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

CM-579 对 DNMT1 的 Kd 为 1.5 nM。 CM-579 同样抑制这些酶，DNMT3A 的 IC50 为 92 nM，DNMT3B 的 IC50 为 1000 nM[1]。

CM-272 (CM-579 类似物)在体内具有抗白血病作用。[1]
将all来源的CEMO细胞（10 × 106）静脉注射于免疫缺陷的Rag2−/−γ - c−/−小鼠，每天给予2.5 mg kg−1 CM-272，注射后3天开始，持续28天。对照动物按相同方案给予生理盐水。与对照动物相比，CM-272治疗诱导小鼠的总生存期（OS）有统计学意义的增加(中位OS；92±5.7天vs 55±10.5天；P=0.0009)（图4a）。测定全肝提取物中H3K9me2和5mC的水平。通过流式细胞术（FACS）分析肝脏匀浆中的肿瘤浸润情况，发现60-80%的人细胞（hCD45+）浸润。治疗1周后，从动物身上获得的白血病细胞中，这两种标记都降低了（补充图10f）。治疗后的动物没有明显的体重减轻（补充图10g）。我们在como -1细胞的第二次体内复制中获得了类似的结果（补充图11a）。为了分析CM-272在体内的剂量依赖性功效，我们重复了同样的研究，给药1 mg kg - 1 CM-272（补充图12）。我们没有观察到动物体重的差异（补充图12a），也没有观察到1 mg kg - 1或2.5 mg kg - 1 CM-272与对照组之间的血液学参数的显著变化（补充图12b）。正如预期的那样，在2.5 mg kg - 1 CM-272处理的小鼠组中，CM-272血浆浓度更高（补充图12c）。然而，与2.5 mg kg - 1 CM-272不同，1 mg kg - 1 CM-272不能延长小鼠的存活时间（补充图12d）。这些结果表明CM-272的有效性是剂量依赖性的，2.5 mg kg - 1剂量的CM-272足以显示出积极的抗肿瘤功效。在第二种异种模型中，将10 × 106个aml来源的MV4-11细胞静脉注射Rag2 - / - γ - c - / -小鼠，14天后的动物用2.5 mg kg -1 CM-272治疗28天。与ALL细胞一样，CM-272治疗延长了小鼠的生存期(治疗组和未治疗组的中位生存期分别为78±12天和57±0.9天；P=0.0005)（图4b）。我们在MV4-11细胞的第二次体内复制中获得了类似的结果（补充图11b），没有任何毒性迹象（补充图10h）。最后，将OCI-Ly10活化的b细胞DLBCL细胞系2.5 × 106个细胞同样静脉注射到Rag2−/−γc−/−小鼠体内。与对照组相比，在8周内用相同剂量的CM-272治疗也延长了治疗小鼠的生存期(中位生存期；59±8天vs 49±6天；P=0.010)（图4c）。我们在OCI-Ly10细胞的第二次体内复制中获得了类似的结果（补充图11c），没有任何毒性迹象（补充图10i）。虽然对淋巴瘤细胞的影响在统计学上是显著的，但其效果不如AML和ALL细胞那么强。这些结果表明，CM-272通过抑制G9a/GLP和dnmt的甲基转移酶活性，在体内对不同类型的血液系统恶性肿瘤具有有效的抗肿瘤活性。除了上述信息外，与其他sam依赖的表观遗传酶相比，最小的混杂性（补充表4a和4b），进一步对癌症中其他药物靶点的脱靶选择性分析（一组97种激酶，补充表12-14）证实了G9a（和GLP）和dnmt是CM-272的主要靶点。

G9a和DNMT1酶活性测定[1]
采用时间分辨荧光能量转移法（TR-FRET）测定G9a和DNMT1活性。对于G9a，在G9a酶促反应后，将生物素化组蛋白单甲基H3K9肽与crypate标记的抗二甲基组蛋白H3K9抗体和链亲和素XL665孵育，观察TR-FRET。对于DNMT1，将Lumi4-Tb（供体）标记的s -腺苷型同型半胱氨酸特异性抗体与d2标记的s -腺苷型同型半胱氨酸（受体）一起孵育，使用EPIgeneous methyltransferase assay，观察到TR-FRET。详情请参阅补充资料。 针对G9a、DNMT1和GLP的放射性配体结合试验由Reaction Biology Corporation （http://www.reactionbiology.com）进行。

