Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a

Article abstract of DOI:10.1021/jm901543m

SAR exploration of the 2,4-diamino-6,7-dimethoxyquinazoline template led to the discovery of 8 (UNC0224) as a potent and selective G9a inhibitor. A high resolution X-ray crystal structure of the G9a-8 complex, the first cocrystal structure of G9a with a small molecule inhibitor, was obtained. The cocrystal structure validated our binding hypothesis and will enable structure-based design of novel inhibitors. 8 is a useful tool for investigating the biology of G9a and its roles in chromatin remodeling.

Full text of DOI:10.1021/jm901543m

7950 J. Med. Chem. 2009, 52, 7950–7953
DOI: 10.1021/jm901543m
accessible state within each cell. The state of chromatin, and
therefore access to the genetic code, is mainly regulated by
covalent and reversible PTMsa to histone proteins and DNA
and by the recognition of these marks by other proteins and
protein complexes. The PTMs of histones and DNA include
histone lysine methylation, arginine methylation, lysine acet-
ylation, sumoylation, ADP-ribosylation, ubiquitination, gly-
cosylation and phosphorylation, and DNA methylation.2
Given the widespread importance of chromatin regulation
to cell biology, the enzymes that produce these modifications
(the “writers”), the proteins that recognize them (the “read-
ers”), and the enzymes that remove them (the “erasers”) are
critical targets for manipulation to further understand the
histone code3,4 and its role in human disease. Indeed, small
molecule histone deacetylase inhibitors5 and DNA methyl-
transferase inhibitors6 have already proven useful in the
treatment of cancer.
Histone lysine methylation refers to covalent methylation of
histone lysine tails to produce mono-, di-, or trimethylated
states. Among a myriad of PTMs, histone lysine methylation
catalyzed by histone lysine methyltransferases (HMTs) has
received great attention because of its essential function in
many biological processes including gene expression and
transcriptional regulation, heterochromatin formation, and
X-chromosome inactivation.7 It is therefore considered to be
one of the most significant PTMs of histones. Since the first
HMT was characterized in 2000,8 more than 50 human histone
methyltransferases have been identified.9 Growing evidence
suggests that HMTs play important roles in the development
of various human diseases including cancer.10,11 For example,
G9a, a H3K9 methyltransferase also known as EHMT2, is
overexpressed in human cancers and knockdown of G9a
inhibits cancer cell growth.12,13
Discovery of a 2,4-Diamino-
7-aminoalkoxyquinazoline as a Potent and
Selective Inhibitor of Histone Lysine
Methyltransferase G9a†
Feng Liu,‡ Xin Chen,‡ Abdellah Allali-Hassani,§
Amy M. Quinn, Gregory A. Wasney,§ Aiping Dong,§
Dalia Barsyte,§ Ivona Kozieradzki,§ Guillermo Senisterra,§
Irene Chau,§ Alena Siarheyeva,§ Dmitri B. Kireev,‡
Ajit Jadhav, J. Martin Herold,‡ Stephen V. Frye,‡
Cheryl H. Arrowsmith,§ Peter J. Brown,§ Anton Simeonov,
Masoud Vedadi,§ and Jian Jin*,‡
‡Center for Integrated Chemical Biology and Drug Discovery,
Division of Medicinal Chemistry and Natural Products, Eshelman
School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, §Structural Genomics
Consortium, University of Toronto, Toronto, Ontario, M5G 1L6,
Canada, and NIH Chemical Genomics Center, National Human
Genome Research Institute, National Institutes of Health, Bethesda,
Maryland 20892
Received October 18, 2009
Abstract: SAR exploration of the 2,4-diamino-6,7-dimethoxyqui-
nazoline template led to the discovery of 8 (UNC0224) as a potent
and selective G9a inhibitor. A high resolution X-ray crystal struc-
ture of the G9a-8 complex, the first cocrystal structure of G9a with
a small molecule inhibitor, was obtained. The cocrystal structure
validated our binding hypothesis and will enable structure-based
design of novel inhibitors. 8 is a useful tool for investigating the
biology of G9a and its roles in chromatin remodeling.
Despite the tremendous progress made in identifying new
HMTs, only two small molecule HMT inhibitors,14-16 which are
not SAM-related analogues, have been reported since the first
HMT was characterized in 2000.8 Therefore, creating multiple,
high quality small molecule HMT inhibitors as research tools for
studying the biological function of HMTs is urgently needed.
