TCF-dependent transcriptional activity (EC50 = 700 nM)
AMBMP hydrochloride targets the Wnt signaling pathway, acting as a potent activator of canonical Wnt signaling. It induces TCF-dependent transcriptional activity, leading to the activation of Wnt target genes. The compound enhances beta-catenin levels and increases transcript and protein levels of P-glycoprotein (P-gp). AMBMP hydrochloride also inhibits tubulin polymerization and suppresses TLR2/4/5-induced inflammatory responses in human monocytes.

In comparison to the NC group, BML-284 (10 µM; 24 hours) dramatically increased the expression of β-catenin. Additionally, as compared to the pizotifen treatment group, it partially counteracted the effects of pizotifen on the expression of N- and E-cadherin in MNK45 and AGS cells [1]. The migration and invasion capacities of MNK45 and AGS cells are markedly enhanced by BML-284 (10 µM; 24 hours), while the migration and invasion capacities of cells inhibited by bentiftine are partially restored [1].
In vitro, AMBMP hydrochloride has been shown to activate canonical Wnt signaling in a concentration-dependent manner. It induces TCF-dependent transcriptional activity with an EC50 of 700 nM. The compound enhances beta-catenin levels and increases the expression of Wnt target genes. AMBMP hydrochloride also inhibits tubulin polymerization. It suppresses TLR2/4/5-induced inflammatory responses in human monocytes.

BML-284 hydrochloride (10 ng) coupled with pyrimethanil (4 mg/L) can partially repair the teratogenic phenotype and heart abnormalities induced by pyrimethanil in Tg (myl7:EGFP) transgenic embryos at 5.5 hpf. Transfer to a plate containing 20 embryos [1].

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AMBMP Targets CaMKIIβ In Vivo, https://pmc.ncbi.nlm.nih.gov/articles/PMC7659555/ Researchers next asked whether AMBMP acts by enhancing CaMKIIβ activity in vivo. C3KO and C57BL/6 WT mice were treated with AMBMP and then their muscles were evaluated for CaMKIIβ and other signaling pathways. The activation of signaling was carried out by western blotting with antibodies specific for the active forms of these signaling pathways. Treatment with AMBMP (daily i.p. injection 7.5 mg/kg) led to CaMKIIβ activation in both WT and C3KO mice (Figures 4C and 4D). The drug appears to engage CaMKIIβ specifically as it does not activate AKT nor AMPK (nor other pathways that control muscle remodeling and oxidative metabolism) (Figures 4E and 4F). Furthermore, the effect of AMBMP on CaMKIIβ was likely post-transcriptional, and there was no significant change in the expression level of the Camk2b gene (Figure 4G). Thus, these studies establish proof of concept for the ability of AMBMP to activate CaMKII and subsequently to promote oxidative metabolism and benefit the LGMDR1 phenotype.
In vivo, AMBMP hydrochloride has been studied in animal models for its effects on Wnt signaling and inflammation. The compound has been shown to modulate immune responses and to have potential therapeutic applications in inflammatory diseases. Further in vivo studies are needed to fully characterize its efficacy and safety profile. AMBMP hydrochloride is primarily used as a research tool.

In vitro cell-based assays for AMBMP hydrochloride typically involve measuring its ability to activate Wnt signaling in cultured cells. Cells are treated with AMBMP hydrochloride at various concentrations, and TCF-dependent transcriptional activity is measured using a luciferase reporter assay. The EC50 for activation is determined from dose-response curves. The effect of AMBMP hydrochloride on beta-catenin levels and Wnt target gene expression is assessed by Western blot and quantitative PCR. The compound's effect on tubulin polymerization and inflammation can also be evaluated.

Western Blot Analysis [1]
Cell Types: Human gastric cancer cell line MNK45 and AGS
Tested Concentrations: 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: β-catenin expression was induced and E-cadherin and N-cadherin expression were retained in MNK45 and AGS cells.
In vitro cell-based studies with AMBMP hydrochloride typically involve cultured cells, such as HEK293 cells or cancer cell lines. Cells are treated with AMBMP hydrochloride at various concentrations. Wnt signaling activation is assessed by measuring TCF-dependent transcriptional activity using a luciferase reporter assay. Beta-catenin levels are measured by Western blot analysis. The effect of AMBMP hydrochloride on cell proliferation, differentiation, and apoptosis can also be evaluated. The compound's anti-inflammatory effects are assessed in monocytes by measuring cytokine production.