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表观遗传选择性面板[1]
选择性的cm - 272和CM-579 对37表观遗传酶目标包括Bromodomain-containing酶(ATAD2A、ATAD2B BAZ2B, BRD1, BRD2(1型+ BD2), BRD4(1型+ BD2), BRDT(1型),CREBBP, TRIM24, TAF1),组蛋白甲基转移酶(EZH1, EZH2, GLP、PRMT1 PRMT3, PRMT4, PRMT5, PRMT6, PRMT8, SETD2, SET7/9, SUV39H1, SUV39H2和MLL-WARD), DNA甲基转移酶(种能阻碍DNMT3B DNMT3A和)和组蛋白demethylase (JMJD2A、JMJD2B JMJD2C, JMJD2D, JMJD2E, JMJD3, JMJD1A, LSD1, Jarid1A,Jarid1B和Jarid1C)由BPS Bioscience （http://www.bpsbioscience.com/index.ph）进行。

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HDAC1和HDAC6酶活性测定[1]
HDAC1和HDAC6酶的活性是用特定的荧光标记的底物来测定的，底物含有乙酰化的赖氨酸侧链，经过hdac去乙酰化。详情请参阅补充资料。

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激酶选择性分析[1]
在DiscoverRx （http://www.discoverx.com/home）上使用KINOMEscan筛选平台，在10 μM的测试浓度下，对分布在kinome中的97个选定的激酶（其中90个是非突变激酶）进行CM-272的选择性分析。

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直接结合分析[1]
采用微尺度热泳术（MST）定量CM-579 与DNMT1（全长）之间的生物分子相互作用。MST分析使用Monolith NT.115仪器进行。

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ADME分析[1]< br > 以下ADME研究：CYP对人肝微粒体5种细胞色素p450 （1A2, 2C9, 2C19, 2D6和3A4, 10 μM）的抑制作用，血浆蛋白结合，动力学溶解度，Pampa渗透性和人和小鼠肝微粒体稳定性的研究由无锡公司（http://www.wuxi.com/）完成。

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hERG阻断试验[1]
使用PredictorTM hERG荧光偏振商业检测试剂盒测定化合物对hERG钾通道的影响。

细胞增殖测定[1]
细胞类型： CEMO-1、MV4-11 和 OCI-Ly10 细胞系
测试浓度： 125 nM、250 nM , 500 nM（CEMO-1 细胞）； 135 nM、270 nM、540 nM（MV4-11 细胞）； 100 nM、400 nM、1000 nM（OCI-Ly10 细胞）
孵育时间：12 小时、24 小时、48 小时和 72 小时
实验结果：以剂量和时间依赖性方式抑制细胞增殖。

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细胞周期分析 [1]
细胞类型： CEMO-1、MV4-11 和 OCI-Ly10 细胞系
测试浓度： > 125 nM、250 nM、500 nM（CEMO-1 细胞）； 135 nM、270 nM、540 nM（MV4-11 细胞）； 100 nM、400 nM、1000 nM（OCI-Ly10 细胞）
孵育时间： 24 小时
实验结果： 阻断细胞周期进程。

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细胞凋亡分析[1]
细胞类型： CEMO-1、MV4-11 和 OCI-Ly10 细胞系
测试浓度： 125 nM、250 nM、500 nM（CEMO-1 细胞）； 135 nM、270 nM、540 nM（MV4-11 细胞）； 100 nM、400 nM、1000 nM（OCI-Ly10 细胞）
孵育时间：12 小时、24 小时、48 小时和 72 小时
实验结果：以剂量和时间依赖性方式诱导 ALL、AML 和 DLBCL 细胞系凋亡。

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.