In this Letter, we report the design and synthesis of novel
compounds to explore the 2,4-diamino-6,7-dimethoxyquina-
zoline template, and biological evaluation of these com-
pounds that led to the discovery of 8 (UNC0224) as a
potent and selective G9a inhibitor. In addition, we disclose a
high resolution (1.7 A) X-ray crystal structure of the G9a-8
complex, the first cocrystal structure of G9a with a small
molecule inhibitor.
The only previously reported small molecule inhibitor of
G9a in the literature is 2,4-diamino-6,7-dimethoxyquinazo-
line 2a (BIX-01294)15,17 (Figure 1), which also inhibited GLP
Multicellular organisms have evolved elaborate mechan-
isms to enable differential and cell-type specific expression of
genes. Epigenetics refers to these heritable changes in how the
genome is accessed in different cell types and during develop-
ment and differentiation. This capability permits specializa-
tion of function between cells even though each cell contains
thesamegenome. Over the past decade, the cellular machinery
that creates these heritable changes has been the subject of
intense scientific investigation, as there is no area of biology,
or for that matter no area of human health, where epigenetics
may not play a fundamental role.1
˚
The template upon which the epigenome is written is
chromatin, the complex of histone proteins, RNA, and
DNA that efficiently package the genome in an appropriately
†The coordinates and structure factors of UNC0224 cocrystallized
with G9a have been deposited in the Protein Data Bank (www.pdb.org,
PDB code 3K5K).
*To whom correspondence should be addressed. Phone: 919-843-
8459. Fax: 919-843-8465. E-mail: jianjin@unc.edu.
a Abbreviations: PTMs, post-translational modifications; HMT, his-
tone lysine methyltransferase; EHMT2, euchromatic histone lysine
methyltransferase 2; H3K9, histone 3 lysine 9; SAM, S-adenosyl-L-
methionine; SAR, structure-activity relationship; GLP, G9a-like pro-
tein; EHMT1, euchromatic histone lysine methyltransferase 1; SET,
suppressor of variegation 3-9, enhancer of zeste, and trithorax; SAH,
S-adenosyl-L-homocysteine; AlphaScreen, amplified luminescence
proximity homogeneous assay; ITC, isothermal titration calorimetry;
FP, fluorescence polarization; DSF, differential scanning fluorimetry.
Figure 1. Structure and reported IC50 of 2a against G9a and
GLP.15,17
r
pubs.acs.org/jmc
Published on Web 11/05/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7951
Table 2. SAR of 2-Amino Moiety
Scheme 1. Synthesis of 2,4-Diamino-6,7-dimethoxyquinazo-
lines 2a
a (a) R0 amines, DMF, DIEA, room temp; (b) R00 amines, i-PrOH,
4 M HCl/dioxane, microwave, 160 °C.
Table 1. SAR of 4-Amino Moiety
no group (2a) with 1-methylpiperidin-4-ylamino (2b) resulted
in no potency loss. This result is consistent with the X-ray
crystal structure of the GLP-2a complex, as the benzyl group
of 2a was outsidethe peptide binding grooveand did not make
any interactions.17 This key SAR finding allowed us to reduce
the molecular weight and lipophilicity of this chemical series.
On the other hand, replacing the 1-methylpiperidin-4-ylamino
(2b) with piperidin-4-ylamino (2c), tetrahydropyran-4-ylami-
no (2d), or cyclohexylamino (2e) led to significant potency
loss, indicating that an alkylated nitrogen is important for
inhibitory activity. Analogues containing a smaller amino
group such as cyclopropylamino (2f) and isopropylamino
(2g) were also significantly less potent compared to 2a and
2b. In general, assay results from the ThioGlo-based assay and
AlphaScreen are consistent. However, the AlphaScreen ap-
pears to be more sensitive for weakly active compounds. The
sensitivity of AlphaScreen for weakly active compounds is
likely due to the assay detecting amplified signal and to the use
of a lower concentration of peptide substrate.
The 2-amino moiety of the quinazoline scaffold was in-
vestigated next. In general, modifications to the 2-amino
region were well tolerated (Table 2). The methylhomopiper-
azine (2b) could be replaced with the methylpiperazine (2h)
and piperidine (2i) without significant potency loss. Analo-
gues containing morpholine (2j), diethylamine (2k), or di-
methylamine (2l) had moderate potency. The 2-chloro
analogue 2m had poor potency in both assays.
