AMPK (Ki = 109 nM); ACVR1; BMPR1A; ALK6; Autophagy

Dorsomorphin 可抑制 AICAR 或二甲双胍导致的 ACC 失活，并减弱 AICAR 和二甲双胍增加脂肪酸氧化或抑制肝细胞中脂肪生成基因的作用。 [1]在 HT-29 细胞中，Dorsomorphin 通过抑制 AMPK 活性几乎完全阻止自噬蛋白水解。 [2]此外，Dorsomorphin 通过特异性抑制 BMP I 型受体 ALK2、ALK3 和 ALK6 来阻断 BMP 介导的 SMAD1/5/8 磷酸化、靶基因转录和成骨分化。 [3]

Dorsomorphin (10 mg/kg) 降低成年小鼠铁调素表达的基础水平并增加血清铁浓度。 [3] Dorsomorphin（0.2 mg/kg，静脉注射）显着降低 LPS 治疗大鼠胸主动脉中的 VCAM-1 和 ICAM-1 表达。 [4]

AMPK激酶检测（ELISA检测）[5]
将HT1080细胞接种在24孔板中（每孔2×104个细胞），在有或没有葡萄糖或10 mM 2DG的情况下用化合物C（Dorsomorphin）处理2小时。通过将质粒DNA（pAMPKα1-wt、pAMPKβ1-D168A和pcFlag作为对照）转染在6孔板中，接种在24孔板中并用UPR抑制剂处理，制备过表达野生型和显性阴性AMPKα1的HT1080细胞。用细胞裂解缓冲液（20 mM Tris-HCl，pH 7.5，250 mM NaCl，10%甘油，0.5%NP-40，1 mM EDTA，1 mM EGTA，0.2 mM PMSF，1µg/mL pepstatin，0.5µg/mL亮肽，5 mM NaF，2 mM Na3Vo4，2 mMβ-甘油磷酸，1 mM DTT）裂解细胞。根据制造商的说明，使用CycLex AMPK激酶测定试剂盒测定对照样品（正常生长条件下的载体或pcFlag）的相对AMPK激酶活性（重复测定的平均值±标准差）。[5]

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碱性磷酸酶活性[3]
将C2C12细胞接种到96孔板中，每孔2000个细胞，在添加了2%FBS的DMEM中。用BMP配体和Dorsomorphin或载体对孔进行四次处理。在用50µl Tris缓冲盐水、1%Triton X-100培养5天后收获细胞。将裂解物加入96孔板中的对硝基苯基磷酸盐试剂中1小时，碱性磷酸酶活性以405 nM的吸光度表示。使用与碱性磷酸酶测量相同的重复孔，分别通过细胞滴度Glo和核染料CyQuant的结合来测量细胞存活率和数量。

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Dorsomorphin diHCl (BML-275 二盐酸盐；化合物 C 二盐酸盐) 是一种有效的、选择性的 ATP 竞争性 AMK 抑制剂，Kiof 109 nM。 Dorsomorphin diHCl 通过靶向 I 型受体 ALK2、ALK3 和 ALK6 抑制 BMP 通路。

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肝脏 AMPK 从雄性 SD 大鼠中部分纯化至蓝色琼脂糖步骤。在 40 mM HEPES、pH 7.0、80 mM NaCl、0.8 mM EDTA、5 mM MgCl2、0.025% BSA 和 0.8 mM DTT 缓冲液中，100 μl AMP、100 μl ATP（每个反应 0.5 μCi 33P-ATP），以及100-l 反应混合物中存在 50 μM SAMS。一旦添加酶，反应就开始。 30°C 孵育 30 分钟后，加入 80 μl 1% H3PO4 终止反应。将等分试样（100 μl）转移至 96 孔 MultiScreen 板。用 1% H3PO4 洗涤板 3 次，然后在 Top-count 中检测。化合物 C — (6-[4-(2-哌啶-1-基-乙氧基)-苯基)]-3-吡啶-4-基-吡唑并[1,5-a]嘧啶获得的体外 AMPK 抑制数据— 在默克研究实验室的 N. Thornberry 编写的计算机程序中，使用最小二乘 Marquardt 算法，通过非线性回归拟合以下方程，以实现竞争性抑制： Vi/Vo = (Km + S)/[S + Km × ( 1 + I/Ki)]，其中 Vi 是抑制速度，Vo 是初始速度，S 是底物 (ATP) 浓度，Km 是 ATP 的米氏常数，I 是抑制剂（化合物 C）浓度，Ki是化合物C的解离常数。

