DNMT1

GSK-3685032（6天）的中位生长IC50值为0.64 μM，这表明它抑制大多数癌细胞系的发育[1]。由于在整个 6 天持续时间内生长 IC50 降低，GSK-3685032（0.1-1000 nM，第 1-6 天）在 3 天后表现出生长抑制 [1]。 GSK3685032（10-1000 nM，第 4 天）可增加免疫相关基因转录，呈剂量依赖性[1]。 DNMT1 蛋白的表达被 GSK3685032（3.2-10,000 nM，2 天）抑制 [1]。 GSK3685032 诱导 DNA 低甲基化和基因激活 [2]。

GSK3685032对DNMT1的抑制显示出体内疗效。[2] 当对小鼠施用GSK3685032时，其清除率低，分布量适中，血液半衰期>1.8小时，剂量比例暴露量为1至45 mg kg−1（扩展数据图6a-c）。基于这些体内吸收、分布、代谢和排泄特性，每天两次皮下给药GSK3685032可实现长期靶点参与（扩展数据图6d）。该给药方案耐受性良好，剂量高达至少45 mg kg−1，持续≥4周，对体重或行为和仪容没有明显不良影响（扩展数据图7a，b）。另一方面，一旦配制，DAC在化学上不稳定，在给药前立即重新配制。此外，由于胞苷脱氨酶的自发水解切割和快速代谢，它表现出较差的药代动力学特性16,17。此外，为了尽量减少重复给药后的毒性，使用了间歇给药方案来实现长期耐受的治疗方案。[2]

荧光耦合破碎光分析。[2] 如前所述，使用半甲基化发夹寡核苷酸检查DNMT的活性39。最终测定浓度由125 nM DNA寡核苷酸和（1）40 nM全长DNMT1、2μM SAM组成；（2） 600 nM DNMT3A/3L，2.5μM SAM；或（3）300 nM DNMT3B/3L，0.15μM SAM。DNMT1的反应在40分钟（26°C）后淬灭，DNMT3A/3L和DNMT3B/3L的反应在120分钟（37°C）后淬灭。将化合物（10点，三倍连续稀释，100%二甲亚砜）预先印在黑色反应板上（最终2%二甲基亚砜）。通过用1:4比例的全甲基/半甲基化发夹寡核苷酸（ATDBio定制合成的5-FAM-ATCTAG5-me-dCG5me-dCATCAGTTTTCTGATG5me-dCTAGA-Dabcyl-3和5-FAM-ATCSAG5-me-dCATCAGTTTCTGATG 5me-dCG5me-dcTAGA-Daccyl-3）代替DNMT反应，进行Gla1计数器筛选。对于可逆性研究，在DNMT1与化合物（10×IC50）预孵育20分钟后，加入底物后，复合物迅速稀释100倍。通过稀释后不同时间点的淬灭，在70分钟内评估DNMT1活性的恢复情况。如Ariazi等人所述，数据符合固定的稳态速度方程。

GSK-3685032 or vehicle (10% captisol adjusted to pH 4.5–5 with 1 M acetic acid, stored for up to 1 week at 4 °C) was administered subcutaneously, twice daily, at a dosing volume of 10 ml kg−1 (0.2 ml per 20 g of body weight). DAC (Sun Pharmaceutical Industries) was administered by intraperitoneal injection, three times per week, at a dosing volume of 10 ml kg−1. DAC was reconstituted with the appropriate amount of manufacturer’s diluent (68 mg of monobasic potassium phosphate and 11.6 mg of sodium hydroxide in 10 ml of water) to yield a dosing solution of 0.04 mg ml−1 immediately before administration (final dose of 0.4 mg kg−1).[1]