Having established initial SAR for the 2- and 4-amino
regions, we next explored the 7-methoxy moiety. The X-ray
crystal structure of the GLP-2a complex revealed that 2a
occupied the histone peptide binding site and did not interact
with the narrow lysine binding channel.17 We hypothesized
that adding a 7-aminoalkoxy side chain to the quinazoline
scaffold would make new interactions with the lysine binding
channel while the rest of molecule maintained interactions
with the peptide binding groove. Thus, we designed 8, which
possesses a 7-dimethylaminopropoxy chain and also com-
bines the best 2- and 4-amino moieties identified previously.
Synthesis of 8 is outlined in Scheme 2. Benzyl protection of
(also known as EHMT1). GLP is another H3K9 methyltrans-
ferase that shares 80% sequence identity with G9a in their
respective SET domains. Because no SAR has been reported
for this quinazoline scaffold, we decided to explore multiple
regions of this template to elucidate the SAR and improve
potency and selectivity as part of our efforts to create multiple
chemical probes for epigenetic targets and make these probes
available to the research community without restrictions on
their use.
An efficient two-step synthetic sequence was developed to
explore the 4-amino and 2-amino regions of the quinazoline
scaffold (Scheme 1). Displacing the 4-chloro of commercially
available 2,4-dichloro-6,7-dimethoxyquinazoline (1) with the
first set of amines at room temperature, followed by displace-
ment of the 2-chloro with the second set of amines under
microwave heating conditions, yielded the desired 2,4-diami-
no-6,7-dimethoxyquinazolines 2 in good yields. Using this
efficient synthesis, we rapidly prepared the compounds listed
in Tables 1 and 2. These compounds were evaluated in two
orthogonal and complementary biochemical assays:18 (1)
ThioGlo-based G9a inhibitory assay for monitoring the con-
version of SAM to SAH;19 (2) G9a AlphaScreen for the
detection of methylated histone peptides.20
The 4-amino moiety of the quinazoline scaffold was first
explored (Table 1). Replacing the 1-benzylpiperidin-4-ylami-

7952 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Scheme 2. Synthesis of Compound 8a
Liu et al.
a (a) BnBr, K2CO3, DMF, room temp; (b) HNO3, Ac2O, 0 °C to room
temp; (c) Fe dust, NH4Cl, i-PrOH-H2O, reflux, 67% over 3 steps;
(d) methyl chloroformate, DIEA, DMF-DCM, 0 °C to room temp;
(e) NaOH, H2O2, EtOH, reflux, 70% over 2 steps; (f) N,N-diethylani-
line, POCl3, reflux, 59%; (g) 4-amino-1-methylpiperidine, DIEA,
THF, room temp; (h) 1-methylhomopiperazine, HCl, i-PrOH, 160 °C,
microwave, 82% over 2 steps; (i) Pd/C, H2, EtOH, room temp; (j)
3-(dimethylamino)propan-1-ol, PPh3, DIAD, THF, 0 °C to room temp,
46% over 2 steps.
Figure 3. 8 showed higher binding affinity to G9a compared to 2a
in an ITC experiment.
Figure 4. 8 (b) displaced fluorescein labeled 15-mer H3 peptide
(1-15) better than 2a (O) (unlabeled 25-mer H3 peptide (1-25) (1)
used as a positive control).
labeled 15-mer H3 peptide (1-15) better than 2a in a G9a FP
assay (Figure 4). In addition, 8 stabilized G9a better com-
pared to 2a in DSF experiments.22 These results together
strongly suggest that 8 is a significantly more potent G9a
inhibitor compared to 2a.
Figure 2. Full concentration response curves of 8 (O) (IC50 = 15 (
10 nM) and 2a (b) (IC50 = 106 ( 20 nM) in the G9a ThioGlo assay.
