concentrated. The residue was treated with aqueous sodium hydroxide (1.5 M, 10 mL) to precipitate the
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Journal of Medicinal Chemistry
crude product. After filtration, the solid was washed with water and dried under reduced pressure to give
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
the title compound (743 mg, 80% over 2 steps). H NMR (600 MHz, CDCl
3
) δ 6.90 (s, 1H), 6.64 (s, 1H),
3.77 (s, 3H), 3.75-3.70 (br, 2H), 2.14 (s, 3H).
2
-Bromo-5-fluoro-4-methoxyaniline (34b). To a suspension of 3-fluoro-4-methoxyaniline (5.06 g, 35.9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mmol) in DCM (160 mL) was added a solution of Br (1.85 mL, 36.0 mmol) in DCM (20 mL) dropwise
2
at -15 °C. After being stirred for 30 min, the reaction was quenched with water (100 mL). The organic
layer was dried with anhydrous Na
2
SO
4
, filtered, and concentrated under reduced pressure. The residue
1
was purified by silica gel chromatograph to yield the desired compound (5.50 g, 70%). H NMR (400
MHz, CDCl ) δ 7.02 (d, J = 8.5 Hz, 1H), 6.55 (d, J = 12.3 Hz, 1H), 3.88-3.80 (br, 2H), 3.79 (s, 3H).
3
2
-Amino-4-methoxy-5-methylbenzonitrile (35a). To a solution of 33a (97 mg, 0.46 mmol) in DMF (2
mL) was added Zn(CN) (111 mg, 0.94 mmol) and Pd(PPh (54 mg, 0.04 mmol). The suspension was
heated in a microwave reactor at 0 °C for 15 min. After cooling to rt, the reaction was diluted with DCM
4 mL) and ammonium hydroxide solution (2 mL). The resulting mixture was extracted with DCM (3 x
mL). The combined organic layer was dried with anhydrous Na SO , filtered, and concentrated under
2
)
3 4
(
4
2
4
reduced pressure. The resulting residue was purified by silica gel chromatograph to yield the desired
1
product (53 mg, 72%). H NMR (400 MHz, CDCl
3
) δ 7.07 (s, 1H), 6.14 (s, 1H), 4.32-4.20 (br, 2H), 3.78
(s, 3H), 2.04 (s, 3H).
2
-Amino-5-fluoro-4-methoxybenzonitrile (35b). The title compound (50% over 2 steps) was prepared
1
according to the synthetic procedure for compound 35a. H NMR (400 MHz, CDCl
3
) δ 7.05 (d, J = 10.5
Hz, 1H), 6.24 (d, J = 7.1 Hz, 2H), 4.38-4.17 (br, 2H), 3.86 (s, 3H).
2-Amino-5-methoxy-4-methylbenzonitrile (36a). The title compound (75%) was prepared from 34a
1
according to the synthetic procedure for compound 35a. H NMR (600 MHz, CDCl
3
) δ 6.74 (s, 1H), 6.58
(s, 1H), 4.16-4.06 (br, 2H), 3.76 (s, 3H), 2.19 (s, 3H).
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Journal of Medicinal Chemistry
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2
-Amino-4-fluoro-5-methoxybenzonitrile (36b). To a solution of 34b (127 mg, 0.58 mmol) in DMF
160 mL) was added CuCN (126 mg, 1.42 mmol). After being stirred for 18h in sealed tube at 120 °C,
the reaction was cooled to rt, poured into ammonium hydroxide solution (3 mL), and extracted with DCM
3 x 3 mL). The combined organic layers were dried with anhydrous Na SO , filtered, and concentrated
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
(
2
4
under reduced pressure. The residue was purified by silica gel chromatograph to yield the desired
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
compound (54 mg, 56%). H NMR (400 MHz, CDCl
3
) δ 6.92 (d, J = 8.7 Hz, 1H), 6.49 (d, J = 12.2 Hz,
1
H), 4.30-4.16 (br, 2H), 3.79 (s, 3H).
