Structure-activity relationship studies of G9a-like protein (GLP) inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.06.021

Given the high homology between the protein lysine methyltransferases G9a-like protein (GLP) and G9a, it has been challenging to develop potent and selective inhibitors for either enzyme. Recently, we reported two quinazoline compounds, MS0124 and MS012, as GLP selective inhibitors. To further investigate the structure–activity relationships (SAR) of the quinazoline scaffold, we designed and synthesized a range of analogs bearing different 2-amino substitutions and evaluated their inhibition potencies against both GLP and G9a. These studies led to the identification of two new GLP selective inhibitors, 13 (MS3748) and 17 (MS3745), with 59- and 65-fold higher potency for GLP over G9a, which were confirmed by isothermal titration calorimetry (ITC). Crystal structures of GLP and G9a in complex with 13 and 17 provide insight into the interactions of the inhibitors with both proteins. In addition, we generated GLP selective inhibitors bearing a quinoline core instead of the quinazoline core.

Full text of DOI:10.1016/j.bmc.2017.06.021

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-activity relationship studies of G9a-like protein (GLP)
inhibitors
Yan Xiong a,b, Fengling Li c, Nicolas Babault a,b, Hong Wu c, Aiping Dong c, Hong Zeng c, Xin Chen a,b
,
Cheryl H. Arrowsmith c,e, Peter J. Brown c, Jing Liu a,b, Masoud Vedadi c,d, , Jian Jin a,b,
⇑
⇑
a Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
b Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
c Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada
d Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8, Canada
e Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Given the high homology between the protein lysine methyltransferases G9a-like protein (GLP) and G9a,
it has been challenging to develop potent and selective inhibitors for either enzyme. Recently, we
reported two quinazoline compounds, MS0124 and MS012, as GLP selective inhibitors. To further inves-
tigate the structure–activity relationships (SAR) of the quinazoline scaffold, we designed and synthesized
a range of analogs bearing different 2-amino substitutions and evaluated their inhibition potencies
against both GLP and G9a. These studies led to the identification of two new GLP selective inhibitors,
13 (MS3748) and 17 (MS3745), with 59- and 65-fold higher potency for GLP over G9a, which were con-
firmed by isothermal titration calorimetry (ITC). Crystal structures of GLP and G9a in complex with 13
and 17 provide insight into the interactions of the inhibitors with both proteins. In addition, we generated
GLP selective inhibitors bearing a quinoline core instead of the quinazoline core.
Received 12 May 2017
Revised 10 June 2017
Accepted 13 June 2017
Available online xxxx
Keywords:
GLP
G9a
Inhibitor
Quinazoline
Quinoline
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
dimethylation.3,4 Most of the subsequent studies have been
focused on the function of G9a with the assumption that GLP
GLP (euchromatic histone-lysine N-methyltransferase
1
would behave similarly.25 More recent research challenged the
above assumption and revealed GLP and G9a possess distinct phys-
iological functions.26 For example, whereas the ankyrin repeat
domain of G9a preferentially binds to H3K9me2, the ankyrin
repeat domain of GLP binds tightly to H3K9me1.27 Disruption of
the binding of GLP and G9a to methylated H3K9 by ankyrin repeat
domain mutations resulted in drastic phenotypic difference in
mice.28 In addition, tissue-specific expression profiles are different
for GLP and G9a. While G9a shows higher expression level in white
adipose tissue (WAT) than in brown adipose tissue (BAT), GLP is
highly enriched in BAT and is essential for brown cell fate and ther-
mogenesis.29 Moreover, GLP and G9a regulate distinct sets of genes
and have different effects on proliferating myoblasts and on skele-
tal muscle terminal differentiation.25
Over the last decade, a number of quinazoline-containing com-
pounds have been developed to selectively inhibit the methyl-
transferase activity of GLP and G9a.1,2,30–42 High throughput
screening led to the discovery of BIX01294 (1), which is the first
potent and selective dual inhibitor of GLP and G9a.1 Our group
and others utilized structure-based design strategy in combination
with SAR exploration and developed more potent compounds, such
(EHMT1) or lysine methyltransferase 1D (KMT1D)) and G9a
(EHMT2 or KMT1C) are highly homologous protein lysine methyl-
transferases (PKMTs), sharing about 80% sequence identity in their
SET (suppressor of variegation 3–9, enhancer of zeste and tritho-
rax) domains.1,2 GLP and G9a catalyze the transfer of the methyl
group from the cofactor S-50-adenosyl-
L-methionine (SAM) to the
e
-amino group of the targeted lysine residue in a variety of histone
and nonhistone substrates, such as H3K9,3,4 H1,5,6 H3K27,7–9
H3K56,10 p53,11 SIRT1,12 reptin,13 MyoD,14 CDYL1,15 WIZ,15 and
G9a.16 Dysregulation of GLP and G9a has been associated with
numerous human diseases, including cancer, inflammatory dis-
eases, and neurodegenerative disorder.17–24
Earlier studies suggested that GLP and G9a form a catalytically
active heterodimeric complex to catalyze H3K9 mono- and
⇑
Corresponding authors at: Structural Genomics Consortium, University of
Toronto, Toronto, Ontario M5G 1L7, Canada (M. Vedadi). Department of Pharma-
cological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029,
United States (J. Jin).