Although 8 also potently inhibited GLP with an IC50 of
20 and 58 nM in the ThioGlo assay and AlphaScreen,
respectively, 8 was more than 1000-fold selective for G9a over
SET7/9 (a H3K4 HMT) and SET8/PreSET7 (a H4K20
HMT) in ThioGlo-based biochemical assays. In addition, 8
was clean (less than 20% inhibitions at 1 μM) against a broad
range of G-protein-coupled receptors, ion channels, and
transporters in a 30-target selectivity panel (tested by MDS
Pharma Services) except hitting muscarinic M2 receptor at
82% inhibition at 1 μM and histamine H1 receptor at 31%
inhibition at 1 μM.
commercially available 2-methoxy-4-cyanophenol (3) fol-
lowed by nitration and subsequent reduction of the nitro
group produced aniline 4. Aniline 4 was then converted to
quinazolinedione 5 via formation of methyl carbamate and
subsequent saponification of the cyano group and ring clo-
sure. POCl3 treatment of intermediate 5 resulted in 2,4-
dichloroquinazoline 6, which underwent two consecutive
chloro displacement reactions to yield 2,4-diaminoquinazo-
line 7. Debenzylation of intermediate 7, followed by Mitsu-
nobu reaction with 3-(dimethylamino)propan-1-ol, produced
the desired 8.
We were pleased to find that 8 was a potent G9a inhibitor
with an IC50 of 15 nM, 7 times more potent compared to 2a
(IC50 = 106 nM), in the G9a ThioGlo assay (Figure 2).
Although 8 (IC50 = 289 nM) had similar potency compared
to 2a (IC50 = 290 nM) in the G9a AlphaScreen (likely due to 8
reaching the IC50 limit of the G9a AlphaScreen), the high
potency of 8 was confirmed in several secondary assays. In
ITC experiments that measure the binding affinity of a small
molecule to the G9a protein,21 8 (Kd = 23( 8 nM (n = 2)) has
about 5-fold higher binding affinity compared to 2a (Kd =
130 ( 18 nM (n = 2)) (Figure 3). 8 also displaced fluorescein
˚
A high resolution (1.7 A) X-ray crystal structure of the
G9a-8 complex, the first crystal structure of a G9a-small
molecule inhibitor complex, was obtained. As shown in
Figure 5, we were pleased to find that the 7-dimethylamino-
propoxy side chain of 8 indeed occupied the lysine binding
channel of G9a nicely, thus validating our binding hypothesis.
The higher potency of 8 compared to 2a can be explained by
these additional interactions between the 7-dimethylamino-
propoxy side chain and the lysine binding channel, and these
interactions were absent in the GLP-2a complex. Other key
features include the following: (1) the distal nitrogen of the

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7953
(3) Strahl, B. D.; Allis, C. D. The language of covalent histone
modifications. Nature 2000, 403, 41–45.
(4) Jenuwein, T.; Allis, C. D. Translating the histone code. Science
2001, 293, 1074–1080.
(5) Esteller, M. Epigenetics in cancer. N. Engl. J. Med. 2008, 358, 1148–
1159.
(6) Lyko, F.; Brown, R. DNA methyltransferase inhibitors and the
development of epigenetic cancer therapies. J. Natl. Cancer Inst.
2005, 97, 1498–1506.
(7) Martin, C.; Zhang, Y. The diverse functions of histone lysine
methylation. Nat. Rev. Mol. Cell Biol. 2005, 6, 838–849.
(8) Rea, S.; Eisenhaber, F.; O’Carroll, D.; Strahl, B. D.; Sun, Z. W.;
Schmid, M.; Opravil, S.; Mechtler, K.; Ponting, C. P.; Allis, C. D.;
Jenuwein, T. Regulation of chromatin structure by site-specific
histone H3 methyltransferases. Nature 2000, 406, 593–599.
(9) Kouzarides, T. Chromatin modifications and their function. Cell
2007, 128, 693–705.
(10) Spannhoff, A.; Sippl, W.; Jung, M. Cancer treatment of the future:
inhibitors of histone methyltransferases. Int. J. Biochem. Cell Biol.
2009, 41, 4–11.
(11) Fog, C. K.; Jensen, K. T.; Lund, A. H. Chromatin-modifying
proteins in cancer. APMIS 2007, 115, 1060–1089.
(12) McGarvey, K. M.; Fahrner, J. A.; Greene, E.; Martens, J.;
Jenuwein, T.; Baylin, S. B. Silenced tumor suppressor genes
reactivated by DNA demethylation do not return to a fully
euchromatic chromatin state. Cancer Res. 2006, 66, 3541–3549.
(13) Kondo, Y.; Shen, L.; Ahmed, S.; Boumber, Y.; Sekido, Y.;
Haddad, B. R.; Issa, J. P. Downregulation of histone H3 lysine
9 methyltransferase G9a induces centrosome disruption and
chromosome instability in cancer cells. PLoS ONE 2008, 3, No.
e2037.
