DMX-5804

MAP4K4 (IC50 = 3 nM); MAP4K4 (pIC50 = 8.55); MINK1/MAP4K6 (pIC50 = 8.18); TNIK/MAP4K7 (pIC50 = 7.96); GCK/MAP4K2 (pIC50 = 6.50); KHS/MAP4K5 (pIC50 = 6.36); GLK/MAP4K3 (pIC50 = 4.95 nM); MLK1/MAP3K9 (pIC50 = 7.19); MLK3/MAP3K11 (pIC50 = 6.99); NUAK (pIC50 = 6.88); VEGFR (pIC50 = 5.72); ABL1 (pIC50 = 5.80 nM); Aurora B (pIC50 = 5.49 nM); FLT3 (pIC50 = 5.31); GSK3β (pIC50 = 4.66)
DMX-5804 exhibits great selectivity at MAP4K4 over other kinases, such as GCK/MAP4K2 (pIC50, 6.50), GLK/MAP4K3 (pIC50, 4.95), GSK3β (pIC50, 4.66), KHS/MAP4K5 (pIC50, 6.36), MLK1/MAP3K9 (pIC50, 7.19), MLK3/MAP3K11 (pIC50, 6.99), NUAK (pIC50, 6.88) and VEGFR (pIC50, 5.72), ABL1 (pIC50, 5.80), Aurora B (pIC50, 5.49), FLT3 (pIC50, 5.31)[1].

与其他激酶相比，DMX-5804 对 MAP4K4 表现出极高的选择性，例如 GCK/MAP4K2 (pIC50, 6.50)、GLK/MAP4K3 (pIC50, 4.95)、GSK3β (pIC50, 4.66)、KHS/MAP4K5 (pIC50, 6.36)、MLK1/ MAP3K9 (pIC50, 7.19)、MLK3/MAP3K11 (pIC50, 6.99)、NUAK (pIC50, 6.88) 和 VEGFR (pIC50, 5.72)、ABL1 (pIC50, 5.80)、Aurora B (pIC50, 5.49)、FLT3 (pIC50, 5.31) ）[1]。

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DMX-5804 能保护人诱导多能干细胞来源的心肌细胞 (hiPSC-CMs) 免受氧化应激诱导的细胞死亡。在 vCor.4U 心室肌细胞中，10 µM 的 DMX-5804 对高达 200 µM H2O2 或 45 µM 甲萘醌诱导的细胞死亡提供了近乎完全的保护，此结果通过 CellTiter-Glo 活力检测和人心脏肌钙蛋白 I 释放实验证实。 [1]
在 iCell 心肌细胞中，针对 400 µM H2O2 诱导的细胞死亡，DMX-5804 的半最大效应浓度 (EC50) 为 500 nM，但该值取决于氧化应激的水平。即使在氧化应激刺激后1小时给予 DMX-5804，仍能观察到部分保护作用。 [1]
DMX-5804 (0.5-2 µM) 在经受亚致死剂量甲萘醌诱导的氧化应激的 hiPSC-CMs 中，能保护钙循环（峰值面积和峰值高度），且保护作用可持续长达96小时。 [1]
在浓度为10 µM时，DMX-5804 能将经甲萘醌处理的 hiPSC-CMs 的最大氧化能力（线粒体呼吸）提高5倍，并部分挽救细胞外酸化率（糖酵解功能）。 [1]
在由 vCor.4U 细胞构建的3D人工心脏组织中，10 µM 的 DMX-5804 能抑制甲萘醌在24小时内诱导的细胞死亡，并在处理后1小时和24小时完全挽救自发性搏动和收缩力产生。 [1]
DMX-5804 在2D或3D培养条件下，对 hiPSC-CMs 的活力、节律性、钙处理、线粒体功能或收缩力产生均未显示不良影响。 [1]
DMX-5804 对克隆的人源离子通道 hERG、hNaV1.5 和 hCaV1.2 无脱靶效应。 [1]

在小鼠心肌缺血-再灌注损伤模型中，口服给予 DMX-5804 (50 mg/kg，两次，间隔10小时) 能显著减小梗死面积。当在缺血前20分钟开始给药时，梗死面积（占危险区域的百分比）从48.5%减少到20.9%。当在再灌注（损伤后）1小时开始给药时，梗死面积从55.1%减少到25.6%。 [1]
在损伤后治疗的研究中，通过 TUNEL 染色检测，DMX-5804 还将梗死区内部及邻近危险心肌中的心肌细胞凋亡分别抑制了39%和52%。 [1]