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/
In vivo animal studies with AMBMP hydrochloride are limited, as the compound is primarily used as a research tool. However, the compound has been studied in animal models of inflammation and cancer. AMBMP hydrochloride is typically administered intraperitoneally or orally. The effect of the compound on Wnt signaling, inflammation, and tumor growth is assessed. Pharmacokinetic studies are performed to evaluate the absorption and distribution of the compound.

The pharmacokinetic properties of AMBMP hydrochloride have not been extensively characterized. The compound has a molecular weight of 386.84 g/mol. It is soluble in DMSO. Detailed PK parameters such as half-life, bioavailability, clearance, and volume of distribution have not been reported. The compound is stable as a powder at -20°C. Further pharmacokinetic studies are needed to evaluate its absorption, distribution, metabolism, and excretion properties.

Toxicological data for AMBMP hydrochloride are limited, as the compound is a research chemical not intended for human therapeutic use. The compound is typically handled with standard laboratory safety precautions. No comprehensive toxicology studies, including acute and chronic toxicity, genotoxicity, or cardiotoxicity assessments, have been reported. The compound's safety in animal models and potential for clinical development would require further toxicological evaluation.

[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-benzodioxane-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine belongs to the pyrimidine class of compounds. Gastric cancer is the fifth most common malignant tumor worldwide and the third leading cause of cancer-related deaths. Therefore, studying the pathogenesis of gastric cancer is crucial. Previous reports have indicated that abnormal activation of the Wnt/β-catenin signaling pathway is closely related to the development and progression of gastric cancer. This study found that pyrazotifen can inhibit the viability of gastric cancer cell lines MNK45 and AGS in a dose-dependent manner. Pyrazotifen treatment inhibited the migration and invasion of MNK45 and AGS cells and induced apoptosis. Western blot analysis showed that pyrazotifen inhibited the expression of Wnt3a, β-catenin, and N-cadherin, while increasing the expression of E-cadherin. In addition, the Wnt signaling pathway activator BML-284 partially reversed the changes in β-catenin, N-cadherin and E-cadherin expression levels induced by pyrazotifen in MNK45 and AGS cells. In summary, these findings suggest that pyrazotifen may become a novel anticancer drug for the treatment of gastric cancer by inhibiting the Wnt/β-catenin pathway. [2]

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Azoxystrobin is a broad-spectrum fungicide commonly used to control gray mold. However, there is little literature on the cardiotoxicity of azoxystrobin. In this study, we used an animal experimental model to investigate the developmental and cardiotoxic effects of azoxystrobin on aquatic vertebrates. We exposed zebrafish embryos to concentrations of 2, 4 and 6 mg/L of azoxystrobin within 5.5 to 72 hours after fertilization. We found that azoxystrobin caused a decrease in hatching rate, heart rate and survival rate of zebrafish embryos. Azoxystrobin exposure also caused pericardial and yolk sac edema, spinal deformities and cardiac circulatory failure. In addition, pyraclostrobin increased reactive oxygen species stress levels and enhanced the activities of superoxide dismutase and catalase. The transcription of apoptosis-related genes (p53, Bax, Bcl2, Casp9, and Casp6l1) and heart development-related genes (Tbx2b, Gata4, Myh6, Vmhc, Nppa, Bmp2b, Bpm4, and Bpm10) was also altered. Our data suggest that BML-284 activation of the Wnt signaling pathway can partially rescue dysmorphic phenotypes induced by pyraclostrobin. Our results provide new evidence for the toxicity of pyraclostrobin and its harmful residues in the environment and agricultural products. [3]
AMBMP hydrochloride is a potent and selective activator of the Wnt signaling pathway. It is a valuable research tool for studying the role of Wnt signaling in development, stem cell biology, and cancer. The compound is also used to study the role of Wnt signaling in inflammation and immune responses. AMBMP hydrochloride is not an FDA-approved drug and is not commercially available as a pharmaceutical product.

C19H19CLN4O3

386.84

386.114

C, 58.99; H, 4.95; Cl, 9.16; N, 14.48; O, 12.41

2095432-75-8

BML-284;853220-52-7

122705993

Solid powder

3

7

5

27

455

0

C1(OC)C=C(C=CC=1)C1=NC(=NC(=C1)NCC1=CC2OCOC=2C=C1)N.Cl

XZOFNDFDGVAIEH-UHFFFAOYSA-N

InChI=1S/C19H18N4O3.ClH/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);1H

4-N-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)pyrimidine-2,4-diamine;hydrochloride

AMBMP Hydrochloride BML284 HCl Wnt Agonist BML284 HCl

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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