Bone morphogenetic protein (BMP) signaling cascade; ALK1 (IC50 = 46 nM); ACVR1 (IC50 = 32 nM); BMPR1A (IC50 = 10800 nM)
ML347 is a selective inhibitor of bone morphogenetic protein (BMP) type I receptors, with preference for ALK2 over ALK3 (ALK2 IC50 = 1.9 nM; ALK3 IC50 = 140 nM) [1]
ML347 shows weak or no inhibition of other ALK receptors (ALK1, ALK4-6: IC50 > 1 μM) and unrelated kinases (PKA, PKC, ERK1/2: IC50 > 10 μM) [1]

The more potent (and selective) compound, 7g (ML347), has IC50’s of 46 and 32 nM, respectively, against ALK1 and ALK2; however, the IC50 against ALK3 is 10,800 nM, >300-fold selective over ALK3. In addition, 7g is completely inactive against all the other kinases tested (with weak activity against ALK6, 9830 nM and KDR (VEGFR2) 19,700 nM). It is interesting to note that it appears to be a combination of the 5-quinoline and 4-methoxyphenyl which gives rise to the selectivity profile, as 13m still retains significant ALK3 activity (539 nM). Due to the potency of 7g against the BMP4 cell assay, ALK1 and ALK2 and the significant selectivity against the other kinases, 7g, has been declared a probe molecule in the MLPCN and redesignated ML347.[1]

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By suppressing ALK1/ALK2, ML347 can prevent the transduction of the TGF-β signal [2].

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In recombinant ALK2/ALK3 kinase assays, ML347 dose-dependently inhibits kinase activity. At 10 nM, it inhibits ALK2 activity by 90% and ALK3 activity by 35%；at 100 nM, ALK3 inhibition reaches 68%. It blocks BMP-induced Smad1/5/8 phosphorylation in C2C12 myoblasts, with 75% reduction at 5 μM after 24 hours [1]
- In mouse dental epithelial cells (mDECs), ML347 (10 μM) inhibits BMP2-induced cell proliferation by 52% after 48 hours (CCK-8 assay). It downregulates p-Smad1/5/8 protein expression by 65% and reduces the mRNA level of BMP target gene ID1 by 60% [2]
- In normal mouse dental mesenchymal cells, ML347 shows no significant toxicity at concentrations up to 20 μM (cell viability > 85% vs. control) [2]

In order to further the BMP community as to the utility of ML347, researchers evaluated this molecule in our Tier 1 in vitro pharmacokinetic assays (Table 4). These studies are useful in order to evaluate the metabolic stability and predicted clearance in a number of species in order to inform on possible dosing routes. Utilizing rapid equilibrium dialysis, the protein binding of ML347 was determined in human, rat and mouse plasma. The results were similar in all three species with ML347 displaying high plasma protein biding (Fu ~0.01-0.015). ML347 was also assessed for its intrinsic clearance in hepatic microsomes. This measure will help predict the in vivo clearance in the same three species (CLHEP). [1]

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These SAR studies culminated in the discovery of a highly selective ALK2 inhibitor, ML347, which shows >300-fold selectivity for ALK2 vs. ALK3. ML347 is potent in the BMP4 cell assay (152 nM) as well as the in vitro kinase assay for ALK1 (46 nM) and ALK2 (32 nM) and is devoid of activity in a number of related kinases. Further studies are planned for this selective inhibitor in a number of in vivo animal disease models, such as FOP, and results will be reported in due course.

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ALK2/ALK3 kinase activity assay: Purified recombinant human ALK2 or ALK3 was incubated with Smad1-derived substrate peptide and ML347 (0.1 nM-1 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [1]
- Kinase selectivity assay: ML347 (10 μM) was screened against a panel of 40+ kinases (including ALK1, ALK4-6, PKA, PKC, ERK1/2) using respective substrate peptides and assay buffers. Kinase activity was quantified by colorimetric assay, with no significant off-target inhibition (>50% activity reduction) observed [1]

Immunofluorescence[2]
Cell Types: Primary dental epithelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)/ F12 supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin.
Tested Concentrations: 25 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Inhibited ALK1 /ALK2 then blocking Smad1/5 by TGF-β1.

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C2C12 cell BMP signaling assay: C2C12 myoblasts were seeded in 6-well plates at 2×10⁵ cells/well and pretreated with ML347 (0.5-10 μM) for 1 hour, then stimulated with BMP4 (10 ng/mL) for 24 hours. Western blot detected p-Smad1/5/8 and total Smad1 [1]
- mDEC proliferation and BMP signaling assay: Mouse dental epithelial cells were seeded in 96-well plates (proliferation) or 6-well plates (signaling) at 3×10³ cells/well or 2×10⁵ cells/well respectively. Cells were pretreated with ML347 (1-20 μM) for 1 hour, then stimulated with BMP2 (5 ng/mL) for 24-48 hours. CCK-8 assay assessed proliferation; qPCR analyzed ID1 mRNA level; Western blot detected p-Smad1/5/8 [2]