(14) Greiner, D.; Bonaldi, T.; Eskeland, R.; Roemer, E.; Imhof, A.
Identification of a specific inhibitor of the histone methyltransfer-
ase SU(VAR)3-9. Nat. Chem. Biol. 2005, 1, 143–145.
(15) Kubicek, S.; O’Sullivan, R. J.; August, E. M.; Hickey, E. R.;
Zhang, Q.; Teodoro, M. L.; Rea, S.; Mechtler, K.; Kowalski, J.
A.; Homon, C. A.; Kelly, T. A.; Jenuwein, T. Reversal of
H3K9me2 by a small-molecule inhibitor for the G9a histone
methyltransferase. Mol. Cell 2007, 25, 473–481.
Figure 5. X-ray crystal structure of the G9a-8 complex (PDB code
3K5K). Compound 8 is in light and dark blue. The superposed
histone backbone trace and the methylated lysine side chain are in
magenta.
homopiperazine interacts with Asp1074; (2) 4-amino group
interacts with Asp1083; (3) the bulk of 8 occupies the histone
peptide binding site. The inhibitor-enzyme interactions re-
vealed by this high resolution cocrystal structure will enable
future structure-based design of novel HMT inhibitors.
In conclusion, 8, a potent and selective inhibitor of histone
lysine methyltransferase G9a, was discovered via SAR
explorationandstructure-baseddesign. Thefirst X-raycrystal
structure of G9a with a small moleculeinhibitor was obtained.
This high resolution cocrystal structure of the G9a-8 com-
plex validated our binding hypothesis and will enable struc-
ture-based design of novel inhibitors. 8 is a potentially
valuable small molecule tool for the research community to
investigate the biology of G9a and its roles in chromatin
regulation.
(16) Cole, P. A. Chemical probes for histone-modifying enzymes. Nat.
Chem. Biol. 2008, 4, 590–597.
(17) Chang, Y.; Zhang, X.; Horton, J. R.; Upadhyay, A. K.; Spannhoff,
A.; Liu, J.; Snyder, J. P.; Bedford, M. T.; Cheng, X. Structural basis
for G9a-like protein lysine methyltransferase inhibition by BIX-
01294. Nat. Struct. Mol. Biol. 2009, 16, 312–317.
(18) The G9a ThioGlo assay IC50 values in this paper are the average
of at least duplicate assay runs with standard deviation (SD)
within 1-fold unless noted otherwise. The G9a AlphaScreen
IC50 values in this paper are the average of at least duplicate
assay runs with standard error of the mean (SEM) <35%. The
observed potency difference for 2a (BIX-01294) in our G9a
ThioGlo and AlphaScreen assays (Table 1) versus the previously
reported IC50 values (shown in Figure 1) is due to use of different
assays.
Acknowledgment. We thank Dr. Yizhou Dong and Pro-
fessor K. H. Lee for HRMS and HPLC support.
(19) Collazo, E.; Couture, J. F.; Bulfer, S.; Trievel, R. C. A coupled
fluorescent assay for histone methyltransferases. Anal. Biochem.
2005, 342, 86–92.
Supporting Information Available: Procedures and character-
ization data for all compounds; procedures for biochemical
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
(20) Quinn, A. M.; Allali-Hassani, A.; Vedadi, M.; Simeonov, A.
A chemiluminescence-based method for identification of histone
lysine methyltransferase inhibitors. Mol. BioSyst., submitted. G9a
AlphaScreen assay protocol is included in Supporting Information.
(21) Min, J.; Allali-Hassani, A.; Nady, N.; Qi, C.; Ouyang, H.; Liu, Y.;
MacKenzie, F.; Vedadi, M.; Arrowsmith, C. H. L3MBTL1 recog-
nition of mono- and dimethylated histones. Nat. Struct. Mol. Biol.
2007, 14, 1229–1230.
References
(1) Bernstein, B. E.; Meissner, A.; Lander, E. S. The mammalian
epigenome. Cell 2007, 128, 669–681.
(2) Gelato, K. A.; Fischle, W. Role of histone modifications in defining
(22) DSF graphs of 8 (UNC0224) and 2a are included in Supporting
Information.
chromatin structure and function. Biol. Chem. 2008, 389, 353–363.