2
,4-Dichloro-7-ethoxy-6-methoxyquinazoline (39a). To a solution of 30a (1.06 g, 5.5 mmol) in DCM
(
12 mL) and DMF (12 mL) were added DIEA (1.8 mL, 10.9 mmol) and methyl chloroformate (0.62 mL,
.2 mmol) at 0 °C. After being stirred for 2 h, the reaction solution was poured into water and extracted
with DCM. The organic layer was dried with anhydrous Na SO , filtered, and concentrated under reduced
pressure to yield 37a as brown solid. To a solution of 37a in EtOH (40 mL) was added H (35 mL)
and NaOH (278 mg, 6.9 mmol). The mixture was refluxed for 2 h before being cooled to rt. The
precipitated solid was collected and dried to yield the intermediate 38a. A suspension of 38a in PhNEt
1.5 mL, 1.4 mmol) and POCl (10 mL) was refluxed for 4 h, before being cooled to rt. After removal of
the solvent, cold water was added, followed by saturated aqueous NaHCO solution. The mixture was
extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried with anhydrous Na SO ,
8
2
4
2
O
2
2
(
3
3
2
4
filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel
1
chromatograph to yield the title compound (450 mg, 30%). H NMR (600 MHz, CDCl
3
) δ 7.38 (s, 1H),
7
.29 (s, 1H), 4.31 (q, J = 7.0 Hz, 2H), 4.09 (s, 3H), 1.60 (t, J = 7.2 Hz, 3H).
2
,4-Dichloro-7-isopropoxy-6-methoxyquinazoline (39b). The title compound (40%) was prepared
1
according to the synthetic procedure for 39a. H NMR (600 MHz, CDCl
3
) δ 7.37 (s, 1H), 7.29 (s, 1H),
4
.83-4.79 (m, 1H), 4.07 (s, 3H), 1.52 (d, J = 6.1 Hz, 6H).
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Journal of Medicinal Chemistry
2
,4-Dichloro-6-ethoxy-7-methoxyquinazoline (39c). The title compound (38%) was prepared according
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
to the synthetic procedure for 39a. H NMR (400 MHz, CDCl
3
) δ 7.27 (s, 1H), 7.26 (s, 1H), 4.28 (q, J =
7.0 Hz, 2H), 4.05 (s, 3H), 1.58 (t, J = 7.0 Hz, 3H).
2
,4-Dichloro-6-isopropoxy-7-methoxyquinazoline (39d). The title compound (40%) was prepared
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
according to the synthetic procedure for 39a. H NMR (400 MHz, CDCl
3
) δ 7.27 (s, 1H), 7.26 (s, 1H),
4.83-4.77 (m, 1H), 4.03 (s, 3H), 1.49 (d, J = 6.1 Hz, 6H).
2
,4-Dichloro-6-methoxy-7-methylquinazoline (39e). The title compound (39%) was prepared
1
according to the synthetic procedure for 39a. H NMR (400 MHz, CDCl ) δ 7.30 (s, 1H), 7.24 (s, 1H),
3
4.01 (s, 3H), 2.43 (s, 3H).
2
,4-Dichloro-7-fluoro-6-methoxyquinazoline (39f). The title compound (31%) was prepared according
1
to the synthetic procedure for 39a. H NMR (600 MHz, CDCl ) δ 7.67 (d, J = 10.8 Hz, 1H), 7.55 (d, J =
3
8.4 Hz, 1H), 4.10 (s, 3H).
2
,4-Dichloro-6-fluoro-7-methoxyquinazoline (39g). The title compound (30%) was prepared
1
according to the synthetic procedure for 39a. H NMR (600 MHz, CDCl
3
) 7.78 (dd, J = 10.3, 0.9 Hz,
1H), 7.33 (d, J = 7.4 Hz, 1H), 4.07 (s, 3H).
2
,4-Dichloro-7-methoxy-6-methylquinazoline (39h). The title compound (34%) was prepared
1
according to the synthetic procedure for 39a. H NMR (600 MHz, CDCl ) δ 7.95 (s, 1H), 7.24 (s, 1H),
3
4.03 (s, 3H), 2.43 (s, 3H).