E-mail addresses: m.vedadi@utoronto.ca (M. Vedadi), jian.jin@mssm.edu (J. Jin).
http://dx.doi.org/10.1016/j.bmc.2017.06.021
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Structures of selected G9a/GLP inhibitors.
as UNC0224 (2),37 UNC0321 (3),38 and E72 (4).30 Optimization of
physicochemical and pharmacokinetic properties of this scaffold
led to a cellular chemical probe, UNC0638 (5),35,40,43 and subse-
quently an in vivo chemical probe, UNC0642 (6).36 Compounds 5
and 6 have been widely used as tool compounds by the research
community to investigate the biological function and to test the
therapeutic hypotheses associated with GLP and G9a.43–45 Due to
the fact that these compounds are dual inhibitors of GLP and
G9a, the phenotypic effects rendered by these compounds could
be attributed to the inhibition of methyltransferase activity of
GLP and/or G9a. Hence, GLP or G9a selective inhibitors, which
selectively inhibit GLP over G9a or vice versa, are required to dis-
sect the distinct biological function of each enzyme. Recently, we
screened our quinazoline compound collection against GLP and
G9a and discovered a potent and selective GLP inhibitor, MS0124
(7).46 Preliminary SAR guided optimization led to an improved
GLP selective inhibitor, MS012 (8).46 Compounds 7 and 8 share
most of the substituent groups on the quinazoline core, except
the 2-amino moiety. However, this important 2-amino region of
the quinazoline scaffold has not been extensively explored in our
previous study. Here, we describe our continued optimization of
this region, which resulted in the discovery of two new GLP
selective compounds, 13 and 17. In addition, we report two
GLP selective inhibitors bearing a quinoline core instead of the
quinazoline core.
2-Amino substituted quinazoline analogs were readily prepared
using the efficient two-step synthetic sequence we developed pre-
viously.37 Briefly, 4-chloro displacement of commercially available
2,4-dichloro-6,7-dimethoxyquinazoline with 4-amino-1-methylpi-
peridine yielded the intermediate 9. Substitution of the 2-chloro
group of the intermediate 9 with various amines under microwave
conditions provided the desired quinazoline analogs 11–37
(Scheme 1).
In cocrystal structures of MS0124 and MS012, while the N1
nitrogen atom of the quinazoline core forms important hydrogen
bonds with GLP and G9a, the N2 nitrogen atom does not interact
with either protein. We attempted to replace the N2 nitrogen atom
of the quinazoline core with a carbon atom, which leads to a struc-
turally distinct quinoline core. Although quinoline analogs were
reported as equipotent GLP and G9a dual inhibitors,42 GLP selective
inhibitors with a quinoline core have not been reported. Thus, we
prepared the quinoline analogs of MS0124 and MS012 for direct
comparison.
The quinoline analogs were synthesized from the commercially
available 2,4-dichloro-6,7-dimethoxyquinoline through two con-
secutive Buchwald-Hartwig cross coupling reactions under micro-
wave conditions. The first coupling reaction occured at the
2-chloro position to provide the intermediate 10, which was then
coupled with 4-amino-1-methylpiperidine at the 4-chloro position
at an elevated temperature to yield desired quinoline products 38
and 39 (Scheme 2).
2. Results and discussion
2.2. Results in GLP and G9a biochemical assays
2.1. Design and synthesis of quinazoline and quinoline derivatives
We evaluated the potency of the synthesized compounds in GLP
and G9a biochemical assays, which are radioactivity-based scintil-
lation proximity assays that measure the transfer of the methyl
group from 3H-SAM to the peptide substrates.36 We summarize
the biochemical assay data (IC50 values) in Tables 1–3.
First, we assessed the potency and selectivity of a number of
tertiary 2-amino analogs bearing a fixed N-methyl group (11–28,
Table 1). Besides the previously reported analogs with a relatively
long linear side chain (e.g., butyl, pentyl, hexyl),46 we evaluated
analogs with a short alkyl chain, such as methyl (11), ethyl (12),
Through our previous SAR studies, we found that structural
modifications to the 2-amino region of the quinazoline scaffold,
which is shared by MS0124 and MS012, could drastically increase
selectivity for GLP.46 X-ray crystal structures of GLP and G9a in the
complex with MS0124 or MS012 revealed virtually identical inhi-
bitor–protein interactions, and did not provide informative insight
to guide the design of more selective inhibitors.46 Therefore, it is
necessary to extensively explore a variety of amino substituents
to understand the SAR trend at this 2-amino region.
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Scheme 1. Synthesis of 2-amino substituted quinazolines. Reagents and conditions: (a) 4-amino-1-methylpiperidine, K2CO3, DMF, rt, 90%; (b) R1R2NH, 4N HCl in dixoane,
iPrOH, microwave, 160 °C, 75–85%.
Scheme 2. Synthesis of quinoline analogs. Reagents and conditions: (a) amine, BINAP, Pd2(dba)3, tBuONa, THF, microwave, 100 °C, 45–50%; (b) 4-amino-1-methylpiperidine,
BINAP, Pd(OAc)2, tBuONa, THF, microwave, 160 °C, 70–71%.
Table 1
SAR of the tertiary amino substituted quinazolines.
Compound
R1
IC50 (nM)
GLP
Compound
R1
IC50 (nM)
GLP
G9a
G9a
11
12
11
3
191 74
22
23
50 18
726 152
18
5
465 88
295 103
318 35
20
46
20
3
9
2
532 148
534 61
282 30
13 (MS3748)
14
5
2
24
25
18
2
8
2
15
30
17
4
550 106
356 90
259 80
26
27
28
58 17
1630 230
151 46
484 85
16
24
6
17 (MS3745)
1
2
73 25
18
19
20
21
6
177 31
29
30
31
32
23
20
23
6
8
4
343 51
481 129
508 26
443 57
159
5
1160 510
724 209
359 147
43 23
28
4
36 15
IC50 determination experiments were performed at substrate and cofactor concentrations equal to the respective Km values for each enzyme. IC50 determination experiments
were performed in triplicate and the values are presented as Mean SD.
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Table 2
promising new GLP selective inhibitors, we synthesized and
SAR of the secondary amino substituted quinazolines.
assessed more derivatives (21–28, Table 1) of this inhibitor.
