DNA Methyltransferase 32 nM (IC50) DNMT1 1.5 nM (Kd) DNMT3A 92 nM (IC50) DNMT3B 1000 nM (IC50) G9a 16 nM (IC50)

CM-579 has a Kd of 1.5 nM for DNMT1. With an IC50 of 92 nM for DNMT3A and 1000 nM for DNMT3B, respectively, CM-579 likewise inhibits these enzymes[1].

CM-272 (CM-579 analog) shows anti-leukaemic effects in vivo. [1]
ALL-derived CEMO cells (10 × 106) were injected i.v. in immunodeficient Rag2−/−γc−/− mice, which were treated with 2.5 mg kg−1 of CM-272 administered daily, starting 3 days after injection and continued during 28 days. Control animals received saline solution under the same protocol. CM-272 therapy induced a statistically significant increase in overall survival (OS) in mice in comparison with control animals (median OS; 92±5.7 days versus 55±10.5 days; P=0.0009) (Fig. 4a). Global H3K9me2 and 5mC levels were measured in the extract from total liver. Tumour infiltration was analysed in liver homogenates by flow cytometry (FACS) analysis and showed an infiltration of 60–80% of human cells (hCD45+). Both marks were reduced in leukaemic cells obtained from animals after 1 week of treatment (Supplementary Fig. 10f). No significant weight loss was observed in treated animals (Supplementary Fig. 10g). We obtained similar results in a second in vivo replicate with CEMO-1 cells (Supplementary Fig. 11a). To analyse the dose-dependent efficacy of CM-272 in vivo, we repeated the same study administering 1 mg kg−1 of CM-272 (Supplementary Fig. 12). We did not observe differences in the body weight of the animals (Supplementary Fig. 12a) nor significant changes in haematological parameters (Supplementary Fig. 12b) between mice treated with 1 mg kg−1 or 2.5 mg kg−1 of CM-272 and the control group. As expected, CM-272 plasma concentration was greater in the mice group treated with 2.5 mg kg−1 of CM-272 (Supplementary Fig. 12c). However, treatment with 1 mg kg−1 of CM-272 was not able to prolong survival of the mice unlike what was observed when the 2.5 mg kg−1 of CM-272 was used (Supplementary Fig. 12d). These results demonstrate dose-dependent efficacy of CM-272 and that a dose of 2.5 mg kg−1 of CM-272 is adequate to demonstrate the positive anti-tumour efficacy. In a second xenogeneic model, 10 × 106 of AML-derived MV4-11 cells were injected i.v. in Rag2−/−γc−/− mice, and 14 days latter animals were treated with 2.5 mg kg−1 of CM-272 for 28 days. As in ALL cells, CM-272 therapy prolonged OS in mice (median OS for treated versus untreated mice, 78±12 days versus 57±0.9 days; P=0.0005) (Fig. 4b). We obtained similar results in a second in vivo replicate with MV4-11 cells (Supplementary Fig. 11b), without any sign of toxicity (Supplementary Fig. 10h). Finally, 2.5 × 106 cells from the OCI-Ly10 activated B-cell DLBCL cell line were similarly i.v. injected into Rag2−/−γc−/− mice. Treatment with CM-272 at the same dose during 8 weeks also prolonged OS of treated mice in comparison to control animals (median OS; 59±8 days versus 49±6 days; P=0.010) (Fig. 4c). We obtained similar results in a second in vivo replicate with OCI-Ly10 cells (Supplementary Fig. 11c), without any sign of toxicity (Supplementary Fig. 10i). Although the effect on lymphoma cells was statistically significant, the effect was less robust than in the case of AML and ALL cells. These results show that CM-272 exerts a potent anti-tumour activity in vivo against different types of haematological malignancies by inhibiting the methyltransferase activity of both G9a/GLP and DNMTs. In addition to the information described above, minimal promiscuity versus other SAM-dependent epigenetic enzymes (Supplementary Tables 4a and 4b), further off-target selectivity profiling against other drug targets in cancer (a panel of 97 kinases, Supplementary Tables 12–14) confirmed G9a (and GLP) and DNMTs as primary main targets for CM-272.