6-Ethoxy-7-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (41). The title
1
compound (79%) was prepared from 39a according to the synthetic procedure for 4. H NMR (400 MHz,
CDCl ) δ 6.87 (s, 1H), 6.77 (s, 1H), 5.09 (d, J = 7.6 Hz, 1H), 4.10-4.05 (m, 3H), 3.879 (s, 3H), 3.80-3.73
3
(m, 8H), 2.84-2.82 (m, 2H), 2.28 (s, 3H), 2.17-2.10 (m, 4H), 1.64-1.54 (m, 2H), 1.44 (t, J = 6.8 Hz, 3H);
MS (ESI) m/z 402.3 [M+H]+.
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6
-Isopropoxy-7-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (42). The
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
title compound (77%) was prepared from 39b according to the synthetic procedure for 4. H NMR (400
MHz, CDCl ) δ 6.88 (s, 1H), 6.86 (s, 1H), 5.09 (d, J = 7.6 Hz, 1H), 4.52-4.46 (m, 1H), 4.12-4.03 (m, 1H),
3
3
1
.87 (s, 3H), 3.79-3.72 (m, 8H), 2.84-2.81 (m, 2H), 2.28 (s, 3H), 2.17-2.08 (m, 4H), 1.64-1.54 (m, 2H),
.32 (d, J = 6.4 Hz, 6H); MS (ESI) m/z 416.3 [M+H]+.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6-Fluoro-7-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (43). The title
1
compound (85%) was prepared from 39c according to the synthetic procedure for 4. H NMR (400 MHz,
CDCl ) δ 7.13 (d, J = 11.6 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 5.04 (d, J = 7.2 Hz, 1H), 4.11-4.02 (m, 1H),
3
3
.91 (s, 3H), 3.81-3.76 (m, 8H), 2.83-2.80 (m, 2H), 2.29 (s, 3H), 2.19-2.07 (m, 4H), 1.64-1.55 (m, 2H);
MS (ESI) m/z 376.2 [M+H]+.
7
-Methoxy-6-methyl-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (44). The title
1
compound (76%) was prepared from 39d according to the synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 7.18 (s, 1H), 6.79 (s, 1H), 5.15 (d, J = 7.2 Hz, 1H), 4.12-4.03 (m, 1H), 3.86 (s, 3H), 3.81-3.76
(m, 8H), 2.83-2.80 (m, 2H), 2.29 (s, 3H), 2.23 (s, 3H), 2.18-2.08 (m, 4H), 1.64-1.554 (m, 2H); MS (ESI):
3
72.3 [M+H]+.
7
-Ethoxy-6-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (45). The title
1
compound (78%) was prepared from 39e according to the synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 6.85 (s, 1H), 6.77 (s, 1H), 5.17 (d, J = 7.6 Hz, 1H), 4.12 (q, J = 7.2 Hz, 2H), 4.09-4.03 (m, 1H),
3
.87 (s, 3H), 3.78-3.74 (m, 8H), 2.84-2.81 (m, 2H), 2.27 (s, 3H), 2.16-2.09 (m, 4H), 1.63-1.53 (m, 2H),
1.07 (t, J = 6.8 Hz, 3H); MS (ESI) m/z 402.3 [M+H]+.
7-Isopropoxy-6-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (46). The
1
title compound (81%) was prepared from 39f according to the synthetic procedure for 4. H NMR (400
MHz, CDCl
3
) δ 6.85 (s, 1H), 6.79 (s, 1H), 5.20 (d, J = 6.8 Hz, 1H), 4.69-4.60 (m, 1H), 4.12-4.03 (m, 1H),
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Journal of Medicinal Chemistry
3
1
.85 (s, 3H), 3.79-3.72 (m, 8H), 2.84-2.81 (m, 2H), 2.26 (s, 3H), 2.15-2.08 (m, 4H), 1.62-1.53 (m, 2H),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
.36 (d, J = 6.0 Hz, 6H); MS (ESI) m/z 416.3 [M+H]+.