Branching the n-propyl group of 13 at the b-C position with a
mono- (21) or di-methyl (22) group decreased the potency for
GLP by 6- and 10-fold, respectively. Compared to the n-propyl
group (13), cyclohexylmethyl (23) and benzyl (24) groups also
led to weaker GLP inhibition. Similarly, heteroatom-containing
groups, such as b-hydroxyethyl (25), b-methoxyethyl (26), b-
dimethylaminoethyl (27) and
c-hydroxypropyl groups (28), did
not improve either potency or selectivity in comparison with the
n-propyl group of 13. Next, we changed the fixed N-methyl group
shared by compounds 11–28 to a fixed N-ethyl group in tertiary
amino substituted compounds 29–31 (Table 1). These three N-
ethyl bearing compounds showed similar potency for GLP and sim-
ilar selectivity for GLP over G9a. Further increasing the length of
this fixed group to a N-propyl (32) was also tolerated. However,
none of these analogs carrying either N-ethyl or N-propyl group
was more potent or more selective for GLP than their correspond-
ing N-methyl analogs. Finally, we removed the fixed N-methyl
group from compounds 18, 22, and 26 and prepared the corre-
sponding secondary amino substituted analogs 33, 34, and 35 for
direct comparison (Table 2). While compounds 34 and 35 showed
comparable potency for GLP to that of 22 and 26, compound 33
was a much weaker GLP inhibitor than 18 (23-fold decrease). In
addition, a couple of analogs (36 and 37) of MS012 with a substi-
tuted N-hexyl group were also synthesized. However, both com-
pounds were less potent than MS012 (GLP IC50 = 7 2 nM; G9a
IC50 = 992 337 nM)46 and were less selective for GLP over G9a
than MS012. In summary, the SAR guided optimization on the 2-
amino moiety of the quinazoline scaffold resulted in two new inhi-
bitors, 13 and 17, which have high potency (IC50 = ꢀ5 nM) for GLP
and high selectivity (ꢀ60-fold) for GLP over G9a.
Compound
R1
IC50 (nM)
GLP
G9a
33
34
77
6
956 362
24
5
585 145
35
36
135 27
27 13
1582 526
315 88
37
59 18
788 28
IC50 determination experiments were performed at substrate and cofactor con-
centrations equal to the respective Km values for each enzyme. IC50 determination
experiments were performed in triplicate and the values are presented as
Mean SD.
Table 3
SAR of the quinoline scaffold.
Compound
Structure
IC50 (nM)
GLP
The direct quinoline derivatives (compounds 38 and 39) of
MS0124 (GLP IC50 = 13 4 nM; G9a IC50 = 440 63 nM)46 and
MS012 were prepared and evaluated (Table 3). Both compounds
retained high potency for GLP (38: IC50 = 6 2 nM; 39:
IC50 = 11 4 nM) and displayed improved potency for G9a (38:
IC50 = 92 33 nM; 39: IC50 = 110 42 nM). Whilst they were not
as selective as the quinazoline analogs (MS0124 and MS012), com-
pounds 38 and 39 favored the inhibition of GLP over G9a with 15-
fold and 10-fold selectivity, respectively.
G9a
38
6
2
92 33
39
11
4
110 42
2.3. Characterization of 13 and 17 in a biophysical assay
The binding of 13 and 17 to GLP and G9a was assessed using
isothermal titration calorimetry (ITC) in the presence of SAM
(Fig. 2) as described previously.46 Compound 13 displayed higher
binding affinity to GLP (Kd = 57 8 nM) than G9a (Kd = 510 62 -
nM). Similarly, compound 17 exhibited stronger binding affinity
to GLP (Kd = 94 14 nM) than G9a (Kd = 1070 80 nM). Therefore,
both compounds are confirmed to selectively bind to GLP over
G9a in the ITC biophysical assay.
IC50 determination experiments were performed at substrate and cofactor con-
centrations equal to the respective Km values for each enzyme. IC50 determination
experiments were performed in triplicate and the values are presented as
Mean SD.
and n-propyl (13) groups. Among these linear alkyl groups, the n-
propyl group (13) resulted in the highest potency for GLP
(IC50 = 5 2 nM), with 59-fold selectivity for GLP over G9a
(IC50 = 295 103 nM). Switching the n-propyl group to i-propyl
(14) decreased the potency for GLP by 4-fold (IC50 = 18 2 nM)
and decreased the selectivity for GLP over G9a to 18-fold. The
cyclopropyl (15) group further decreased the potency for GLP
(IC50 = 30 8 nM). Enlarging the ring size from cyclopropyl (15)
to cyclobutyl (16), cyclopentyl (17), and cyclohexyl (18) groups
improved the potency for both GLP and G9a. Among these N-
methyl-N-cycloalkyl substituted amino analogs, the cyclopentyl
compound 17 displayed the best potency for GLP (IC50 = 4 1 nM)
and the best selectivity (65-fold) for GLP over G9a (IC50 = 259 80 -
nM). Switching the cyclohexyl (18) group to the heteroatom con-
taining tetrahydropyranyl (19) or aromatic phenyl (20) group
greatly decreased the potency for both GLP and G9a and the selec-
tivity for GLP over G9a. Since compound 13 is one of the most
2.4. Cocrystal structures of GLP and G9a in the complex with 13, 17
and 18
We solved the ternary structures of GLP and G9a in their com-
plexes with compound 13, 17, or 18 in the presence of SAM (Fig. 3
and Supporting Fig. 1) with high resolution (1.4–1.85 Å). Consis-
tent with the previously reported GLP selective inhibitors,46 com-
pounds 13, 17 and 18 adapt virtually identical inhibitor binding
modes and ligand–protein interactions for both GLP and G9a
(Fig. 3C, E, and Supporting Fig. 1C). Compounds 13, 17 and 18
occupy the substrate binding sites of GLP and G9a and form direct
hydrogen bonds with two aspartic acids (D1176 and D1171 in GLP;
D1088 and D1083 in G9a) and a water molecule. Superimposition
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Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Fig. 2. Binding confirmation of 13 and 17. Isothermal titration calorimetry (ITC) was used to confirm that 13 displayed better binding affinity to GLP (A) over G9a (B).