G9a and DNMT1 enzyme activity assays [1]
G9a and DNMT1 activities were measured using a time-resolved fluorescence energy transfer (TR-FRET). For G9a, TR-FRET is observed when biotinylated histone monomethyl-H3K9 peptide is incubated with cryptate-labelled anti-dimethyl-histone H3K9 antibody and streptavidin XL665 after enzymatic reaction of G9a. For DNMT1, TR-FRET is observed when antibody specific to S-adenosylhomocysteine labelled with Lumi4-Tb (donor) is incubated with d2-labelled S-adenosylhomocysteine (acceptor), using the EPIgeneous methyltransferase assay. Details are provided in Supplementary Information. The radioligand binding assay against G9a, DNMT1 and GLP was performed by Reaction Biology Corporation (http://www.reactionbiology.com).

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Epigenetics selectivity panel[1]
Selectivity of CM-272 and CM-579 against 37 epigenetic enzyme targets including Bromodomain-containing enzymes (ATAD2A, ATAD2B, BAZ2B, BRD1, BRD2(BD1+BD2), BRD4(BD1+BD2), BRDT(BD1), CREBBP, TRIM24, TAF1), Histone methyltransferases (EZH1, EZH2, GLP, PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT8, SETD2, SET7/9, SUV39H1, SUV39H2 and MLL-WARD), DNA methyltransferases (DNMT3A and DNMT3B) and histone demethylase (JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD2E, JMJD3, JMJD1A, LSD1, Jarid1A, Jarid1B and Jarid1C) was performed by BPS Bioscience (http://www.bpsbioscience.com/index.ph).

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HDAC1 and HDAC6 enzyme activity assays[1]
HDAC1 and HDAC6 enzyme activities were measured with a specific fluorescence-labelled substrate, containing an acetylated lysine side chain, after its deacetylation by HDACs. Details are provided in Supplementary Information.

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Kinase selectivity profiling[1]
The selectivity profiling of CM-272 against a selected panel of 97 kinases distributed through the kinome (out of which 90 are non-mutant kinases) was performed at DiscoverRx (http://www.discoverx.com/home) using the KINOMEscan screening platform at a test concentration of 10 μM.

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Direct binding analysis[1]
MicroScale Thermophoresis (MST) was performed to quantify biomolecular interactions between CM-579 and DNMT1 (full length). The MST analysis was performed using the Monolith NT.115 instrument.

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ADME profiling[1]
The following ADME studies: CYP inhibition on five human cytochrome P450s (1A2, 2C9, 2C19, 2D6 and 3A4 at 10 μM) in human liver microsomes, plasma protein binding, kinetic solubility, Pampa permeability and human and mouse liver microsomal stability were performed by Wuxi (http://www.wuxi.com/).

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hERG blockade assay[1]
The effect of the compound on hERG potassium channels was determined using the PredictorTM hERG fluorescence polarization commercial assay kit.

Cell Proliferation Assay[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4- 11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Inhibited cell proliferation in a dose- and time-dependent manner.

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Cell Cycle Analysis [1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells) ; 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 24 hrs (hours)
Experimental Results: Blocked cell cycle progression.

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Apoptosis Analysis[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Induced apoptosis in ALL, AML and DLBCL cell lines in a dose- and time-dependent manner.

Animal/Disease Models: Female BALB/Ca-Rag2−/−γc−/− mice (6–8weeks old) with CEMO-1 cells[1]
Doses: 2.5 mg/kg
Route of Administration: intravenous (iv) injection; daily; for 28 days
Experimental Results: Induced a statistically significant increase in overall survival (OS) in mice.