7-Fluoro-6-methoxy-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (47). The title
1
compound (85%) was prepared from 39g according to the synthetic procedure for 4. H NMR (400 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CDCl
3
) δ 7.15 (d, J = 12.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 5.28 (d, J = 7.6 Hz, 1H), 4.19-4.10 (m, 1H),
3.95 (s, 3H), 3.83-3.74 (m, 8H), 2.97-2.94 (m, 2H), 2.38 (s, 3H), 2.56 (t, J = 11.2 Hz, 2H), 2.18-2.15 (m,
H), 1.79-1.69 (m, 2H); MS (ESI) m/z 376.2 [M+H]+.
2
6
-Methoxy-7-methyl-N-(1-methylpiperidin-4-yl)-2-morpholinoquinazolin-4-amine (48). The title
1
compound (75%) was prepared from 39h according to the synthetic procedure for 4. H NMR (400 MHz,
CDCl
3
) δ 7.26 (s, 1H), 6.65 (s, 1H), 5.19 (d, J = 7.2 Hz, 1H), 4.13-4.05 (m, 1H), 3.83 (s, 3H), 3.79-3.71
(m, 8H), 2.84-2.82 (m, 2H), 2.28 (s, 3H), 2.24 (s, 3H), 2.16-2.11 (m, 4H), 1.64-1.55 (m, 2H); MS (ESI)
m/z 372.3 [M+H]+.
N-(2-Cyano-4,5-dimethoxyphenyl)tetrahydro-2H-pyran-4-carboxamide (50a). To a solution of 2-
amino-4,5-dimethoxybenzonitrile (372 mg, 2.1 mmol) in DCM (10 mL) were added DIEA (0.55 mL, 3.3
mmol), and tetrahydro-2H-pyran-4-carbonyl chloride (374 mg, 2.5 mmol) at 0 °C. After being stirred for
2
h, the reaction was concentrated and the resulting residue was purified by silica gel chromatography to
1
yield the title compound (577 mg, 95%). H NMR (400 MHz, CDCl
3
) δ 8.49 (s, 1H), 6.92 (s, 1H), 5.92-
5
3
.51 (br, 1H), 4.07 (t, J = 3.5 Hz, 1H), 4.04 (t, J = 3.5 Hz, 1H), 4.00 (dd, J = 7.3, 1.2 Hz, 1H), 3.96 (s,
H), 3.89 (s, 3H), 3.52-3.41 (m, 2H), 1.96-1.85 (m, 4H).
4
-Chloro-6,7-dimethoxy-2-(tetrahydro-2H-pyran-4-yl)quinolone (52a). To a solution of 50a (405 mg,
1.4 mmol) in EtOH (10 mL) were added H
mixture was heated under reflux for 2 h before being cooled to rt. The precipitated solid was collected
and dried under vacuum to yield intermediate 51a as a yellow solid. A mixture of 51a, PhNEt (1.5 mL,
.4 mmol), and POCl (10 mL) was refluxed for 4 h, before being cooled to rt. After removal of the
2
2
O (10 mL) and NaOH (160 mg, 4.0 mmol). The resulting
2
1
3
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Page 22 of 50
solvent, cold water was added, followed by saturated aqueous NaHCO
3
solution. The mixture was
SO ,
1
2
3
4
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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4
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4
4
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5
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5
5
6
extracted with ethyl acetate (3 x 30 mL). The combined organic layers were dried with anhydrous Na
2
4
filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel
1
chromatograph to yield the title compound (240 mg, 55%). H NMR (600 MHz, CDCl
3
) δ 7.40 (s, 1H),
7
3
.33 (s, 1H), 4.14 (dd, J = 11.4, 3.2 Hz, 2H), 4.09 (s, 3H), 4.09 (s, 3H), 3.61 (t, J = 11.0 Hz, 2H), 3.34-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
.11 (m, 1H), 2.11 (m 2H), 2.03 (m, 2H).