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Efficacy ofGSK-3685032 in an MV4–11 human systemic AML model in female NOD.CB17-Prkdcscid/NCrCrl mice was evaluated at Charles River Laboratories. To ablate bone marrow, animals (10 weeks old) were dosed with cyclophosphamide (150 mg kg−1) starting 3 d before injection of MV4–11 cells intravenously into the tail vein. Randomization by body weight and dosing commenced 21 d after implant. Animals (10 per group, 70 total) were dosed over 30 study days, where GSK-3685032 or vehicle was administered subcutaneously twice daily while DAC was dosed intraperitoneally two times per week. Body weight measurements were taken three times per week. After a single observation of >30% body weight loss or consecutive measurements of >25% body weight loss, the animal was euthanized. Clinical signs associated with tumor progression such as impairment of hind limb function or ocular proptosis also resulted in euthanasia. The study endpoint was 76 d.[1]

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A separate pharmacokinetic study was conducted in naïve animals (3 mice per group, 9 mice total), where mice received a single intravenous or subcutaneous dose of 2 mg kg−1 (intravenously, male CD-1 mice), 2 mg kg−1 (subcutaneously, male C57/BL6 mice) or 30 mg kg−1 (subcutaneously, female Nu/Nu mice) GSK-3685032 and composite blood samples were collected over 24 h post-dose. Blood concentrations were determined by HPLC–MS/MS and pharmacokinetic parameters were estimated from the mean blood concentration–time profiles using noncompartmental analysis with Phoenix WinNonlin v.6.3 (Certara). Area under the blood concentration–time curve was calculated using the linear trapezoidal rule for each incremental trapezoid up to the maximal concentration (Cmax), and the linear or log interpolation rule for each trapezoid thereafter. The dose-normalized area under the curve (AUC) was calculated by dividing the AUC0–8 h by the dose.[1]

[1]. Substituted pyridines as inhibitors of DNMT1 and their preparation. WO2017216727A1.

[2]. Discovery of a first-in-class reversible DNMT1-selective inhibitor with improved tolerability and efficacy in acute myeloid leukemia. Nat Cancer. 2021;2(10):1002-1017.

DNA methylation is a key epigenetic driver of transcriptional silencing and is abnormally regulated in cancer. Reversing DNA methylation using demethylating agents (such as cytidine analogs decitabine or azacitidine) has shown clinical efficacy in hematologic malignancies. These nucleoside analogs can be incorporated into replicating DNA and inhibit DNA cytosine methyltransferases DNMT1, DNMT3A, and DNMT3B through irreversible covalent interactions. These drugs are significantly toxic to normal blood cells, thus limiting their clinical dosage. This article reports the discovery of GSK3685032, a highly potent first-in-class selective inhibitor of DNMT1. Crystallographic studies show that GSK3685032 competes with the active site loop of DNMT1 for access to the hemimethylated DNA region between two CpG base pairs. GSK3685032 significantly reduces DNA methylation levels in vitro, activates transcription, and inhibits cancer cell growth. Compared with decitabine, GSK3685032 has better in vivo tolerability, and therefore can achieve better tumor regression and survival in mouse models of acute myeloid leukemia. [2]

C22H24N6OS

420.53

420.173

C, 62.83; H, 5.75; N, 19.98; O, 3.80; S, 7.62

2170140-50-6

GSK-3685032;2170137-61-6;(S)-GSK-3685032;2170142-58-0

132233544

White to off-white solid powder

2.6

158

2

7

6

30

684

1

C(C1C(=C(C#N)C(S[C@H](C2C=CC=CC=2)C(=O)N)=NC=1N1CCC(N)CC1)CC)#N

KNKHRZYILDZLRE-LJQANCHMSA-N

InChI=1S/C22H24N6OS/c1-2-16-17(12-23)21(28-10-8-15(25)9-11-28)27-22(18(16)13-24)30-19(20(26)29)14-6-4-3-5-7-14/h3-7,15,19H,2,8-11,25H2,1H3,(H2,26,29)/t19-/m1/s1

(2R)-2-[6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl]sulfanyl-2-phenylacetamide

(R)-GSK-3685032; 2170140-50-6; R-GSK3685032; (2R)-2-{[6-(4-aminopiperidin-1-yl)-3,5-dicyano-4-ethylpyridin-2-yl]sulfanyl}-2-phenylacetamide; SCHEMBL19717300; BDBM491438; US10975056, Example 412;

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/受潮和光照。

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

DMSO : ~60 mg/mL (~142.68 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。