Before performing PK studies, researchers identified the maximum tolerated dose (MTD) for CM-272, which it was at 2.5 mg kg−1 (intravenous, i.v.); however, for CM-579 we could not administer a dose higher than 1 mg kg−1 (i.v.). PK studies that researchers performed using the MTD dose (Supplementary Tables 9–11), showed clearance levels for CM-579 (5.7 l h−1 kg−1) and CM-272 (0.91 l h−1 kg−1).

Before evaluating the in vivo efficacy of our dual inhibitors, researchers examined the therapeutic window achieved by these two molecules and their pharmacokinetic (PK) parameters. Thus, we studied toxicity of CM-272 and CM-579 using the non-tumoural hepatic cell line THLE-2 (LC50s were 1.78 and 1.30 μM) as well as peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (LC50s were 1.90 and 7.39 μM) (Supplementary Table 2) and compared with the in vitro activity against tumour cell lines (Supplementary Table 3). Researchers detected that CM-272 and CM-579 showed an acceptable therapeutic window (around 1 log units). Before performing PK studies, we identified the maximum tolerated dose (MTD) for CM-272, which it was at 2.5 mg kg−1 (intravenous, i.v.); however, for CM-579 researchers could not administer a dose higher than 1 mg kg−1 (i.v.). PK studies that we performed using the MTD dose (Supplementary Tables 9–11), showed clearance levels for CM-579 (5.7 l h−1 kg−1) and CM-272 (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 is a member of the class of aminoquinolines that is is quinoline substituted by 5-methylfuran-2-yl, (1-methylpiperidin-4-yl)amino, methoxy, and 3-(pyrrolidin-1-yl)propoxy groups at positions 2, 4, 6 and 7, respectively. It is a dual G9a/DNA methyltransferases inhibitor with antitumor activity. It inhibits G9a, DNMT1, DNMT3A, DNMT3B and GLP (IC50 = 8 nM, 382 nM, 85 nM, 1200 nM and 2 nM, respectively). It has a role as an apoptosis inducer, a ferroptosis inducer, an antineoplastic agent, an EC 2.1.1.43 (enhancer of zeste homolog 2) inhibitor and an EC 2.1.1.37 [DNA (cytosine-5-)-methyltransferase] inhibitor. It is a N-alkylpyrrolidine, a member of furans, an aminoquinoline, an aromatic ether, a member of piperidines, a tertiary amino compound, a secondary amino compound and a diether.

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The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.[1]

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The results of the transcriptomic analysis performed after treatment with CM-272 consistently suggest a tumour interferon type 1 response with expression of ISGs and induction of immunogenic cell death (ICD) in AML, ALL and DLBCL cells, pointing to a common mechanism of anti-tumour effect. Despite the fact that ICD had not been described as a mechanism of action for epigenetic drugs, these results might have been at least partially predicted, because recent studies have shown that expression of ISGs is epigenetically regulated by H3K9me2 (ref. 30), supporting the role of G9a inhibition in the activation of type I interferon responses and ICD. Thus, we speculate that the use of immunocompromised mice unable to develop anti-tumour immune responses may have underestimated the efficacy of CM-272 against tumour cells, prompting the evaluation of immune-competent models to explore the full potential therapeutic effects of our compounds33. On the basis of recent studies demonstrating that type I interferon responses contribute to the efficacy of chemotherapeutic agents32, the use of CM-272 in combination with such drugs and/or with immune modulators such as checkpoint inhibitors might also represent an attractive therapeutic strategy.
In summary, CM-272 is a potent novel first-in-class dual reversible inhibitor of G9a (GLP) and DNMTs that prolongs survival in in vivo models of haematological malignancies by at least in part inducing immunogenic cell death. These compounds represent a novel approach for targeting cancer safely and efficiently, paving the way for treating a broad series of human tumours with poor prognosis.[1]

C29H43CL3N4O3

602.04

600.2400

2448471-08-5

CM-579;1846570-40-8

Light yellow to yellow solid powder

63Ų

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.15 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.15 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 16.67 mg/mL (27.69 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。