4
-Chloro-2-cyclohexyl-6,7-dimethoxyquinoline (52b). The title compound (41% over 3 steps) was
1
prepared according to synthetic procedure for 52a. H NMR (600 MHz, CDCl
3
) δ 7.39 (s, 1H), 7.32 (s,
1
3
H), 4.08 (s, 6H), 2.97 (tt, J = 11.9, 3.4 Hz, 1H), 2.16-2.01 (m, 2H), 1.92-1.90 (m, 2H), 1.83-1.67 (m,
H), 1.52-1.41 (m, 2H), 1.40-1.30 (m, 1H).
2
-Cyclohexyl-6,7-dimethoxy-N-(1-methylpiperidin-4-yl)quinazolin-4-amine (53). A mixture of 52a
(60 mg, 0.2 mmol), 4-methylpiperidin-1-amine (0.05 mL, 0.40 mmol) and DIEA (0.06 mL, 0.36 mmol)
in i-PrOH (1 mL) was heated by microwave irradiation at 160 °C for 15 min. After concentration, the
1
crude product was purified by preparative HPLC to yield the title compound (69 mg, 90%). H NMR (400
MHz, CDCl ) δ 7.13 (s, 1H), 6.86 (s, 1H), 5.31 (d, J = 6.8 Hz, 1H), 4.25-4.16 (m, 1H), 3.90 (s, 3H), 3.89
3
(s, 3H), 2.86-2.83 (m, 2H), 2.70 (tt, J = 11.6, 3.6 Hz, 1H), 2.29 (s, 3H) 2.21-2.12 (m, 4H), 1.97-1.94 (m,
2H), 1.82-1.79 (m, 2H), 1.72-1.54 (m, 5H), 1.42-1.22 (m, 3H); MS (ESI) m/z 385.3 [M+H]+.
6
,7-Dimethoxy-N-(1-methylpiperidin-4-yl)-2-(tetrahydro-2H-pyran-4-yl)quinazolin-4-amine (54).
1
The title compound (80%) was prepared according to synthetic procedure for 53. H NMR (400 MHz,
CDCl
Hz, 2H), 3.92 (s, 6H), 3.52 (td, J = 12.0, 1.6 Hz, 2H), 2.93 (tt, J = 12.0, 4.0 Hz, 1H), 2.86-2.83 (m, 2H),
.29 (s, 3H) 2.20-2.12 (m, 4H), 2.04 (qd, J = 12.0, 4.4 Hz, 2H), 1.87-1.84 (m, 2H), 1.59 (qd, J = 12.0, 4.0
3
) δ 7.12 (s, 1H), 6.83 (s, 1H), 5.26 (d, J = 7.6 Hz, 1H), 4.25-4.16 (m, 1H), 4.05 (dd, J = 11.2, 4.4
2
Hz, 2H); MS (ESI) m/z 387.3 [M+H]+.
GLP and G9a IC50 Determination. Methyltransferase activity assays for G9a and GLP were performed
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by monitoring the incorporation of tritium-labeled methyl group to lysine 9 of H3 (1-25) peptide using
Scintillation Proximity Assay (SPA). The enzymatic reactions were performed at 23 ºC with 20 min
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9
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1
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4
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5
5
6
incubation of 10 µl reaction mixture in 25 mM potassium phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl ,
2
3
0
.01% Triton X-100 containing 8 µM of cold SAM and 2 µM of H-SAM (Cat.# NET155V250UC; Perkin
Elmer; www.perkinelmer.com), 1 µM of biotinylated H3 (1-25), 5 nM G9a or GLP, and compound
titrations from 1.5 nM to 25 µM. To stop the reactions, 10 µL of 7.5 M Guanidine hydrochloride was
added, followed by 60 µl of buffer (20 mM Tris, pH 8.0), mixed and transferred to a 384-well Streptavidin
coated Flash-plate (PerkinElmer, http://www.perkinelmer.ca). After mixing, the mixtures in Flash-plate
were incubated for 2 h and the CPM counts were measured using Topcount plate reader (Perkin Elmer,
www.perkinelmer.com). The CPM counts in the absence of compound for each dataset were defined as
100% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
0
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9
0
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
(0%). All enzymatic reactions were performed in triplicate and IC50 values were determined by fitting the
data to Four Parameter Logistic equation using GraphPad Prism 7 software.
