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

Abstract

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.

1. Introduction

GLP (euchromatic histone-lysine N-methyltransferase 1 (EHMT1) or lysine methyltransferase 1D (KMT1D)) and G9a (EHMT2 or KMT1C) are highly homologous protein lysine methyltransferases (PKMTs), sharing about 80% sequence identity in their SET (suppressor of variegation 3–9, enhancer of zeste and trithorax) domains.^1,2 GLP and G9a 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 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 diseases, and neurodegenerative disorder.^17–24

Earlier studies suggested that GLP and G9a form a catalytically active heterodimeric complex to catalyze H3K9 mono- and dimethylation.^3,4 Most of the subsequent studies have been focused on the function of G9a with the assumption that GLP would behave similarly.²⁵ More recent research challenged the above assumption and revealed GLP and G9a possess distinct physiological 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 thermogenesis.²⁹ Moreover, GLP and G9a regulate distinct sets of genes and have different effects on proliferating myoblasts and on skeletal muscle terminal differentiation.²⁵

Over the last decade, a number of quinazoline-containing compounds have been developed to selectively inhibit the methyltransferase 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 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 subsequently 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 dissect 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. Results and discussion

2.1. Design and synthesis of quinazoline and quinoline derivatives

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 inhibitor–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.

2-Amino substituted quinazoline analogs were readily prepared using the efficient two-step synthetic sequence we developed previously. ³⁷ Briefly, 4-chloro displacement of commercially available 2,4-dichloro-6,7-dimethoxyquinazoline with 4-amino-1-methylpiperidine 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).

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%.

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 structurally 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 consecutive Buchwald-Hartwig cross coupling reactions under microwave 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).

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%

2.2. Results in GLP and G9a biochemical assays

We evaluated the potency of the synthesized compounds in GLP and G9a biochemical assays, which are radioactivity-based scintillation 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.

Table 1.

SAR of the tertiary amino substituted quinazolines.

Compound	R1	IC50 (nM)
		GLP	G9a
11		11 ± 3	191 ± 74
12		18 ± 5	465 ± 88
13 (MS3748)		5 ± 2	295 ± 103
14		18 ± 2	318 ± 35
15		30 ± 8	550 ± 106
16		17 ± 2	356 ± 90
17 (MS3745)		4 ± 1	259 ± 80
18		6 ± 2	177 ± 31
19		159 ± 5	1160 ± 510
20		43 ± 23	724 ± 209
21		28 ± 4	359 ± 147
22		50 ± 18	726 ± 152
23		20 ± 3	532 ± 148
24		46 ± 9	534 ± 61
25		20 ± 2	282 ± 30
26		58 ± 17	1630 ± 230
27		24 ± 6	151 ± 46
28		73 ± 25	484 ± 85
29		23 ± 6	343 ± 51
30		20 ± 8	481 ± 129
31		23 ± 4	508 ± 26
32		36 ± 15	443 ± 57

IC₅₀ determination experiments were performed at substrate and cofactor concentrations equal to the respective K_m 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	G9a
38		6 ± 2	92 ± 33
39		11 ± 4	110 ± 42

IC₅₀ determination experiments were performed at substrate and cofactor concentrations equal to the respective K_m values for each enzyme. IC₅₀ determination experiments were performed in triplicate and the values are presented as Mean ± SD.

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), 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 containing tetrahydropyranyl (19) or aromatic phenyl (20) group greatly decreased the potency for both GLP and G9a and the selectivity for GLP over G9a. Since compound 13 is one of the most promising new GLP selective inhibitors, we synthesized and assessed more derivatives (21–28, Table 1) of this inhibitor. Branching the n-propyl group of 13 at the β-C position with a mono- (21) or dimethyl (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 β-hydroxyethyl (25), β-methoxyethyl (26), β-dimethylaminoethyl (27) and γ-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 similar 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 corresponding N-methyl analogs. Finally, we removed the fixed N-methyl group from compounds 18, 22, and 26 and prepared the corresponding 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 substituted N-hexyl group were also synthesized. However, both compounds 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 inhibitors, 13 and 17, which have high potency (IC₅₀ = ~5 nM) for GLP and high selectivity (~60-fold) for GLP over G9a.