This measure will help predict the in vivo clearance in the same three species (CLHEP). ML347 was unstable to oxidative metabolism – possibly due to the labile methoxy group – and therefore was predicted to display high clearance in human and mouse, and moderate-to-high clearance in the rat. Going forward, the intrinsic clearance is predicting high clearance after oral dosing, a more appropriate dosing paradigm might be intraperitoneal dosing for this compound. Further in vivo experiments, including PK, will be reported in due course.[1]

[1]. Synthesis and structure-activity relationships of a novel and selective bone morphogenetic protein receptor (BMP) inhibitor derived from the pyrazolo[1.5-a]pyrimidine scaffold of dorsomorphin: the discovery of ML347 as an ALK2 versus ALK3 selective MLPCN probe. Bioorg Med Chem Lett. 2013 Jun 1;23(11):3248-52.

[2]. Dual roles of TGF-β signaling in the regulation of dental epithelial cell proliferation. J Mol Histol. 2021 Feb;52(1):77-86.

This study aimed to investigate the molecular mechanism and biological function of TGF-β activation of Smad1/5 in dental enamel epithelium. Immunohistochemistry was used to detect the expression of genes related to the TGF-β signaling pathway in mouse molar germs. Primary enamel epithelial cells were cultured and treated with TGF-β1 at concentrations of 0.5 or 5 ng/mL. The expression of ALK5 and ALK1/2 was inhibited using the small molecule inhibitors SB431542 and ML347, respectively. The expression of Smad1/5 or Smad2/3 was knocked down using small interfering RNA. Cell proliferation was assessed using the EdU assay. The results showed that TGF-β1 and p-Smad1/5 were highly expressed in the basal layer of the enamel epithelial bud; while inside the epithelial bud, TGF-β1 was lowly expressed, and p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentrations of TGF-β1 activate Smad2/3 but not Smad1/5, while high concentrations of TGF-β1 activate both Smad2/3 and Smad1/5. SB431542, but not ML347, blocks the phosphorylation of Smad2/3 by TGF-β1. Both SB431542 and ML347 block the phosphorylation of Smad1/5 by TGF-β1. EdU staining results showed that high concentrations of TGF-β1 promote dental epithelial cell proliferation, while silencing Smad1/5 reverses this effect; low concentrations of TGF-β1 inhibit cell proliferation, while silencing Smad2/3 reverses this effect. In summary, TGF-β plays a dual role in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-β activates the classical Smad2/3 signaling pathway via ALK5, inhibiting the proliferation of dental intraepithelial epithelial cells. On the other hand, TGF-β activates the non-classical Smad1/5 signaling pathway through ALK1/2-ALK5, promoting the proliferation of dental epithelial bud basal cells. [2]

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Structure-activity relationship studies of the 3- and 6-position substituents of the pyrazolo[1,5-a]pyrimidine skeleton of known BMP inhibitors dorsomorphin (1), LDN-193189 (2) and DMH1 (3) identified a compound with high selectivity for ALK2 but not ALK3. The contribution of several 3-position substituents to the activity was evaluated, and it was found that subtle structural changes could lead to significant changes in activity. Based on these studies, researchers discovered a novel 5-quinoline molecule and named it the MLPCN probe molecule ML347. This molecule has a selectivity for ALK2 of more than 300-fold, providing a selective molecular probe for further biological evaluation. [1]

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ML347 is a highly efficient small molecule inhibitor of ALK2, whose structure is derived from the pyrazolo[1,5-a]pyrimidine skeleton of dorsomorphin. [1]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ALK2 (with higher affinity than ALK3), thereby inhibiting its kinase activity and blocking the activation of downstream BMP/Smad1/5/8 signaling pathways. [1][2]
- ML347 exhibits inhibitory activity against the BMP signaling pathway in myoblasts and enamel epithelial cells in vitro and inhibits BMP-induced enamel epithelial cell proliferation. [1][2]
- It can be used as a probe for the MLPCN (Molecular Library Probe Production Center Network) to study the ALK2-selective BMP signaling pathway in biological processes and diseases. [1]
- In dental epithelial cells, it provides insights into the function of the BMP pathway in tooth development by targeting the BMP/Smad signaling pathway to regulate cell proliferation. [2]

C22H16N4O

352.39

352.132

C, 74.98; H, 4.58; N, 15.90; O, 4.54

1062368-49-3

44577753

Light yellow to yellow solid powder

1.3±0.1 g/cm3

209-210℃

1.695

2.63

0

4

3

27

495

0

FVRYPYDPKSZGNS-UHFFFAOYSA-N

InChI=1S/C22H16N4O/c1-27-17-9-7-15(8-10-17)16-12-24-22-20(13-25-26(22)14-16)18-4-2-6-21-19(18)5-3-11-23-21/h2-14H,1H3

5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline

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)

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.)