Selectivity Assays. Effect of compound on methyltransferase activity of SUV39H1, SUV39H2,
SETDB1, SETD8, SUV420H1, SUV420H2, SETD7, MLL1 trimeric complex, MLL3 pentameric
complex, EZH2 trimeric complex, PRMT1, PRMT3, PRMT4, PRMT5/MEP50 complex, PRMT6,
PRMT7, PRMT8, PRMT9, PRDM9, SETD2, BCDIN3D, METTL3-14, SMYD2, SMYD3 and DNMT1
was assessed by monitoring the incorporation of tritium-labeled methyl group to lysine or arginine
residues of peptide substrates using Scintillation Proximity Assay (SPA). Assays were performed in a 10
3
µl reaction mixture containing H-SAM (Cat.# NET155V250UC; Perkin Elmer; www.perkinelmer.com)
and substrate at concentrations close to K values for each enzyme. Two concentrations (1µM, and
m
10µM) of compound were used in all selectivity assays. To stop the enzymatic reactions, 10 µl of 7.5 M
Guanidine hydrochloride was added, followed by 180 µl of buffer (20 mM Tris, pH 8.0), mixed and then
transferred to a 96-well FlashPlate (Cat.# SMP103; Perkin Elmer; www.perkinelmer.com). After mixing,
the reaction mixtures in Flash plates were incubated for 1 hour and the CPM were measured using
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Topcount plate reader (Perkin Elmer, www.perkinelmer.com). The CPM counts in the absence of
compound for each data set were defined as 100% activity. In the absence of the enzyme, the CPM counts
in each data set were defined as background (0%).
1
2
3
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9
1
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4
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6
For DNMT1 the dsDNA substrate was prepared by annealing two complementary strands
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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7
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9
0
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2
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9
0
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3
4
5
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7
8
9
0
(biotintlated forward strand: B-GAGCCCGTAAGCCCGTTCAGGTCG and reverse strand:
CGACCTGAACGGGCTTACGGGCTC), synthesized by Eurofins MWG Operon.
For DOT1L, ASH1L, DNMT3A/3L, and DNMT3B/3L, a filter-based assay was used. In this
assay, 10 µl of reaction mixtures were incubated at RT for 1 hour, 50 µl of 10% TCA was added, mixed
and transferred to filter-plates (Millipore; cat.# MSFBN6B10; www.millipore.com). Plates were
centrifuged at 2000 rpm (Allegra X-15R - Beckman Coulter, Inc.) for 2 min followed by 2 additional 10%
TCA wash and one ethanol wash (180 µl) followed by centrifugation. Plates were dried and 100 µl
MicroO (MicroScint-O; Cat. # 6013611, Perkin Elmer; www.perkinelmer.com) was added to each well,
centrifuged and removed. 70 µl of MicroO was added again and CPM was measured using Topcount plate
reader.
Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) measurements were made at
2
5 °C on a MicroCal ITC200 Instrument (Malvern Instruments). Co-concentrated G9a−SAM and
GLP−SAM (protein/SAM molar ratio of 1:5) were diluted at 35 M in ITC buffer [50 mM Tris (pH 8.0),
50 mM NaCl] supplemented with 1% DMSO. Compound 4 and 18 were prepared in DMSO at 50 mM
1
and diluted to 0.5 mM in ITC buffer with a final DMSO concentration of 1%. Binding constants were
calculated by fitting the data using the ITC data analysis module in Origin 7.0 (OriginLab Corp.).