在体外研究中，将约1×106/mL RAW264.7细胞接种在48孔板中。细胞用AICAR或/和Dorsomorphin（化合物C）处理不同的时间点。或者，在100ng/mL或1µg/mL脂多糖（LPS，来自大肠杆菌0111:B4）攻击之前，用AICAR或/和Dorsomorphin（化合物C）预处理细胞15分钟至1小时。[4]

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活性氧（ROS）检测[4]
在10µM二苯碘鎓氯化物（DPI）、AICAR或Dorsomorphin（化合物C）存在或不存在的情况下，用2µM羧基-2′，7′-二氯二氢荧光素二乙酸酯（H2DCFDA）预处理细胞15分钟，然后用LPS（1µg/mL）刺激。15分钟后，通过流式细胞术分析确定ROS的产生。未经H2DCFDA处理的细胞被定义为阴性对照。

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Dorsomorphin (compound C)（0-10 μM，18 h）以剂量依赖性方式抑制人纤维肉瘤 HT1080 细胞中 2DG 诱导的 GRP78 启动子活性，但对 tunicamycin 诱导的 GRP78 启动子活性几乎没有影响。 Dorsomorphin（化合物 C） 还可抑制葡萄糖戒断诱导的 GRP78 启动子活性。 Dorsomorphin（化合物 C）对 2DG 诱导的 PERK 激活没有影响，并降低 HT1080 细胞中基础和 2DG 诱导的 AMPK 磷酸化水平。
细胞用过氧化氢处理，并在完全培养基中孵育3天，无论是否用AMPK激活剂二甲双胍（10μM）、AICAR（10μM）单独或加AMPK抑制剂化合物C（CC，10μM）处理。

Iron-replete mice
~10 mg/kg
i.v.

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12-week-old C57BL/6 mice raised on a standard diet are injected via the tail vein with 0.2 g/kg of Dextran or 0.2 g/kg of iron-dextran USP. Dextran is injected with vehicle only, whereas iron-dextran is injected with either vehicle or Dorsomorphin (10 mg/kg). 1 h after injection, mice are killed and liver segments are collected in 500 µL of SDS-lysis buffer and mechanically homogenized. 20 µL of liver extracts are resolved by SDS-PAGE and immunoblotted. Total RNA is harvested using Trizol from mechanically homogenized mouse livers (6 h after injection with a single intraperitoneal dose of Dorsomorphin (10 mg/kg) or DMSO).[3]

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Zebrafish bone mineralization[3]
WT zebrafish embryos were raised in E3 buffer containing phenylthiourea. At 1 day post fertilization (d.p.f.), embryos were treated with dorsomorphin (1–4 µM) or DMSO vehicle. At 5 d.p.f. and onward, larvae were fed for 1 h every other day. Following each feeding, residual food was washed out and medium was replaced with E3 containing dorsomorphin or vehicle. At 10 d.p.f., larvae were immersed in 0.2% calcein for 30 min. Embryos were washed repeatedly in E3 buffer for 3 h to remove unbound calcein and anesthetized with tricaine. Calcified skeletal structures were visualized by green fluorescence, and the number of vertebral bodies were counted. Iron-dextran injections[3]
Adult fish were anesthetized with tricaine and injected with 10 µl of iron-dextran solution (100 mg ml−1, average dextranMW= 5,000) into the abdominal cavity with dorsomorphin (23 µg/g) or vehicle (DMSO). Control fish were injected with 10 µl of dextran (average MW = 5,000, Sigma). Fish were revived in water. 1 h after injection, fish were anesthetized on ice, and livers were collected into 200 µl SDS-lysis buffer and homogenized mechanically. 15 µl of protein extract was fractionated by SDS-PAGE and immunoblotted, as described above. 3 h after injection, total RNA was extracted from mechanically homogenized zebrafish livers using Trizol reagent.[3]
12-week-old C57BL/6 mice raised on a standard diet are injected via the tail vein with 0.2 g/kg of Dextran or 0.2 g/kg of iron-dextran USP. Dextran is injected with vehicle only, whereas iron-dextran is injected with either vehicle or Dorsomorphin (10 mg/kg). 1 h after injection, mice are killed and liver segments are collected in 500 µL of SDS-lysis buffer and mechanically homogenized. 20 µL of liver extracts are resolved by SDS-PAGE and immunoblotted. Total RNA is harvested using Trizol from mechanically homogenized mouse livers (6 h after injection with a single intraperitoneal dose of Dorsomorphin (10 mg/kg) or DMSO).[3]