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PK study of CM-272 and CM-579 in plasma samples [1]
CM-272 and CM-579 were measured in plasma samples using a Xevo-TQ MS triple quadropole mass spectrometer with an electrospray ionization (ESI) source and an Acquity UPLC. CM-272 and CM-579 solutions were prepared by dissolving the solid in saline. A drug dosage of 1 mg kg−1 or 2.5 mg kg−1 (CM-272) or 1 mg kg−1 (CM-579 ) was administered as a single intravenous injection. Blood was collected at predetermined times over 24 h post injection (0.25, 2, 4, 6, 8 and 24 h for CM-272) and (0.25, 1, 2, 4 and 8 h for CM-579). Chromatographic separation was performed by gradient elution at 0.6 ml min−1 using an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm particle size). The PK parameters were obtained by fitting the blood concentration-time data to a non-compartmental model with the WinNonlin software. Details are provided in Supplementary Information.

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In vivo experiments[1]
The human ALL CEMO-1 (control group with saline solution n=6; treated group with CM-272, n=6), AML MV4-11 (control group with saline solution n=8; treated group with CM-272 n=8) and DLBCL OCI-Ly10 (control group with saline solution n=6; treated group with CM-272 n=6) xenograft mice models were generated by i.v. injection of cells diluted in 100 μl of saline solution in the tail vein of a 6–8-week-old female BALB/cA−Rag2−/−γc−/− mice as described in Supplementary Information. CM-272 administration was detailed in Supplementary Information. Statistical results were calculated using the statistical software medcalc.

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CM-272 toxicity assay: haematological and liver parameters[1]
After treating Rag2−/−γc−/− mice with daily i.v. 2.5 mg kg−1 of CM-272 during 4 weeks, followed by a 7 days washout period, haematological and liver parameters were measured as described in Supplementary Information.

Prior to conducting pharmacokinetic studies, the researchers determined the maximum tolerated dose (MTD) of CM-272 to be 2.5 mg kg⁻¹ (intravenous injection); however, for CM-579, doses higher than 1 mg kg⁻¹ (intravenous injection) could not be administered. Pharmacokinetic studies conducted using the MTD dose (Supplementary Tables 9-11) showed a clearance of 5.7 L h⁻¹ kg⁻¹ for CM-579 and 0.91 L h⁻¹ kg⁻¹ for CM-272.

Before evaluating the in vivo efficacy of our dual inhibitors, the researchers examined the therapeutic window and pharmacokinetic (PK) parameters of both molecules. Therefore, we investigated the toxicity of CM-272 and CM-579 using the non-tumor hepatocyte cell line THLE-2 (LC50 of 1.78 and 1.30 μM, respectively) and peripheral blood mononuclear cells (PBMCs) from healthy donors (LC50 of 1.90 and 7.39 μM, respectively) (Supplementary Table 2) and compared it with the activity of in vitro antitumor cell lines (Supplementary Table 3). The researchers found that both CM-272 and CM-579 had acceptable therapeutic windows (approximately 1 log unit). Prior to conducting pharmacokinetic studies, we determined the maximum tolerated dose (MTD) of CM-272 to be 2.5 mg kg⁻¹ (intravenous injection); however, for CM-579, the researchers were unable to administer doses exceeding 1 mg kg⁻¹ (intravenous injection). Pharmacokinetic studies using the MTD dose (Supplementary Tables 9-11) showed that the clearance of CM-579 was 5.7 L h⁻¹ kg⁻¹, and the clearance of CM-272 was 0.91 L h⁻¹ kg⁻¹. [1]

[1]. Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies. Nat Commun. 2017 May 26;8:15424.