Mechanism of Action (MOA) Studies. IC50 values were determined for compound 4 and 18 at various
concentrations of SAM (3.1, 6.2,12.5, 25, 50 and 100 µM), 5 nM of GLP, 5 µM of biotinylated H3 (1-25)
peptide; or at various concentrations of biotinylated H3 (1-25) peptide substrate (0.31, 0.62,
1.2,
2
.5, 5 and 10 µM), 5 nM of GLP, 50 µM SAM. The reaction mixtures were incubated for 15 min at 23
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Journal of Medicinal Chemistry

C. To stop the enzymatic reactions, equal volume of 7.5 M guanidine hydrochloride was added, followed
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by 180 µl of buffer (20 mM Tris, pH 8.0), mixed and then transferred to a 96-well FlashPlate (Cat.#
SMP103; Perkin Elmer; www.perkinelmer.com). After mixing, the reaction mixtures in Flash plates were
incubated for 2 h and the CPM were measured using Topcount plate reader (Perkin Elmer,
www.perkinelmer.com). The CPM counts in the absence of compound for each data set were defined as
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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9
0
1
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0
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4
5
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9
0
1
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4
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9
0
1
00% activity. In the absence of the enzyme, the CPM counts in each data set were defined as background
(0%). The IC50 values were determined using GraphPad Prism 7 software.
Protein Expression, Purification, Crystallization, Data Collection and Structure Determination for
Compound 4. The recombinant proteins GLP catalytic SET domain (982-1266) and G9a catalytic SET
domain (913-1193), from plasmids given by Alexander Plotnikov, were overexpressed in E. coli BL21
(DE3) codon plus RIL strain (Stratagene) by addition of 0.3 mM isopropyl-1-thio-D-galactopyranoside
with a supplement of 1mM zinc sulfate and incubated overnight at 15 °C. Harvested cells were
resuspended in 50 mM sodium phosphate buffer, pH 7.5, supplemented with 0.5mM sodium chloride and
5
% glycerol and lysed using a microfluidizer (Microfluidics) at 10,000 psi. After clarification of the crude
extract by high-speed centrifugation, the lysate was loaded onto a 5 ml HiTrap chelating column (GE
Healthcare) charged with Ni2+. The column was washed, and the protein was eluted with 50 mM Tris
buffer, pH 8.0, 250 mM sodium chloride, 250 mM imidazole and supplemented with 0.5 mM of TCEP.
The protein was next purified on a Superdex200 column (26/600; GE Healthcare) equilibrated with 50
mM Tris-HCl buffer, pH 8.0, and 150 mM sodium chloride, the elution fractions were pooled and
supplemented with 0.5 mM of TCEP. All purification steps were performed at 4 °C and in the presence
of a protease inhibitor AEBSF (Goldbio). The size and the purity of the recombinant G9a and GLP
proteins were checked by SDS-PAGE and mass spectroscopy.
Purified GLP and G9a were mixed in the presence of S-adenosyl-L-methionine (Sigma) and
compound 4 (protein/SAM/4 molar ratio of 1:5:10), co-concentrated to about 20mg/mL and crystallized
using the hanging drop vapor diffusion method at 17 °C by mixing equal volume of the protein solution
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with the reservoir solution. The GLP−SAM−4 ternary complex was crystallized in 20% PEG 20,000, 2%
v/v) 1,4-Dioxane, 0.1 M Bicine (pH 9.0). The G9a−SAM−4 ternary complex was crystallized in 20%
1
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7
8
9
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5
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5
6
(
(w/v) PEG 3,350, 0.2 M NaF, 0.1 M Bis-Tris propane (pH 6.5). GLP−SAM−4 crystals were soaked in the
corresponding mother liquor supplemented with 20% ethylene glycol and the G9a−SAM−4 complex
crystals in a mixture of parafin70% and silicon30% as cryoprotectant before flash freezing in liquid
nitrogen.
0
1
2
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8
9
0
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2
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0
X-Ray diffraction data were collected at 100K at NE-CAT beamline 24-ID-E of Advanced Photon
Source (APS) at Argonne National Laboratory. The data were processed with MOSFLM, SCALA and
5
8
other programs from the CCP4 suite. The structures of the G9a and GLP complexes were solved by
59
molecular replacement using the PHASER and the atomic model of the G9a and GLP SET domains
PDB code: 3K5K and 3HNA respectively). The locations of the bound molecules were determined from
(
60
61 62
a Fo-Fc difference electron density map. REFMAC and phenix.refine
were used for structure
refinement. Graphic program COOT63 was used for model building and visualization. The overall
assessment of model quality was performed using MOLPROBITY.64
Protein Expression, Purification, Crystallization, Data Collection and Structure Determination for
Compound 18. Human G9a and GLP catalytic domains were cloned, expressed and purified as
65
previously described. Purified G9a and GLP proteins were pre-mixed with compound 18 at 1:2 molar
ratio of protein: compound. The protein-compound complexes were crystallized using sitting drop vapor
diffusion method at 20 °C by mixing equal volume of the protein solution with the reservoir solution
containing 25 % PEG3350, 0.2 M NaCl, 0.1 M Bis-Tris pH6.5 (for G9a) and 20 % isopropanol, 20 %
PEG 4K, 0.1 M NaCitrate, pH5.6 (for GLP).
