ML347

Bone morphogenetic protein (BMP) signaling cascade; ALK1 (IC50 = 46 nM); ACVR1 (IC50 = 32 nM); BMPR1A (IC50 = 10800 nM)

更有效（和选择性）的化合物7g（ML347）对ALK1和ALK2的IC50分别为46和32 nM；然而，对ALK3的IC50为10800 nM，比ALK3的选择性高出300倍以上。此外，7g对所有其他测试的激酶完全无活性（对ALK6、9830 nM和KDR（VEGFR2）19700 nM的活性较弱）。值得注意的是，它似乎是5-喹啉和4-甲氧基苯基的组合，这导致了选择性分布，因为13m仍然保留了显著的ALK3活性（539 nM）。由于7g对BMP4细胞测定的效力，ALK1和ALK2以及对其他激酶的显著选择性，7g已被宣布为MLPCN中的探针分子，并重新命名为ML347。[1]

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通过抑制 ALK1/ALK2，ML347 可以阻止 TGF-β 信号的转导 [2]。

为了使BMP社区进一步了解ML347的效用，研究人员在我们的Tier 1体外药代动力学测定中评估了该分子（表4）。这些研究有助于评估许多物种的代谢稳定性和预测清除率，从而为可能的给药途径提供信息。利用快速平衡透析法，测定了人、大鼠和小鼠血浆中ML347的蛋白结合。三种物种的结果相似，ML347显示出高血浆蛋白结合率（Fu~0.01-0.015）。还评估了ML347在肝微粒体中的内在清除率。该措施将有助于预测同三种物种（CLHEP）的体内清除率。[1]

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这些SAR研究最终发现了一种高选择性的ALK2抑制剂ML347，它对ALK2的选择性是ALK3的300倍以上。ML347在BMP4细胞测定（152 nM）以及ALK1（46 nM）和ALK2（32 nM）的体外激酶测定中是有效的，在许多相关激酶中没有活性。计划在许多体内动物疾病模型（如FOP）中进一步研究这种选择性抑制剂，结果将在适当的时候发表。

免疫荧光[2]
细胞类型： 原代牙上皮细胞在补充有 20% 胎牛血清和 1% 青霉素/链霉素的 Dulbecco 改良 Eagle 培养基 (DMEM)/F12 中培养。
测试浓度： 25 μM
孵育时间： 2 小时
实验结果： 抑制 ALK1 /ALK2然后通过 TGF-β1 阻断 Smad1/5。

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本研究旨在探讨TGF-β激活的Smad1/5在牙上皮中的分子机制和生物学功能。采用免疫组织化学方法检测TGF-β信号传导相关基因在小鼠磨牙胚中的表达。培养原代牙上皮细胞，用浓度为0.5或5 ng/mL的TGF-β1处理。分别使用小分子抑制剂SB431542和ML347抑制ALK5和ALK1/2。使用小干扰RNA敲除Smad1/5或Smad2/3。通过EdU法评估细胞的增殖率。TGF-β1和p-Smad1/5在牙上皮芽的基底层高度表达，TGF-β1在上皮芽的内部低表达，而p-Smad2/3高表达。在原代培养的牙上皮细胞中，低浓度的TGF-β1激活Smad2/3，但不激活Smad1/5，而高浓度的TGF-α1能够激活Smad2/3和Smad1/5。SB431542而非ML347能够阻断TGF-β1对Smad2/3的磷酸化。SB431542或ML347均能阻断TGF-β1对Smad1/5的磷酸化。EdU染色显示，高浓度的TGF-β1促进了牙上皮细胞的增殖，这可以通过沉默Smad1/5来逆转，而低浓度的TGF-α1抑制了细胞增殖，这可通过沉默Smad2/3来逆转。总之，TGF-β通过两种途径在调节牙上皮细胞增殖中发挥双重作用。一方面，TGF-β通过ALK5激活典型的Smad2/3信号传导，抑制内部牙上皮细胞的增殖。另一方面，TGF-β通过ALK1/2-ALK5激活非正则Smad1/5信号传导，促进牙上皮芽中基底细胞的增殖。[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.

The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-β-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-β signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-β1 at a concentration of 0.5 or 5 ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-β1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-β1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-β1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-β1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-β1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-β1. EdU staining showed that high concentration of TGF-β1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-β1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-β exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-β activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-β activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.[2]

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A structure-activity relationship of the 3- and 6-positions of the pyrazolo[1,5-a]pyrimidine scaffold of the known BMP inhibitors dorsomorphin, 1, LDN-193189, 2, and DMH1, 3, led to the identification of a potent and selective compound for ALK2 versus ALK3. The potency contributions of several 3-position substituents were evaluated with subtle structural changes leading to significant changes in potency. From these studies, a novel 5-quinoline molecule was identified and designated an MLPCN probe molecule, ML347, which shows >300-fold selectivity for ALK2 and presents the community with a selective molecular probe for further biological evaluation.[1]

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

52.31

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)

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;
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