Table 2.

SAR of the secondary amino substituted quinazolines.

Compound	R1	IC50 (nM)
		GLP	G9a
33		77 ± 6	956 ± 362
34		24 ± 5	585 ± 145
35		135 ± 27	1582 ± 526
36		27 ± 13	315 ± 88
37		59 ± 18	788 ± 28

IC₅₀ determination experiments were performed at substrate and cofactor concentrations equal to the respective K_m values for each enzyme. IC₅₀ determination experiments were performed in triplicate and the values are presented as Mean ± SD.

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), compounds 38 and 39 favored the inhibition of GLP over G9a with 15-fold and 10-fold selectivity, respectively.

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 (K_d = 57 ± 8 nM) than G9a (K_d = 510 ± 62 - nM). Similarly, compound 17 exhibited stronger binding affinity to GLP (K_d = 94 ± 14 nM) than G9a (K_d = 1070 ± 80 nM). Therefore, both compounds are confirmed to selectively bind to GLP over G9a in the ITC biophysical assay.

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.

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 complexes 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). Consistent with the previously reported GLP selective inhibitors,⁴⁶ compounds 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 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-(3′-dimethylamino)propyl group of E72 is located (Fig. 3F). This is consistent with our previous hypothesis⁴⁶ that the N-(3′-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.

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.

3. Conclusion

Extensive SAR studies around the 2-amino group of the quinazoline scaffold have led to the discovery of two new potent and 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 complexes with 13 and 17. The structures revealed that the larger N-propyl group of 13 or N-cyclopentyl group of 17 occupies a sterically 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.

4. Experiment protocols

4.1. Chemistry

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), 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). 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 × 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) 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)

The title compound (80% yield) was prepared according to synthetic procedures reported previously.^{46 1}H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.3. 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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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) δ 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.

4.1.4. N²-Isopropyl-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (14)

The title compound (76% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.5. N²-Cyclopropyl-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (15)

The title compound (79% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.6. N²-Cyclobutyl-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (16)

The title compound (79% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

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 synthetic procedures for 12. ¹H NMR (600 MHz, CD₃OD) δ 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) δ 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 (ESITOF) m/z: [M+H]⁺ calcd for C₂₂H₃₄N₅O₂, 400.2707, found 400.2705.

4.1.8. N²-Cyclohexyl-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (18)

The title compound (83% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.9. 6,7-Dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)-N²-(tetrahydro-2H-pyran-4-yl)quinazoline-2,4-diamine (19)

The title compound (80% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.10. 6,7-Dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)-N²-phenylquinazoline-2,4-diamine (20)

The title compound (85% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.11. N²-Isobutyl-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (21)

The title compound (79% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.12. 6,7-Dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)-N²-neopentylquinazoline-2,4-diamine (22)

The title compound (75% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.13. N²-(Cyclohexylmethyl)-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (23)

The title compound (81% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.14. 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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.15. 2-((6,7-Dimethoxy-4-((1-methylpiperidin-4-yl)amino) quinazolin-2-yl)(methyl)amino)ethan-1-ol (25)

The title compound (78% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.16. 6,7-Dimethoxy-N²-(2-methoxyethyl)-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (26)

The title compound (75% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.17. N²-(2-(dimethylamino)ethyl)-6,7-dimethoxy-N²-methyl-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (27)

The title compound (80% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.18. 3-((6,7-Dimethoxy-4-((1-methylpiperidin-4-yl)amino)quinazolin-2-yl)(methyl)amino)propan-1-ol (28)

The title compound (78% yield) was prepared according to synthetic procedures for 12. ¹H NMR (600 MHz, CD₃OD) δ 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]⁺.

4.1.19. N²,N²-Diethyl-6,7-dimethoxy-N⁴-(1-methylpiperidin-4-yl) quinazoline-2,4-diamine (29)

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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.21. 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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.22. 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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.23. N²-Cyclohexyl-6,7-dimethoxy-N⁴-(1-methylpiperidin-4-yl) quinazoline-2,4-diamine (33)

The title compound (77% yield) was prepared according to synthetic procedures for 12.

¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.24. 6,7-Dimethoxy-N⁴-(1-methylpiperidin-4-yl)-N²-neopentylquinazoline-2,4-diamine (34)

The title compound (81% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.25. 6,7-Dimethoxy-N²-(2-methoxyethyl)-N⁴-(1-methylpiperidin-4-yl)quinazoline-2,4-diamine (35)

The title compound (83% yield) was prepared according to synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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)

The title compound (80% yield) was prepared according to synthetic procedures for 12. ¹H NMR (600 MHz, CD₃OD) δ 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]⁺.

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 synthetic procedures for 12. ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.28. 4-(4-Chloro-6,7-dimethoxyquinolin-2-yl)morpholine

Morpholine (32 μL, 0.38 mmol), 2,4-dichloro-6,7-dimethoxyquinoline (102 mg, 0.39 mmol), Pd₂(dba)₃ (6 mg, 6.5 μmol), BINAP (6 mg, 9.6 μmol), and sodium tert-butoxide (63 mg, 0.65 mmol) were mixed·THF (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) δ 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).

4.1.29. 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 mixed·THF (2 mL) was added and the resulting suspension was heated at 120 °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 (46 mg, 71% yield). ¹H NMR (400 MHz, CDCl₃) δ 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]⁺.

4.1.30. N²-Hexyl-6,7-dimethoxy-N⁴-(1-methylpiperidin-4-yl) quinoline-2,4-diamine (39)

The title compound (35% over 2 steps) was prepared according to synthetic procedures for 38. ¹H NMR (600 MHz, CD₃OD) δ 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]⁺.

4.2. GLP and G9a IC₅₀ determination⁴⁶

Methyltransferase activity assays for G9a and GLP were performed 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 μl reaction mixture in 25 mM potassium phosphate pH 8.0, 1 mM EDTA, 2 mM MgCl₂, 0.01% Triton X-100 containing 8 μM of cold SAM and 2 μM of ³H-SAM (Cat.# NET155-V250UC; 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%). 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.

4.3. Isothermal titration calorimetry⁴⁶

Isothermal titration calorimetry (ITC) measurements were made at 25 °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 μM in ITC buffer [50 mM Tris (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 constants were calculated by fitting the data using the ITC data analysis module in Origin 7.0 (OriginLab Corp.).

4.4. Protein expression, purification, crystallization, data collection and structure determination for compounds 13 and 17

Human G9a and GLP catalytic domains were cloned, expressed and purified as previously described.⁴⁶ Purified G9a and GLP proteins at about 20 mg/ml were mixed with compound 13 and 17 at 1:4 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 7% PEG 20 K, 1.4% v/v 1,4-dioxane, 10% glycerol, 0.07 M bicine, pH 9.0 (for GLP-13); 10% PEG 20 K, 1.4% v/v 1,4-dioxane, 10% glycerol, 0.1 M bicine, pH 9.0 (for GLP-17); 20% PEG3350, 10% glycerol, 0.2 M sodium bromide (for G9a-13); 20% PEG3350, 0.2 M sodium fluoride, 10% glycerol, 0.1 M Bis-Tris propane pH 6.5 (for G9a-17). All crystals were soaked in the corresponding mother liquor supplemented with 20% glycerol as cryoprotectant before flash freezing in liquid nitrogen.

X-ray diffraction data for the different complexes were all collected at 100 K at beamline 24ID-E of Advanced Photon Source (APS), Argonne National Laboratory. Data sets were processed using MOSFLM and SCALA from the CCP4 suite (1994). The structures of the G9a and GLP complexes were solved by molecular replacement using the PHASER⁴⁷ and the atomic model of the G9a and GLP SET domains (PDB code 5TUY and 5TUZ respectively). The locations of the bound molecules were determined from a Fo-Fc difference electron density map. REFMAC⁴⁸ and phenix.refine^49,50 were used for structure refinement. Graphic program COOT⁵¹ was used for model building and visualization. Crystal diffraction data and refinement statistics for the structure are displayed in Supporting Tables 1 and 2.

Fig. 1.

Structures of selected G9a/GLP inhibitors.

A. Supplementary data

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.