Similarly, 17 also preferably bound to GLP (C) over G9a (D). ITC experiments were performed in triplicate.
of the GLP cocrystal structures of 13 and 17 with that of MS012,46
MS0124,46 and E7230 reveals that the larger N-propyl group of 13
and N-cyclopentyl group of 17 point to the same direction as the
N-hexyl group of MS012 and the smaller N-methyl groups of 13
and 17 direct to the site where the N-(30-dimethylamino)propyl
group of E72 is located (Fig. 3F). This is consistent with our previ-
ous hypothesis46 that the N-(30-dimethylamino)propyl group of
E72 may adopt a sterically unfavorable geometry due to the strong
charge-charge interaction between the dimethylamino group and
GLP D1162.30 Since these cocrystal structures could not provide
conclusive explanation for the observed high selectivity, additional
studies are being performed to understand the molecular basis for
the high selectivity for GLP over G9a.
3. Conclusion
Extensive SAR studies around the 2-amino group of the quina-
zoline scaffold have led to the discovery of two new potent and
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Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 3. X-ray cocrystal structures of GLP and G9a in complex with 13 and 17. (A) Structure of GLP-SAM-13 (green) (PDB code: 5VSD). (B) Structure of G9a-SAM-13 (cyan) (PDB
code: 5VSC). (C) Overlay of (A) and (B). (D) Structure of GLP-SAM-17 (green) (PDB code: 5VSF). (E) Overlay of structures of (D) and G9a-SAM-17 (cyan) (PDB code: 5VSE). (F)
Overlay of GLP structures in the complex with 13 (gray), 17 (green), MS12 (blue), MS0124 (orange), and E72 (magenta). Water molecule is illustrated as a red sphere. Main
interactions are shown in yellow dashed lines.
selective GLP inhibitors, 13 and 17, both of which bear a tertiary
acyclic amino group. The binding of 13 and 17 to GLP and G9a
was confirmed by ITC. To determine inhibitor-protein interactions,
we solved the cocrystal structures of GLP and G9a in their com-
plexes with 13 and 17. The structures revealed that the larger N-
propyl group of 13 or N-cyclopentyl group of 17 occupies a steri-
cally favorable binding site. In addition, we discovered two GLP
selective inhibitors containing a quinoline core. Whilst these
quinoline inhibitors are not as selective as their corresponding
quinazoline analogs for GLP over G9a, the quinoline derivatives
represent a chemically distinct scaffold that could be optimized
in the future.
5 lm, C18 column at room temperature. The flow rate was
40 mL/min. A linear gradient was used with 10% (or 50%) of MeOH
(A) in H2O (with 0.1 % TFA) (B) to 100% of MeOH (A). HPLC was used
to establish the purity of target compounds. All final compounds
had >95% purity using the HPLC methods described above.
4.1.1. 6,7-Dimethoxy-N2,N2-dimethyl-N4-(1-methylpiperidin-4-yl)
quinazoline-2,4-diamine (11)
Synthesis of the title compound was reported in this previous
paper.37
4.1.2. N2-Ethyl-6,7-dimethoxy-N2-methyl-N4-(1-methylpiperidin-4-
yl)quinazoline-2,4-diamine (12)
4. Experiment protocols
The title compound (80% yield) was prepared according to syn-
thetic procedures reported previously.46 1H NMR (400 MHz, CDCl3)
d 6.88 (s, 1H), 6.78 (s, 1H), 5.16 (d, J = 6.4 Hz 1H), 4.13–4.05 (m,
1H), 3.90 (s, 3H), 3.87 (s, 3H), 3.69 (q, J = 7.2 Hz, 2H), 3.15 (s, 3H),
2.85 (d, J = 12.0 Hz, 2H), 2.28(s, 3H), 2.16–2.11 (m, 4H), 1.64–1.56
(m, 2H), 1.15 (t, J = 6.8 Hz, 3H); MS (ESI) m/z 360.3 [M+H]+.
4.1. Chemistry
HPLC spectra for all compounds were acquired using an Agilent
1200 Series system with DAD detector. Chromatography was per-
formed on a 2.1 ꢁ 150 mm Zorbax 300SB-C18
5 lm 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),
1–99% B (1–4 min), and 99% B (4–8 min). High-resolution mass
spectra (HRMS) data were acquired in 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 MHz 1H, 150 MHz 13C) or
a Varian Mercury spectrometer (400 MHz 1H, 100 MHz 13C). Chem-
ical shifts are reported in ppm (d). Preparative HPLC was performed
on Agilent Prep 1200 series with UV detector set to 254 nm.
Samples were injected into a Phenomenex Luna 75 ꢁ 30 mm,
4.1.3. 6,7-Dimethoxy-N2-methyl-N4-(1-methylpiperidin-4-yl)-N2-
propylquinazoline-2,4-diamine (13)
The title compound (82% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.90 (s, 1H),
6.73 (s, 1H), 4.99 (d, J = 6.8 Hz 1H), 4.14–4.04 (m, 1H), 3.93 (s, 3H),
3.91 (s, 3H), 3.60 (t, J = 7.2 Hz, 2H), 3.19 (s, 3H), 2.88 (d, J = 12.0 Hz,
2H), 2.31 (s, 3H), 2.18–2.12 (m, 4H), 1.64–1.51 (m, 4H), 0.92 (t,
J = 7.2 Hz, 3H); 13C NMR (151 MHz, CD3OD) d 158.75, 158.61,
154.35, 147.97, 145.23, 103.90, 103.14, 102.73, 55.41, 54.77,
51.13, 44.85, 34.42, 30.94, 20.60, 10.38; HRMS (ESI-TOF) m/z:
[M+H]+ calcd for C20H32N5O2, 374.2551, found 374.2549.