CM-272 belongs to the aminoquinoline class of compounds, with its quinoline derivatives having 5-methylfuran-2-yl, (1-methylpiperidin-4-yl)amino, methoxy, and 3-(pyrrolidone-1-yl)propoxy substitutions at positions 2, 4, 6, and 7, respectively. It is a dual G9a/DNA methyltransferase inhibitor with antitumor activity. It inhibits G9a, DNMT1, DNMT3A, DNMT3B, and GLP (IC50 values of 8 nM, 382 nM, 85 nM, 1200 nM, and 2 nM, respectively). It exhibits apoptosis-inducing, ferroptosis-inducing, antitumor, EC 2.1.1.43 (enhancer Zeste homolog 2), and EC 2.1.1.37 (DNA (cytosine-5-)methyltransferase) activities. It is an N-alkylpyrrolidine compound, belonging to the furans, aminoquinolines, aromatic ethers, piperidines, tertiary amines, secondary amines, and diethers. Epigenetics plays an undeniable role in cancer, and the reversibility of epigenetic alterations has facilitated the development of epigenetic drugs. This study designed and synthesized a highly efficient, novel, selective, and reversible chemical probe that can simultaneously inhibit the activity of G9a and DNMTs methyltransferases. In vitro experiments showed that the lead compound CM-272 can inhibit the proliferation of hematological malignancies (acute myeloid leukemia-AML, acute lymphoblastic leukemia-ALL, and diffuse large B-cell lymphoma-DLBCL) cells and promote apoptosis, inducing interferon-stimulated gene expression and immunogenic cell death. CM-272 significantly prolonged the survival of xenograft models of AML, ALL, and DLBCL. Our findings represent the first discovery of a dual G9a/DNMTs inhibitor and confirm that this series of compounds holds promise for solving a pressing problem in the treatment of hematological malignancies. [1] Transcriptome analysis consistently showed that CM-272 treatment induced tumor type I interferon responses in AML, ALL, and DLBCL cells, accompanied by ISG expression and induction of immunogenic cell death (ICD), suggesting a common anti-tumor mechanism. Although immunogenic cell death (ICD) has not been described as the mechanism of action of epigenetic drugs, these results may at least be partially predictable, as recent studies have shown that the expression of interferon-stimulating genes (ISG) is epigenetically regulated by H3K9me2 (reference 30), which supports the role of G9a inhibition in activating type I interferon responses and ICD. Therefore, we speculate that the use of immunodeficient mice that cannot produce anti-tumor immune responses may underestimate the efficacy of CM-272 against tumor cells, and therefore, immunologically normal models need to be evaluated to explore the full potential therapeutic effects of our compound. Based on recent studies showing that type I interferon response helps improve the efficacy of chemotherapy drugs³², the combination of CM-272 with such drugs and/or immunomodulators (e.g., checkpoint inhibitors) may be an attractive treatment strategy. In summary, CM-272 is a potent, novel, first-in-class, dual reversible inhibitor that inhibits G9a (GLP) and DNMTs, and it prolongs survival in in vivo models of hematologic malignancies at least in part by inducing immunogenic cell death. These compounds represent a new approach to safely and effectively targeting cancer, paving the way for the treatment of a variety of poorly prognostic human tumors. [1]

C29H43CL3N4O3

602.04

600.2400

2448471-08-5

CM-579;1846570-40-8

138454770

Light yellow to yellow solid powder

4

7

10

39

654

0

XKSDREMVFCQUPC-UHFFFAOYSA-N

InChI=1S/C29H40N4O3.3ClH/c1-21-7-8-27(36-21)26-18-24(30-20-22-9-14-32(2)15-10-22)23-17-28(34-3)29(19-25(23)31-26)35-16-6-13-33-11-4-5-12-33;;;/h7-8,17-19,22H,4-6,9-16,20H2,1-3H3,(H,30,31);3*1H

6-methoxy-2-(5-methylfuran-2-yl)-N-[(1-methylpiperidin-4-yl)methyl]-7-(3-pyrrolidin-1-ylpropoxy)quinolin-4-amine;trihydrochloride

CM-579 trihydrochloride; CM579 triHCl; CM 579 3HCl; 2448471-08-5;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~37.5 mg/mL (~62.29 mM)
DMSO :~33.33 mg/mL (~55.36 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 16.67 mg/mL (27.69 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)