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Animal Studies[4]
Male BALB/c mice at 6–7 weeks of age weighing 20–22 g were fed with food and water ad libitum, and housed in a standard animal facility with 12 h light/dark cycle and 50%–70% humidity) for 3 days before the study.
BALB/c mice were randomly divided into five experimental groups: Control (intraperitoneally (i.p.) injection of PBS), LPS (i.p. injection of 2 mg/kg body weight), LPS+AICAR (i.p. injection, 500 mg/kg body weight 60 min before LPS injection), LPS+Compound C (CC or Dorsomorphin) (i.p. injection, 25 mg/kg body weight 60 min before LPS challenge), and LPS+AICAR+Compound C (CC or Dorsomorphin) (i.p. injection of 500 mg of AICAR and 25 mg of Compound C (CC or Dorsomorphin) per kilogram of body weight 60 min before i.p. injection of LPS). Six or twelve hours after injection of LPS, the mice were anesthetized with pentobarbital and euthanized thereafter by cervical dislocation, and blood and tissues were collected for analysis.
For survival experiment, the grouped mice as mentioned above were injected i.p. with LPS (20 mg/kg body weight). To investigate the effect of AICAR or compound C/Dorsomorphin, the mice received injection (i.p.) of 500 mg of AICAR or/and 25 mg of compound C per kilogram of body weight 60 min before administration of LPS. Survival of animals was monitored every 2 hours for up to 24 hours. Severity of sepsis was monitored according to general appearance, breathing frequency, and provoked behavior. The mice were euthanized by cervical dislocation under deep anaesthesia, if the mice exhibited a disease point of no return. After 24 hours, the number of the survival mice of each group was recorded: 8 mice survived in total 8 Control mice (8/8), 0/20, 8/19, 12/19, and 3/19 surviving mice in LPS, LPS+AICAR, LPS+CC, LPS+AICAR+CC respectively. All surviving mice were anesthetized and euthanized with the same protocol described above.

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Sprague-Dawley (SD) of 9–12 weeks old female rats (220–270 g) were used in this research. The mechanistic group was given Dorsomorphin (0.2 mg/kg, i.p dissolved in 1% DMSO solution, for the next 21 days) 30 min prior to fisetin HD (40 mg/kg, p.o.). Dorsomorphin was employed as an inhibitor of the activity of AMPK and SIRT1. [6]

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The rats were randomised into four groups, that is control, leptin-, leptin + dorsomorphin (AMPK inhibitor)- and leptin + LY294002 (PI3K inhibitor)-treated groups, with each group consisting of six rats. Leptin was given once daily for 14 days via the intraperitoneal (i.p.) route at a dose of 60 µg/kg body weight (Recombinant Rat Leptin; Purity >95%, Biovision, USA). In the leptin- and inhibitor-treated groups, the animals were given either Dorsomorphin (5 mg kg−1 day−1) or LY294002 (PI3K inhibitor; 1.2 mg kg−1 day−1) i.p. together with leptin for 14 days. Control rats received 0.1 ml of normal saline i.p. for 14 days. Body weight of both the control and experimental animals was recorded weekly. The doses of leptin and dorsomorphin used in this study were according to Almabhouh et al., 2015, and Pachori et al., 2010, respectively. The dose of LY294002 used was according to Shan et al., 2008. The duration of treatment was based on Haron et al., 2010.[7]

[1]. J Clin Invest. 2001 Oct;108(8):1167-74.

[2]. J Biol Chem. 2006 Nov 17;281(46):34870-9.

[3]. Nat Chem Biol. 2008 Jan;4(1):33-41.

[4]. PLoS One. 2014 Jan 24;9(1):e86881.

[5]. PLoS One. 2012;7(9):e45845.

[6]. Naunyn Schmiedebergs Arch Pharmacol. 2024 Jul 4. doi: 10.1007/s00210-024-03257-7

[7]. Andrologia. 2019 Apr;51(3):e13196.

Dorsomorphin is a pyrazolopyrimidine that is pyrazolo[1,5-a]pyrimidine which is substituted at positions 3 and 6 by pyridin-4-yl and p-[2-(piperidin-1-yl)ethoxy]phenyl groups, respectively. It is a potent, selective, reversible, and ATP-competitive inhibitor of AMPK (AMP-activated protein kinase, EC 2.7.11.31) and a selective inhibitor of bone morphogenetic protein (BMP) signaling. It has a role as an EC 2.7.11.31 {[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase} inhibitor and a bone morphogenetic protein receptor antagonist. It is a pyrazolopyrimidine, a member of piperidines, an aromatic ether and a member of pyridines.
Dorsomorphin has been reported in Trigonella foenum-graecum.