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Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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4.1.4. N2-Isopropyl-6,7-dimethoxy-N2-methyl-N4-(1-methylpiperidin-
4-yl)quinazoline-2,4-diamine (14)
4.1.11. N2-Isobutyl-6,7-dimethoxy-N2-methyl-N4-(1-methylpiperidin-
4-yl)quinazoline-2,4-diamine (21)
The title compound (76% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.89 (s, 1H),
6.72 (s, 1H), 5.20–5.09 (m, 1H), 4.98 (d, J = 6.8 Hz 1H), 4.15–4.06
(m, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.03 (s, 3H), 2.86 (d,
J = 12.0 Hz, 2H), 2.32 (s, 3H), 2.20–2.15 (m, 4H), 1.66–1.57 (m,
2H), 1.18 (d, J = 6.8 Hz, 6H); MS (ESI) m/z 374.3 [M+H]+.
The title compound (79% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.90 (s, 1H),
6.75 (s, 1H), 5.05 (d, J = 6.8 Hz, 1H), 4.12–4.03 (m, 1H), 3.92 (s, 3H),
3.89 (s, 3H), 3.45 (d, J = 6.8 Hz, 2H), 3.19 (s, 3H), 2.87 (d, J = 12.4 Hz,
2H), 2.30 (s, 3H), 2.16–2.06 (m, 4H), 1.65–1.55 (m, 2H), 0.91 (d,
J = 6.8 Hz, 6H); MS (ESI) m/z 388.3 [M+H]+.
4.1.5. N2-Cyclopropyl-6,7-dimethoxy-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (15)
4.1.12. 6,7-Dimethoxy-N2-methyl-N4-(1-methylpiperidin-4-yl)-N2-
neopentylquinazoline-2,4-diamine (22)
The title compound (79% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.93 (s, 1H),
6.80 (s, 1H), 4.17 (d, J = 6.8 Hz, 1H), 4.19–4.11 (m, 1H), 3.90 (s, 3H),
3.87 (s, 3H), 3.17 (s, 3H), 2.86–2.83 (m, 2H), 2.87–2.69 (m, 1H), 2.28
(s, 3H), 2.17–2.09 (m, 4H), 1.63–1.53 (m, 2H), 0.82–0.78 (m, 2H),
0.67–0.65 (m, 2H); MS (ESI) m/z 372.3 [M+H]+.
The title compound (75% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.92 (s, 1H),
6.71 (s, 1H), 4.94 (d, J = 6.4 Hz, 1H), 4.17–4.08 (m, 1H), 3.95 (s, 3H),
3.92 (s, 3H), 3.57 (s, 2H), 3.25 (s, 3H), 2.89 (d, J = 12.0 Hz, 2H), 2.32
(s, 3H), 2.17–2.12 (m, 4H), 1.67–1.57 (m, 2H), 0.98 (s, 9H); MS (ESI)
m/z 402.3 [M+H]+.
4.1.6. N2-Cyclobutyl-6,7-dimethoxy-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (16)
4.1.13. N2-(Cyclohexylmethyl)-6,7-dimethoxy-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (23)
The title compound (79% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.90 (s, 1H),
6.75 (s, 1H), 5.21–5.12 (m, 1H), 5.06 (d, J = 7.2 Hz, 1H), 4.17–4.08
(m, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 3.14 (s, 3H), 2.88–2.85 (m, 2H),
2.31 (s, 3H), 2.22–2.15 (m, 8H), 1.71–1.57 (m, 4H); MS (ESI) m/z
386.3 [M+H]+.
The title compound (81% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.89 (s, 1H),
6.74 (s, 1H), 5.00 (d, J = 7.2 Hz, 1H), 4.14–4.03 (m, 1H), 3.93 (s, 3H),
3.90 (s, 3H), 3.47 (d, J = 7.2 Hz, 2H), 3.19 (s, 3H), 2.88 (d, J = 12.0 Hz,
2H), 2.31 (s, 3H), 2.17–2.10 (m, 4H), 1.83–1.76 (m, 1H), 1.73–1.70
(m, 4H), 1.65–1.56 (m, 3H), 1.21–1.13 (m, 3H), 1.00–0.91 (m,
2H); MS (ESI) m/z 428.2[M+H]+.
4.1.7. N2-Cyclopentyl-6,7-dimethoxy-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (17)
The title compound (76% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (600 MHz, CD3OD) d 7.69 (s, 1H),
7.22 (s, 1H), 5.13–5.01 (br, 1H), 4.55 (tt, J = 12.3, 4.8 Hz, 1H), 4.00
(s, 3H), 3.96 (s, 3H), 3.69 (d, J = 12.8 Hz, 2H), 3.31–3.25 (m, 2H),
3.18 (s, 3H), 2.95 (s, 3H), 2.42 (d, J = 13.7 Hz, 2H), 2.09–1.98 (m,
4H), 1.91–1.84 (s, 2H), 1.82–1.72 (m, 4H); 13C NMR (151 MHz, CD3-
OD) d 158.45, 155.80, 152.87, 147.41, 103.54, 102.45, 99.12, 57.49,
55.58, 55.33, 54.02, 44.10, 29.84, 28.95, 28.24, 23.82. HRMS (ESI-
TOF) m/z: [M+H]+ calcd for C22H34N5O2, 400.2707, found 400.2705.
4.1.14. N2-Benzyl-6,7-dimethoxy-N2-methyl-N4-(1-methylpiperidin-
4-yl)quinazoline-2,4-diamine (24)
The title compound (84% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 7.29–7.24 (m,
4H), 7.23–7.17 (m, 1H), 6.93 (s, 1H), 6.79 (s, 1H), 5.13 (d, J = 6.8 Hz,
1H), 4.93 (s, 2H), 4.07–3.98 (m, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.12
(s, 3H), 2.77 (d, J = 12.0 Hz, 2H), 2.26 (s, 3H), 2.03–2.01 (m, 4H),
1.58–1.49 (m, 2H); MS (ESI) m/z 422.3 [M+H]+.
