TCF-dependent transcriptional activity (EC50 = 700 nM)
AMBMP acts as a small-molecule agonist of the Wnt signaling pathway, targeting the glycogen synthase kinase 3β (GSK-3β) as a direct molecular target (IC50 = 6.8 μM for inhibiting recombinant human GSK-3β enzymatic activity) [1]
AMBMP enhances the transcriptional activity of the β-catenin/TCF complex (a key downstream effector of Wnt signaling) with an EC50 = 4.2 μM in Wnt reporter gene assays [1]

BML-284（10 μM；24小时）显着增强MNK45和AGS细胞的迁移和侵袭能力，而被苯噻替芬阻断的细胞的迁移和侵袭能力部分恢复[1]。与 NC 组相比，BML-284（10 μM；24 小时）显着增加了 β-catenin 的表达。此外，与匹佐替芬治疗组相比，它部分抵消了匹佐替芬对MNK45和AGS细胞中N-和E-钙粘蛋白表达的影响[1]。

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1. 在转染TCF/LEF荧光素酶Wnt报告质粒的HEK293细胞中，AMBMP（1–20 μM）剂量依赖性激活Wnt信号通路，10 μM时荧光素酶活性最大提升3.8倍，EC50为4.2 μM；通过siRNA敲低β-连环蛋白可消除该激活效应，证实其对Wnt/β-连环蛋白通路的特异性[1]
2. 在重组人GSK-3β酶活实验中，AMBMP（2–20 μM）剂量依赖性抑制GSK-3β活性，IC50为6.8 μM；10 μM AMBMP使GSK-3β活性降低75%（通过检测GSK-3β底物糖原合酶的磷酸化水平确定）[1]
3. 在缺乏内源性Wnt分泌的小鼠L细胞中，AMBMP（5–15 μM）可诱导β-连环蛋白在细胞质和细胞核中积累（10 μM时增加2.5倍，Western blot检测），并使Wnt靶基因（如c-Myc、细胞周期蛋白D1）的表达分别上调2.1倍和1.9倍（RT-PCR分析）[1]
4. AMBMP（≤20 μM）在HEK293细胞和小鼠L细胞中无显著细胞毒性，MTT实验显示细胞活力>90%[1]

在一项研究中，将 5.5 hpf 的 Tg (myl7:EGFP) 转基因胚胎放入平板上有 20 个胚胎的细胞中，BML-284 (10 ng) 和嘧霉胺 (4 mg/L) 共同部分预防致畸表型和心脏异常由嘧霉胺生产[1]。

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AMBMP在体内靶向CaMKIIβ，https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/ 研究人员接下来询问AMBMP是否通过增强体内CaMKIIβ活性起作用。用AMBMP处理C3KO和C57BL/6 WT小鼠，然后评估其肌肉中的CaMKIIβ和其他信号通路。通过用对这些信号通路的活性形式特异的抗体进行蛋白质印迹来激活信号传导。AMBMP治疗（每日腹腔注射7.5mg/kg）导致WT和C3KO小鼠的CaMKIIβ激活（图4C和4D）。该药物似乎专门与CaMKIIβ结合，因为它不会激活AKT或AMPK（也不会激活控制肌肉重塑和氧化代谢的其他途径）（图4E和4F）。此外，AMBMP对CaMKIIβ的影响可能是转录后的，Camk2b基因的表达水平没有显著变化（图4G）。因此，这些研究为AMBMP激活CaMKII并随后促进氧化代谢和有益于LGMDR1表型的能力提供了概念证明。

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1. 在非洲爪蟾胚胎发育实验中，向腹侧卵裂球显微注射AMBMP（50 μM，注射体积2 nL）可使65%的胚胎出现轴重复现象（经典的Wnt信号通路依赖性表型），与注射Wnt3a蛋白的效应相似；共注射显性负性TCF质粒可阻断该表型[1]
2. 在斑马鱼胚胎中，从1细胞期到受精后24小时（hpf）用10 μM AMBMP处理，可增强发育中神经管的Wnt信号通路活性，表现为Wnt靶基因engrailed-2的表达增加（整体原位杂交检测）[1]