使用均相时间分辨荧光竞争性免疫分析法测定化合物对重组人 MAP4K4 激酶结构域的抑制活性。在该实验中，激酶反应产生 ADP，ADP 与染料标记的 ADP 示踪剂竞争结合铕穴状化合物标记的抗 ADP 抗体。信号通过测定665 nm和620 nm处的发射光比率来读取。反应在 ATP 处于其 Km 浓度 (10 µM)、MAP4K4 激酶结构域和生物素化髓鞘碱性蛋白底物存在下进行。抑制百分比是相对于含 DMSO 的对照反应（100%活性）和无酶反应（0%活性）计算的。通过绘制剂量反应曲线来确定 IC50 值。 [1]
针对包含 376 种人源激酶的 panel 评估了 DMX-5804 更广泛的激酶选择性谱。该化合物以其 MAP4K4 IC50 值的30倍浓度（即90 nM）进行测试。测定并报告了残留的激酶活性。 [1]

细胞活力与死亡： 将 hiPSC-CMs（iCell 或 vCor.4U 系）铺板于适当的维持培养基中。对于活力检测，在加入死亡诱导剂（H2O2 或甲萘醌）前1小时，用 DMX-5804（DMSO 中的系列稀释液，终浓度0.1% DMSO）处理细胞。24小时后，使用 CellTiter-Glo 发光法评估细胞活力，该方法通过量化 ATP 来反映活细胞数量。或者，通过使用 AlphaLISA 免疫分析法测量释放到培养基中的人心脏肌钙蛋白 I 来量化细胞死亡。在 iCell 心肌细胞中，也使用细胞不通透性染料 DRAQ7 在高内涵成像分析中评估膜完整性损失，并特异性分析通过 Myh6-RFP 报告基因识别的成功转导的 (GFP+) 心肌细胞。 [1]
钙循环： 用 DMX-5804 和甲萘醌处理 vCor.4U hiPSC-CMs。2-3小时后，用荧光钙指示剂染料加载细胞。使用荧光成像微孔板检测仪监测细胞内钙振荡。计算搏动频率、峰值高度、峰值宽度和总峰值面积等参数，并与对照组进行比较。 [1]
线粒体功能： 将 vCor.4U hiPSC-CMs 接种于专用板中。用 DMX-5804 和甲萘醌处理后，将培养基更换为不含碳酸氢盐的检测培养基。使用 Seahorse 细胞外通量分析仪实时测量耗氧率（OCR，线粒体呼吸指标）和细胞外酸化率（ECAR，糖酵解指标）。依次注入寡霉素（ATP合酶抑制剂）、FCCP（线粒体解偶联剂）以及抗霉素 A 和鱼藤酮的混合物（复合物 III 和 I 抑制剂）以评估线粒体功能的不同参数。 [1]
3D 人工心脏组织： 将 vCor.4U 心肌细胞与纤维蛋白原和凝血酶混合，注入包含柔性硅胶柱的模具中形成3D组织。成熟12天后，在存在或不存在 DMX-5804 的情况下用甲萘醌处理 hEHTs。评估细胞死亡。通过光学跟踪硅胶柱的偏转，并根据柱的尺寸和材料特性计算力，每天测量自发性收缩力。 [1]
细胞内的靶点结合： 为确认对内源性 MAP4K4 的抑制，用先导化合物 F1386-0303 (10 µM) 处理 HEK293T 细胞。1小时后，通过免疫复合物激酶实验测量内源性 MAP4K4 活性，显示70%的抑制。 [1]

Myocardial Infarction Model in Mice: Female CD-1 mice (9-10 weeks old) were used. Under general anesthesia and mechanical ventilation, a left thoracotomy was performed. The left anterior descending coronary artery (LAD) was ligated for 45 minutes to induce ischemia, followed by release of the ligature to allow reperfusion. DMX-5804 was formulated in 1.5% Captisol (sulfobutylether-β-cyclodextrin) solution. Mice received two oral gavage doses of DMX-5804 (50 mg/kg), spaced 10 hours apart. In the "pre-injury" protocol, the first dose was given 20 minutes before LAD occlusion. In the "post-injury" protocol, the first dose was given 1 hour after the initiation of reperfusion. After 24 hours of reperfusion, infarct size was quantified. Mice were re-anesthetized, the LAD was re-occluded, and Evans blue dye was perfused to delineate the non-ischemic area. The heart was excised, sectioned, stained with triphenyltetrazolium chloride (TTC) to visualize viable (red) and infarcted (white) tissue within the area at risk (unstained by blue). Infarct size was expressed as a percentage of the area at risk. Apoptosis was assessed in separate heart sections by TUNEL staining co-labeled with an antibody against sarcomeric myosin to identify cardiomyocytes. [1]
Pharmacokinetics Study: For pharmacokinetic profiling, DMX-5804 was formulated similarly. Female CD-1 mice received a single oral dose (50 mg/kg). Blood samples were collected at multiple time points (e.g., 10 min, 30 min, 1 h, 2.5 h, 5 h, 10 h, 20 h) post-dose. Plasma concentrations of DMX-5804 were determined. [1]
Target Engagement In Vivo: Mice were orally dosed with DMX-5804. Hearts were harvested at various times or after different doses. Cardiac lysates were prepared and incubated with a desthiobiotin-ATP probe, which covalently labels active kinase ATP-binding sites. MAP4K4 was subsequently immunoprecipitated, and the level of probe labeling was assessed by western blot to measure the degree of target occupancy by the inhibitor. [1]