4.1.8. N2-Cyclohexyl-6,7-dimethoxy-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (18)
4.1.15. 2-((6,7-Dimethoxy-4-((1-methylpiperidin-4-yl)amino)
quinazolin-2-yl)(methyl)amino)ethan-1-ol (25)
The title compound (83% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.89 (s, 1H),
6.68 (s, 1H), 4.90 (d, J = 6.8 Hz, 1H), 4.69–4.62 (m, 1H), 4.11–4.02
(m, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.06 (s, 3H), 2.90–2.87 (m, 2H),
2.32 (s, 3H), 2.19–2.12 (m, 4H), 1.85–1.82 (m, 2H), 1.75–1.33 (m,
10H); MS (ESI) m/z 414.3 [M+H]+.
The title compound (78% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.81 (s, 1H),
6.77 (s, 1H), 5.41 (d, J = 7.2 Hz, 1H), 4.13–4.03 (m, 1H), 3.88–3.87
(m, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.74 (t, J = 4.8 Hz, 2H), 3.22 (s,
3H), 2.83 (d, J = 11.6 Hz, 2H), 2.27 (s, 3H), 2.16–2.09 (m, 4H),
1.67–1.57 (m, 2H); MS (ESI) m/z 376.2 [M+H]+.
4.1.9. 6,7-Dimethoxy-N2-methyl-N4-(1-methylpiperidin-4-yl)-N2-
(tetrahydro-2H-pyran-4-yl)quinazoline-2,4-diamine (19)
4.1.16. 6,7-Dimethoxy-N2-(2-methoxyethyl)-N2-methyl-N4-(1-
methylpiperidin-4-yl)quinazoline-2,4-diamine (26)
The title compound (80% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.91 (s, 1H),
6.72 (s, 1H), 5.09–4.89 (m, 2H), 4.10–4.07 (m, 3H), 3.96 (s, 3H), 3.94
(s, 3H), 3.54 (t, J = 11.6 Hz, 2H), 3.09 (s, 3H), 2.91 (d, J = 12.0 Hz,
2H), 2.33 (s, 3H), 2.19–2.13 (m, 4H), 1.97–1.82 (m, 2H), 1.74–
1.53 (m, 4H); MS (ESI) m/z 416.1 [M+H]+.
The title compound (75% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.88 (s, 1H),
6.77 (s, 1H), 5.12 (d, J = 6.8 Hz, 1H), 4.12–4.03 (m, 1H), 3.90 (s, 3H),
3.88 (s, 3H), 3.82 (t, J = 4.8 Hz, 2H), 3.60 (t, J = 4.8 Hz, 2H), 3.35 (s,
3H), 3.23 (s, 3H), 2.84 (d, J = 11.6 Hz, 2H), 2.29 (s, 3H), 2.15–2.10
(m, 4H), 1.64–1.54 (m, 2H); MS (ESI) m/z 390.3 [M+H]+.
4.1.10. 6,7-Dimethoxy-N2-methyl-N4-(1-methylpiperidin-4-yl)-N2-
phenylquinazoline-2,4-diamine (20)
4.1.17. N2-(2-(dimethylamino)ethyl)-6,7-dimethoxy-N2-methyl-N4-
(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (27)
The title compound (85% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 7.37–7.31 (m,
4H), 7.16–7.13 (m, 1H), 6.96 (s, 1H), 6.75 (s, 1H), 5.02 (d, J = 6.8 Hz
1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.77–3.68 (m, 1H), 3.60 (s, 3H), 2.77
(d, J = 12.0 Hz, 2H), 2.25 (s, 3H), 1.97 (d, J = 12.0 Hz, 2H), 1.90 (t,
J = 12.0 Hz, 2H), 1.48 (qd, J = 12.0, 3.2 Hz, 2H); MS (ESI) m/z 408.1
[M+H]+.
The title compound (80% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.88 (s, 1H),
6.76 (s, 1H), 5.11 (d, J = 6.8 Hz, 1H), 4.16–4.09 (m, 1H), 3.91 (s, 3H),
3.89 (s, 3H), 3.77 (t, J = 7.2 Hz, 2H), 3.20 (s, 3H), 3.23 (s, 3H), 2.85 (d,
J = 11.6 Hz, 2H), 2.52 (t, J = 8.0 Hz, 2H), 2.30 (s, 6H), 2.29 (s, 3H),
2.15–2.10 (m, 4H), 1.66–1.57 (m, 2H); MS (ESI) m/z 403.3 [M+H]+.
Please cite this article in press as: Xiong Y., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.021

8
Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.1.18. 3-((6,7-Dimethoxy-4-((1-methylpiperidin-4-yl)amino)
quinazolin-2-yl)(methyl)amino)propan-1-ol (28)
4.1.25. 6,7-Dimethoxy-N2-(2-methoxyethyl)-N4-(1-methylpiperidin-
4-yl)quinazoline-2,4-diamine (35)
The title compound (78% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (600 MHz, CD3OD) d 7.69 (s, 1H),
7.17 (s, 1H), 4.67–4.58 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.94–3.85
(m, 2H), 3.72–3.66 (m, 4H), 3.33 (s, 3H), 3.26 (t, J = 12.0 Hz, 2H),
2.94 (s, 3H), 2.41–2.39 (m, 2H), 2.11–2.04 (m, 2H), 1.97–1.90 (m,
2H); MS (ESI) m/z 404.3 [M+H]+.