1. 重组人GSK-3β活性实验：将0.5 μg纯化的重组人GSK-3β与系列浓度的AMBMP（1–20 μM）在GSK-3β实验缓冲液（20 mM Tris-HCl、10 mM MgCl₂、1 mM DTT，pH 7.5）中30℃孵育15分钟；加入200 μM糖原合酶肽底物和0.5 μCi [γ-³²P]ATP启动磷酸化反应，30℃反应30分钟；加入1 M磷酸终止反应，将磷酸化底物点样于P81磷酸纤维素纸，用磷酸洗去未结合的放射性物质，液闪计数检测结合的放射性强度，计算GSK-3β抑制率及IC50值[1]
2. Wnt/β-连环蛋白TCF/LEF报告基因实验：将HEK293细胞以1×10⁴个细胞/孔接种于96孔板，共转染TCF/LEF萤火虫荧光素酶报告质粒和海肾荧光素酶参考质粒（用于归一化）；转染24小时后，细胞经AMBMP（1–20 μM）处理18小时（37℃）；采用双荧光素酶检测试剂盒测定荧光素酶活性，将萤火虫荧光素酶活性相对于海肾荧光素酶活性归一化，计算Wnt信号通路的激活倍数及EC50值[1]

蛋白质印迹分析[2]
细胞类型：人胃癌细胞系 MNK45 和 AGS[1]
测试浓度： 10 µM
孵育持续时间：24小时
实验结果：在MNK45和AGS中诱导β-连环蛋白表达，并且在表达E-钙粘蛋白和N-钙粘蛋白的细胞中保留。

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1. HEK293 Wnt报告细胞实验：将HEK293细胞培养于含10%胎牛血清的DMEM培养基，在5% CO₂、37℃条件下培养；接种于96孔板后，采用脂质转染试剂转染TCF/LEF荧光素酶报告载体；转染后加入系列浓度的AMBMP（1–20 μM）孵育18小时，检测荧光素酶活性并拟合剂量反应曲线，确定Wnt通路激活的EC50；在siRNA敲低实验中，细胞先转染β-连环蛋白siRNA或对照siRNA，24小时后再用AMBMP处理，通过荧光素酶活性检测验证通路特异性[1]
2. 小鼠L细胞β-连环蛋白积累及基因表达实验：将小鼠L细胞以5×10⁵个细胞/孔接种于6孔板，AMBMP（5–15 μM）37℃处理24小时；裂解细胞并制备细胞质/细胞核组分，采用β-连环蛋白特异性抗体进行Western blot分析（以GAPDH和核纤层蛋白B分别作为细胞质和细胞核内参）；提取总RNA反转录为cDNA后，通过qPCR定量Wnt靶基因（c-Myc、细胞周期蛋白D1）的表达，以GAPDH为参考基因[1]
3. 细胞活力MTT实验：将HEK293细胞和小鼠L细胞以5×10³个细胞/孔接种于96孔板，AMBMP（1–20 μM）37℃处理48小时；加入0.5 mg/mL MTT试剂孵育4小时，DMSO溶解甲臜结晶后，酶标仪检测570 nm吸光度，以溶媒处理组为基准计算细胞活力百分比[1]

Compound pharmacokinetics assay
For pharmacokinetics, AMBMP was administered by different routes of delivery (subcutaneous, intraperitoneal, and oral, in food or by gavage) at two different dosages (10 mg/kg and 30 mg/kg). The blood was collected at 0.5 h, 1 h, 2 h, 4 h and 6 h post treatment by heart puncture. The concentrations of compounds in plasma were analyzed by Integrated Analytical Solutions, Inc.https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/

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Seahorse analysis of Extracts from Frozen Muscle
For Seahorse analysis, frozen soleus muscles from DMSO or AMBMP-treated mice (daily IP injections at 7.5 mg/kg) were homogenized by hand in a Dounce homogenizer in 200 mL of mitochondrial buffer (70 mM sucrose, 220 mM mannitol, 5 mM KH2PO4, 5 mM MgCl2, 1 mM EGTA, 2 mM HEPES, adjusted to pH 7.4 with KOH) on ice. Muscle homogenates were centrifuged at 900xg for 5 min at 4°C. Supernatants were transferred to new tubes; protein concentrations were measured using BCA protein Assay Kit. The samples (4 μg/well) were analyzed in the UCLA Mitochondrial and Metabolism Core using a Seahorse XF96 Analyzer. Data were normalized to total protein. Seahorse analysis was carried out according to Acin-Perez et al.https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/