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Activation of the NF-κB and mitogen activated protein (MAP) kinases plays an important role in the expression of inflammatory genes such as adhesion molecules. Although compound C (Dorsomorphin) is known as an AMPK inhibitor, AMPK-independent action of it has been recognized. Effects on the expression of ICAM-1 and VCAM-1 by compound C (Dorsomorphin) were investigated in TNF-α-activated human umbilical vein endothelial cells (HUVECs) in vitro and in thoracic aorta of rats treated with lipopolysaccharide (LPS) in vivo. Compound C inhibited ICAM-1 and VCAM-1 expression at the transcriptional as well as translational level in TNF-α-activated HUVECs. In both DN-AMPK- and AMPKα(1)-siRNA-transfected HUVECs, compound C still inhibited TNF-α-induced VCAM-1 and ICAM-1 expression, indicating that this is AMPK-independent action. Interestingly, compound C significantly inhibited NF-κB activity and translocation of p65 to nucleus in HUVECs when activated with TNF-α. Importantly, administration of compound C (0.2 mg/kg) significantly reduced expression of both ICAM-1 and VCAM-1 in LPS-treated rat thoracic aortas. In addition, compound C significantly inhibited iNOS and production of NO in both TNF-α- and LPS-activated RAW 264.7 cells. Finally, compound C significantly inhibited phosphorylation of Akt and p-38MAPK but not protein kinase c or ERK1/2 in HUVECs. Taken together, we conclude that adhesion molecules (ICAM-1, VCAM-1) are to be the novel targets of compound C in preventing inflammatory insult to endothelial cells independent of AMPK inhibition via inhibition of NF-κB activity along with inhibition of phosphorylation of PI3K and P38 MAPK.[Atherosclerosis. 2011 Nov;219(1):57-64. ]

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Inhibiting the unfolded protein response (UPR) can be a therapeutic approach, especially for targeting the tumor microenvironment. Here, we show that compound C (Dorsomorphin) (also known as dorsomorphin), a small-molecule inhibitor of AMP-activated protein kinase (AMPK) and bone morphogenetic protein (BMP) signaling, inhibit the UPR-induced transcription program depending on the glucose deprivation conditions. We found that compound C prevented UPR marker glucose-regulated protein 78 (GRP78) accumulation and exerted enhanced cytotoxicity during glucose deprivation. Gene expression profiling, together with biochemical analysis, revealed that compound C (Dorsomorphin) had a unique mode of action to suppress the transcriptional activation of UPR-targeted genes, as compared with the classic UPR inhibitors versipelostatin and biguanides. Surprisingly, the UPR-inhibiting activity of compound C was not associated with either AMPK or BMP signaling inhibition. We further found that combination treatments of compound C and the classic UPR inhibitors resulted in synergistic cell death with UPR suppression during glucose deprivation. Our findings demonstrate that compound C could be a unique tool for developing a UPR-targeted antitumor therapy.[PLoS One. 2012;7(9):e45845.]

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Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-Dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin- and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.[3]

C24H27CL2N5O

472.41008

471.159

C, 61.02; H, 5.76; Cl, 15.01; N, 14.82; O, 3.39

1219168-18-9

Dorsomorphin;866405-64-3;Dorsomorphin dihydrochloride;1219168-18-9

49761481

Light yellow to yellow solid powder

5.864

55.55

2

5

6

32

514

0

[H]Cl.[H]Cl.C12=C(C3=CC=NC=C3)C=NN1C=C(C4=CC=C(OCCN5CCCCC5)C=C4)C=N2

RJDVIJJQKMGPMV-UHFFFAOYSA-N

InChI=1S/C24H25N5O.2ClH/c1-2-12-28(13-3-1)14-15-30-22-6-4-19(5-7-22)21-16-26-24-23(17-27-29(24)18-21)20-8-10-25-11-9-20;;/h4-11,16-18H,1-3,12-15H2;2*1H

6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine;dihydrochloride

Compound C; CpdC; BML275; Dorsomorphin dihydrochloride; 1219168-18-9; Dorsomorphin 2HCl; Dorsomorphin (dihydrochloride); 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine;dihydrochloride; 6-(4-(2-(piperidin-1-yl)ethoxy)phenyl)-3-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidine dihydrochloride; Dorsomorphin (dihydrochloride) (GMP); Pyrazolo[1,5-a]pyrimidine, 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-, hydrochloride (1:2); BML-275; BML 275

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

DMSO: ~54 mg/mL (~11 mM)
Water: ~100 mg/mL (212 mM) mg/mL

配方 1 中的溶解度: 20 mg/mL (42.34 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。 (<60°C).

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配方 2 中的溶解度: PBS: 15mg/mL

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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。