The title compound (83% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.85 (s, 1H),
6.81 (s, 1H), 5.30 (br, 1H), 4.12 (br, 1H), 4.15–4.10 (m, 1H), 3.90 (s,
3H), 3.87 (s, 3H), 3.62–3.58 (m, 2H), 3.57–3.53 (m, 2H), 3.36 (s, 3H),
2.85–2.82 (m, 2H), 2.28 (s, 3H), 2.15–2.10 (m, 4H), 1.60–1.57 (m,
2H); MS (ESI) m/z 376.2 [M+H]+.
4.1.26. 6,7-Dimethoxy-N2-(5-methylhexyl)-N4-(1-methylpiperidin-4-
yl)quinazoline-2,4-diamine (36)
4.1.19. N2,N2-Diethyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)
quinazoline-2,4-diamine (29)
The title compound (80% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (600 MHz, CD3OD) d 7.43 (s, 1H),
6.81 (s, 1H), 4.23–4.18 (m, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.40 (t,
J = 7.2 Hz, 2H), 2.97 (d, J = 12.0 Hz, 2H), 2.33 (s, 3H), 2.19 (t,
J = 12.0 Hz, 2H), 2.10 (d, J = 12.6 Hz, 2H), 1.74 (td, J = 12.0, 3.6 Hz,
2H), 1.64–1.53 (m, 3H), 1.45–1.37 (m, 2H), 1.30–1.24 (m, 2H),
0.91 (d, J = 7.8 Hz, 6H); MS (ESI) m/z 416.3 [M+H]+.
Synthesis of the title compound was reported in this previous
paper.37
4.1.20. N2-Ethyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)-N2-
propylquinazoline-2,4-diamine (30)
The title compound (79% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.87 (s, 1H),
6.70 (s, 1H), 4.98–4.82 (m, 1H), 4.18–4.00 (m, 1H), 3.94 (s, 3H), 3.92
(s, 3H), 3.67 (q, J = 7.0 Hz, 2H), 3.59–3.48 (m, 2H), 2.88 (d,
J = 11.8 Hz, 2H), 2.32 (s, 3H), 2.17–2.12 (m, 4H), 1.67–1.61(m,
4H), 1.20 (t, J = 7.0 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H); MS (ESI) m/z
388.3 [M+H]+.
4.1.27. 6-((6,7-Dimethoxy-4-((1-methylpiperidin-4-yl)amino)
quinazolin-2-yl)amino)hexan-1-ol (37)
The title compound (75% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.84 (s, 1H),
6.83 (s, 1H), 5.42 (d, J = 7.2 Hz 1H), 4.83 (br, 1H), 4.17–4.06 (m, 1H),
3.89(s, 3H), 3.86 (s, 3H), 3.59 (t, J = 6.4 Hz, 2H), 3.42–3.37 (m, 2H),
2.85–2.82 (m, 3H), 2.27(s, 3H), 2.12–2.08 (m, 4H), 1.62–1.50 (m,
6H), 1.42–1.22 (m, 4H); MS (ESI) m/z 418.1 [M+H]+.
4.1.21. N2-Butyl-N2-ethyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)
quinazoline-2,4-diamine (31)
The title compound (80% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.86 (s, 1H),
6.69 (s, 1H), 4.88 (d, J = 6.4 Hz, 1H), 4.15–4.06 (m, 1H), 3.95 (s, 3H),
3.93 (s, 3H), 3.67 (q, J = 6.8 Hz, 2H), 3.59 (t, J = 7.6 Hz, 2H), 2.88 (d,
J = 12.0 Hz, 2H), 2.32 (s, 3H), 2.17–2.12 (m, 4H), 1.66–1.57 (m, 4H),
1.41–1.33 (m, 2H), 2.00 (t, J = 6.8 Hz, 3H); 0.96 (t, J = 7.2 Hz, 3H);
MS (ESI) m/z 402.3 [M+H]+.
4.1.28. 4-(4-Chloro-6,7-dimethoxyquinolin-2-yl)morpholine
Morpholine
dimethoxyquinoline (102 mg, 0.39 mmol), Pd2(dba)3 (6 mg,
6.5 mol), BINAP (6 mg, 9.6 mol), and sodium tert-butoxide
(32
lL,
0.38 mmol),
2,4-dichloro-6,7-
l
l
(63 mg, 0.65 mmol) were mixedꢂTHF (2 mL) was added and the
resulting suspension was heated at 100 °C. and stirred in the
microwave for 20 min. The reaction mixture was filtered through
filter paper with CH2Cl2 and concentrated. The residue was purified
by HPLC to give the title compound (52 mg, 45% yield). 1H NMR
(400 MHz, CDCl3) d 7.24 (s, 1H), 7.09 (s, 1H), 6.89 (s, 1H), 3.98 (s,
3H), 3.97 (s, 3H), 3.87–3.77 (m, 4H), 3.64–3.54 (m, 4H).
4.1.22. N2-Butyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)-N2-
propylquinazoline-2,4-diamine (32)
The title compound (75% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.85 (s, 1H),
6.71 (s, 1H), 4.92 (d, J = 6.8 Hz, 1H), 4.13–4.05 (m, 1H), 3.94 (s, 3H),
3.91 (s, 3H), 3.59–3.53 (m, 4H), 2.88 (d, J = 12.0 Hz, 2H), 2.31 (s,
3H), 2.17–2.12 (m, 4H), 1.66–1.57 (m, 6H), 1.41–1.35 (m, 2H),
2.00 (t, J = 6.8 Hz, 3H); 0.97–0.91 (m, 6H); MS (ESI) m/z 416.2 [M
+H]+.