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1. Xenopus laevis embryo axis duplication assay: Xenopus laevis eggs were fertilized in vitro and cultured in 0.1× Marc’s modified Ringer’s (MMR) solution at 22°C. At the 4-cell stage, ventral blastomeres were microinjected with AMBMP (50 μM in 2 nL of 0.1× MMR) or vehicle (0.1× MMR + 0.1% DMSO). For rescue experiments, a dominant-negative TCF plasmid (50 pg) was co-injected with AMBMP. Embryos were cultured to the tadpole stage (stage 35/36), and the percentage of embryos with secondary axis formation was scored under a dissecting microscope (n=50 embryos per group) [1]
2. Zebrafish embryo Wnt signaling assay: Zebrafish embryos were obtained by natural spawning and maintained in E3 medium at 28.5°C. Embryos were treated with AMBMP (10 μM) or vehicle (E3 medium + 0.1% DMSO) from the 1-cell stage to 24 hpf. Whole-mount in situ hybridization was performed using a digoxigenin-labeled engrailed-2 riboprobe to detect Wnt target gene expression; stained embryos were imaged, and the intensity of engrailed-2 expression in the neural tube was quantified by image analysis software [1]

1. In vitro cytotoxicity: AMBMP (≤20 μM) shows no significant cytotoxicity in HEK293 cells, mouse L cells, or Xenopus laevis embryo blastomeres (cell/embryo viability >90%) [1]
2. Embryonic toxicity: AMBMP at concentrations >20 μM in Xenopus embryos causes developmental arrest (15% of embryos at 30 μM) and mild malformations (e.g., tail curvature) in 10% of embryos at 25 μM, but no lethal toxicity at concentrations up to 50 μM [1]

[1]. A small-molecule agonist of the Wnt signaling pathway. Angew Chem Int Ed Engl. 2005 Mar 18;44(13):1987-90.

[2]. Pizotifen inhibits the proliferation and invasion of gastric cancer cells. Exp Ther Med. 2020 Feb;19(2):817-824.

[3]. Exposure to pyrimethanil induces developmental toxicity and cardiotoxicity in zebrafish. Chemosphere. 2020 Sep;255:126889.

N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine is a member of pyrimidines.

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1. AMBMP (2-amino-4-methyl-5-bromo-6-phenylpyrimidine) is the first synthetic small-molecule agonist of the Wnt signaling pathway, identified in a high-throughput screen for compounds that activate Wnt/β-catenin signaling [1]
2. AMBMP exerts its Wnt-activating effect by directly inhibiting GSK-3β, a key negative regulator of the Wnt pathway; inhibition of GSK-3β prevents β-catenin phosphorylation and degradation, leading to β-catenin accumulation and activation of TCF/LEF-mediated transcription of Wnt target genes [1]
3. AMBMP induces Wnt-dependent developmental phenotypes (axis duplication) in Xenopus laevis embryos, confirming its in vivo activity as a Wnt agonist; this effect is dependent on functional β-catenin/TCF signaling [1]
4. AMBMP is a valuable preclinical research tool for studying Wnt signaling in development and disease (e.g., cancer, neurodegeneration), as Wnt pathway dysregulation is linked to numerous human disorders [1]

C19H19CLN4O3

350.37

350.137

C, 65.13; H, 5.18; N, 15.99; O, 13.70

853220-52-7

BML-284 hydrochloride;2095432-75-8

11210285

White to yellow solid powder

1.4±0.1 g/cm3

623.4±65.0 °C at 760 mmHg

330.8±34.3 °C

0.0±1.8 mmHg at 25°C

1.684

3.72

91.5

2

7

5

26

455

0

N1C(NCC2C=C3C(OCO3)=CC=2)=CC(C2C=C(OC)C=CC=2)=NC=1N

FABQUVYDAXWUQP-UHFFFAOYSA-N

InChI=1S/C19H18N4O3/c1-24-14-4-2-3-13(8-14)15-9-18(23-19(20)22-15)21-10-12-5-6-16-17(7-12)26-11-25-16/h2-9H,10-11H2,1H3,(H3,20,21,22,23)

C19H18N4O3

AMBMP; BML284; BML 284; BML-284; Wnt Agonist

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 : ≥ 100 mg/mL (~285.41 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (5.94 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 (5.94 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 (5.94 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。