X-ray diffraction data for G9a-18 and GLP-18 were both collected at 100K at beamline 19ID of
Advanced Photon Source (APS), Argonne National Laboratory. Both data sets were processed using the
6
6
HKL-3000 suite. The structures of G9a and GLP with 18 were solved by molecular replacement using
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Journal of Medicinal Chemistry
6
7
60
MOLREP, with PDB entry 3K5K and 4I51 as search template respectively. REFMAC was used for
structure refinement. Geometry restraints for the compound refinement were prepared with by GRADE
Developed at Global Phasing Ltd. (O.S.Smart, T.O.Womack, A. Sharff,C. Flensburg, P. Keller, W.
Paciorek, C. Vonrhein, and G. Bricogne, (2011) grade v1.102. Cambridge, United Kingdom, Global
Phasing Led.) Graphics program COOT63 was used for model building and visualization.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
4
MOLPROBITY was used for structure validation. Crystal diffraction data and refinement statistics for
the structure are displayed in Table 1.
ASSOCIATED CONTENT
Supporting Information
This material is available free of charge via the internet at http://pubs.acs,org.
1
13
H and C NMR spectra of compound 4 and 18; Crystallography data and refinement statistics; Molecular
formula strings.
Accession Codes
The structure of GLP in complex with 4 has been deposited under PDB ID 5TUZ. The structure of G9a
in complex with 4 has been deposited under PDB ID 5TUY. The structure of GLP in complex with 18
has been deposited under PDB ID 5TTG. The structure of G9a in complex with 18 has been deposited
under PDB ID 5TTF. Authors will release the atomic coordinates and experimental data upon article
publication.
AUTHOR INFORMATION
Corresponding Authors
*
J.J.: e-mail: jian.jin@mssm.edu; phone: (212) 659-8699.
*
M.V.: e-mail: m.vedadi@utoronto.ca; phone: (416) 976-0897.
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Notes
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The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
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We thank Albina Bolotokova for compound management and Taraneh Hajian for protein purification.
The research described here was supported by the grant R01GM103893 (to J.J.) from the U.S. National
Institutes of Health. The SGC is a registered charity (number 1097737) that receives funds from AbbVie,
Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no.
1
15766], Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and
Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust. We thank
the staff at the 24-ID-E beamline of Advanced Photon Source (APS) at Argonne National Laboratory for
facilitating x-ray data collection.
ABBREVIATIONS USED
GLP, G9a-like protein 1; PKMT, protein lysine methyltransferase; PRMT, protein arginne
methyltransferase; H3K9me1, monomethylation of lysine 9 on histone H3; H3K9me2, dimethylation of
lysine 9 on histone H3; SAM, S-5′-adenosyl-L-methionine; SAH, SAH, S-5′-adenosyl-L-homocysteine;
SAR, structure–activity relationship; DNMT, DNA methyltransferase; RNMT, RNA methyltransferases;
SET: suppressor of variegation 3−9 enhancer-of-zeste trithorax; SIRT1, sirtuin 1; MyoD, myogenic
differentiation 1; CDYL1, chromodomain Y-like protein; WIZ, widely interspaced zinc finger motifs
protein; RMSD, root-mean-square deviation; METTL3-14, N6-adenosine-methyltransferase subunit
METTL3 and 14 complex; ITC, isothermal titration calorimetry.
REFERENCES
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Journal of Medicinal Chemistry
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