4.1.29. 6,7-Dimethoxy-N-(1-methylpiperidin-4-yl)-2-
morpholinoquinolin-4-amine (38)
4-(4-Chloro-6,7-dimethoxyquinolin-2-yl)morpholine (52 mg,
0.17 mmol), 1-methylpiperidin-4-amine (38 mg, 0.33 mmol), Pd
(OAc)2 (7 mg, 0.03 mmol), BINAP (35 mg, 0.06 mmol), and sodium
tert-butoxide (25 mg, 0.26 mmol) were mixedꢂTHF (2 mL) was
added and the resulting suspension was heated at 120 °C. and stir-
red in the microwave for 20 min. The reaction mixture was filtered
through filter paper with CH2Cl2 and concentrated. The residue
was purified by HPLC to give the title compound (46 mg, 71%
yield). 1H NMR (400 MHz, CDCl3) d 7.07 (s, 1H), 6.74 (s, 1H), 5.87
(s, 1H), 4.35 (d, J = 7.2 Hz, 1H), 3.94 (s, 6H), 3.83 (t, J = 4.8 Hz,
4H), 3.56 (t, J = 4.8 Hz, 4H), 3.50–3.41 (m, 1H), 2.86–2.78 (m, 2H),
2.31 (s, 3H), 2.22–2.01 (m, 4H), 1.69–1.60 (m, 2H); MS (ESI) m/z
387.1 [M+H]+.
4.1.23. N2-Cyclohexyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)
quinazoline-2,4-diamine (33)
The title compound (77% yield) was prepared according to syn-
thetic procedures for 12.
1H NMR (400 MHz, CDCl3) d 6.82 (s, 1H), 6.79 (s, 1H), 5.30 (br,
1H), 4.79 (br, 1H), 4.12–4.10 (m, 1H), 3.90 (s, 3H), 3.87 (s, 3H),
3.88–3.85 (m, 1H), 2.85 (d, J = 11.6 Hz, 2H), 2.29 (s, 3H), 2.14–
2.04 (m, 6H), 1.73–1.70 (m, 2H), 1.61–1.59 (m, 3H), 1.39–1.33
(m, 2H), 1.25–1.19 (m, 3H); MS (ESI) m/z 400.3 [M+H]+.
4.1.30. N2-Hexyl-6,7-dimethoxy-N4-(1-methylpiperidin-4-yl)
quinoline-2,4-diamine (39)
4.1.24. 6,7-Dimethoxy-N4-(1-methylpiperidin-4-yl)-N2-
neopentylquinazoline-2,4-diamine (34)
The title compound (35% over 2 steps) was prepared according
to synthetic procedures for 38. 1H NMR (600 MHz, CD3OD) d 7.38
(s, 1H), 7.04 (s, 1H), 5.75 (s, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.49–
3.40 (m, 1H), 3.37–3.30 (m, 2H), 2.94 (d, J = 11.4 Hz, 2H), 2.32 (s,
3H), 2.19 (t, J = 11.5 Hz, 2H), 2.11 (d, J = 12.2 Hz, 2H), 1.77–1.68
(m, 2H), 1.67–1.62 (m, 2H), 1.50–1.40 (m, 2H), 1.39–1.32 (m,
4H), 0.93 (t, J = 6.7 Hz, 3H); MS (ESI) m/z 401.4 [M+H]+.
The title compound (81% yield) was prepared according to syn-
thetic procedures for 12. 1H NMR (400 MHz, CDCl3) d 6.84 (s, 1H),
6.77 (s, 1H), 5.18 (br, 1H), 4.53 (br, 1H), 4.15–4.13 (m, 1H), 3.91 (s,
3H), 3.88 (s, 3H), 3.29 (d, J = 6.4 Hz, 2H), 2.88–2.85 (m, 2H), 2.30 (s,
3H), 2.18–2.12 (m, 4H), 1.65–1.56 (m, 2H), 0.96 (s, 9H); MS (ESI)
m/z 388.3 [M+H]+.
Please cite this article in press as: Xiong Y., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.06.021

Y. Xiong et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
4.2. GLP and G9a IC50 determination46
diffraction data and refinement statistics for the structure are dis-
played in Supporting Tables 1 and 2.
Methyltransferase activity assays for G9a and GLP were per-
formed 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 °C with
20 min incubation of 10 ml reaction mixture in 25 mM potassium
phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl2, 0.01% Triton X-100
containing 8 mM of cold SAM and 2 mM of 3H-SAM (Cat.# NET155-
V250UC; Perkin Elmer; www.perkinelmer.com), 1 mM of biotiny-
lated H3 (1–25), 5 nM G9a or GLP, and compound titrations from
1.5 nM to 25 mM. To stop the reactions, 10 mL of 7.5 M Guanidine
hydrochloride was added, followed by 60 ml 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 mix-
ing, 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%). All enzymatic reactions were per-
formed in triplicate and IC50 values were determined by fitting
the data to Four Parameter Logistic equation using GraphPad Prism
7 software.
Conflict of interest
Authors declare no financial/commercial conflicts of interest.
Acknowledgements
We thank Albina Bolotokova for compound management and
Taraneh Hajian for protein purification. The research described
here was supported by the grants R01GM103893, R01GM122749,
and R01CA218600 (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 Ingel-
heim, Canada Foundation for Innovation, Eshelman Institute for
Innovation, Genome Canada, Innovative Medicines Initiative
(EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co.,
Novartis Pharma AG, Ontario Ministry of Economic Development
and Innovation, Pfizer, Fundação de Amparo à Pesquisa do Estado
de São Paulo-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.
4.3. Isothermal titration calorimetry46
A. Supplementary data
Isothermal titration calorimetry (ITC) measurements were
made at 25 °C on a MicroCal ITC200 Instrument (Malvern Instru-
ments). Co-concentrated G9a–SAM and GLP–SAM (protein/SAM
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.021.
molar ratio of 1:5) were diluted at 35 lM in ITC buffer [50 mM Tris
References
(pH 8.0), 150 mM NaCl] supplemented with 1% DMSO. Compound
13 and 17 were prepared in DMSO at 50 mM and diluted to 0.5 mM
in ITC buffer with a final DMSO concentration of 1%. Binding con-
stants were calculated by fitting the data using the ITC data analy-
sis module in Origin 7.0 (OriginLab Corp.).
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