Following a single intravenous dose (1 mg/kg) in mice, DMX-5804 had a clearance (Cl) of 2.50 L hr⁻¹ kg⁻¹, a volume of distribution (Vd) of 1.22 L kg⁻¹, a half-life (t₁/₂) of 0.6 hours, and a peak plasma concentration (Cmax) of 1590 nM. [1]
Following a single oral dose (50 mg/kg) in mice, DMX-5804 had an area under the plasma concentration-time curve (AUC₀→∞) of 63733 nMhr, a Cmax of 13847 nM, a time to Cmax (Tmax) of 1 hour, and a half-life of 1.8 hours. [1]
Plasma concentrations of DMX-5804 were 334 nM and 8 nM at 1 and 10 hours, respectively, after a 50 mg/kg oral dose. A second dose at 10 hours maintained plasma levels above the in vitro EC50 for cardiomyocyte protection for nearly a full day. [1]
The oral bioavailability and plasma exposure (AUC and Cmax) of DMX-5804 were substantially improved (29.5-fold and 46.9-fold, respectively) compared to its predecessor compound F1386-0303. [1]

DMX-5804 showed no adverse effects on hiPSC-CM viability, contractility, calcium handling, or mitochondrial function at tested concentrations. [1]
DMX-5804 showed no significant inhibition of key cardiac ion channels (hERG, hNaV1.5, hCaV1.2) in electrophysiological safety screening. [1]
DMX-5804 was tested in an Ames genotoxicity reverse mutation assay using Salmonella typhimurium strains TA98 and TA100, with and without metabolic activation (S9). No mutagenic potential was observed at tested concentrations up to 250 µg/mL. [1]
DMX-5804 was screened against a panel of 44 off-target safety-related proteins (SafetyScreen44 panel). The only hit reported was activity against metallothionein 3. [1]

[1]. MAP4K4 Inhibition Promotes Survival of Human Stem Cell-Derived Cardiomyocytes and Reduces Infarct Size In Vivo. Cell Stem Cell. 2019 Mar 1. pii: S1934-5909(19)30013-X.

DMX-5804 (5-[4-(2-methoxy-ethoxy)-phenyl]-7-phenyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-4-one) is a highly selective small-molecule inhibitor of MAP4K4, developed through pharmacophore modeling, virtual screening, and medicinal chemistry optimization from a lead compound F1386-0303. [1]
MAP4K4 is activated in failing human hearts and promotes cardiomyocyte death in response to oxidative stress, which is a hallmark of cardiac ischemia-reperfusion injury. [1]
This study utilized human iPSC-derived cardiomyocytes (hiPSC-CMs) as a critical human-relevant platform for target validation and drug discovery, highlighting their predictive value over traditional animal models alone for cardiac drug development. [1]
The protection conferred by MAP4K4 inhibition in hiPSC-CMs was comparable to that of the clinically effective beta-blocker metoprolol and superior to inhibition of the p38 MAPK pathway, which failed in clinical trials, suggesting the predictive fidelity of the hiPSC-CM model. [1]
The chemical series containing DMX-5804 provided valuable tool compounds for validation but was noted to have solubility and pharmacokinetic properties insufficient for direct development as an intravenous drug candidate for acute ischemic injury, though it supports the target's rationale for further drug development. [1]

C21H19N3O3

361.393864870071

361.14

C, 69.79; H, 5.30; N, 11.63; O, 13.28

2306178-56-1

2306178-56-1

137334091

Off-white to yellow solid powder

2.9

64.8

1

4

6

27

527

0

FUSHFOGORKZNNQ-UHFFFAOYSA-N

InChI=1S/C21H19N3O3/c1-26-11-12-27-17-9-7-15(8-10-17)18-13-24(16-5-3-2-4-6-16)20-19(18)21(25)23-14-22-20/h2-10,13-14H,11-12H2,1H3,(H,22,23,25)

5-[4-(2-methoxyethoxy)phenyl]-7-phenyl-3H-pyrrolo[2,3-d]pyrimidin-4-one

DMX-5804; DMX 5804; DMX5804

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO: 72~125 mg/mL (199.2~345.9 mM)
Ethanol: ~1.5 mg/mL (~4.2 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.76 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (5.76 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 20.8 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: ≥ 2.08 mg/mL (5.76 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。