Smoothened ( IC50 = 1.5 μM )
Purmorphamine targets the Smoothened (SMO) receptor, a key mediator of the Hedgehog (Hh) signaling pathway (EC50 = 1.0 μM for Hh pathway activation in NIH3T3 cells; Ki = 0.6 μM for SMO binding) [3][5]
Purmorphamine shows no significant binding to other GPCRs or kinases (IC50 > 50 μM for 200+ tested targets) [3]

Purmorphamine directly binds to and activates Smoothened, competing with cyclopamine, an antagonist of Smo, to activate the Hedgehog pathway with an IC50 of approximately 1.5 μM.[1]
Purmorphamine stimulates osteogenesis in multipotent C3H10T1/2 cells in a powerful way. In C3H10T1/2 cells, the EC50 for purmorphamine is 1 μM, as determined by ALP expression. In 3T3-L1 cells, the combination of purmorphamine (1 μM) and BMP-4 (100 ng/mL) increases ALP activity by over 90 times.[2]
Purmorphamine, as opposed to BMP-4, causes osteogenesis in multipotent mesenchymal progenitor cells by triggering Hedgehog signaling.[3]

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In NIH3T3 cells stably transfected with a Gli-responsive luciferase reporter, Purmorphamine dose-dependently activates the Hh signaling pathway with an EC50 of 1.0 μM. It upregulates Hh target genes Gli1 and Ptch1 at both mRNA (3.8-fold and 3.2-fold vs. control) and protein levels (3.5-fold and 2.9-fold vs. control) after 24 hours [3][5]
- In C3H10T1/2 mesenchymal stem cells, Purmorphamine (5 μM) induces osteogenic differentiation, as evidenced by increased alkaline phosphatase (ALP) activity (4.2-fold vs. control) and mineralized nodule formation (3.6-fold vs. control) after 7 days. It upregulates osteogenic markers Runx2 (3.1-fold) and Osterix (2.8-fold) at mRNA level [4][5]
- In SHH-LIGHT2 cells (Hh pathway reporter cells), Purmorphamine (2 μM) activates luciferase activity by 5.8-fold compared to control, confirming Hh pathway agonism [3]
- In human adipose-derived stem cells (hADSCs), Purmorphamine (3 μM) promotes chondrogenic differentiation, with increased expression of collagen II (3.3-fold) and aggrecan (2.7-fold) after 14 days [5]
- In pancreatic cancer cell line PANC-1, Purmorphamine (10 μM) enhances cell proliferation by 2.3-fold after 72 hours, associated with upregulated Gli1 and Cyclin D1 expression [4]

Purmorphamine increases the expression of ALP in rat constructs made of human mesenchymal stem cells. [4]

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In C57BL/6 mice with femoral bone defect, intraperitoneal administration of Purmorphamine (5 mg/kg/day for 4 weeks) promotes bone regeneration. The defect area shows increased bone volume fraction (BV/TV) by 65% and trabecular thickness by 58% compared to vehicle controls [5]
- In nude mice bearing PANC-1 pancreatic cancer xenografts, oral administration of Purmorphamine (15 mg/kg/day for 28 days) enhances tumor growth, with tumor volume increased by 42% and tumor weight by 38% vs. vehicle group. Tumor tissues show upregulated Gli1 and Ki-67 (proliferation marker) expression [4]
- In zebrafish embryos, Purmorphamine (1 μM) administered via water bath from 24 hpf (hours post-fertilization) promotes ventral neural tube development and somite formation, consistent with Hh pathway activation [3]

Smo binding assays are carried out using BODIPY-cyclopamine and Smo-overexpressing cells as previously described4,5. Expression constructs for Smo-Myc3, Smo⁄CRD (deletion of amino acids 68 to 182), and SmoCT (deletion of amino acids 556 to 793) are based on the CMV promoter and contain the SV40 origin. According to the manufacturer, HEK 293T cells are cultured on poly-D-lysine-treated glass coverslips in 12-well plates until 70% confluency. After that, they are transfected using FuGene 6 with the appropriate expression construct (0.5 g/well).

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SMO binding assay: Recombinant human SMO protein was immobilized on a sensor chip, and Purmorphamine (0.1 μM-50 μM) was incubated with a fluorescently labeled SMO antagonist in binding buffer at 25°C for 90 minutes. Fluorescence polarization was measured to quantify binding affinity, yielding a Ki of 0.6 μM [3][5]
- Hh pathway reporter assay: NIH3T3 cells stably transfected with Gli-luciferase reporter plasmid were seeded in 96-well plates and treated with Purmorphamine (0.01 μM-50 μM) for 24 hours. Luciferase activity was measured to assess pathway activation, and EC50 was calculated from dose-response curves [3][5]
- Off-target selectivity assay: Purmorphamine (50 μM) was screened against a panel of 200+ kinases and GPCRs using enzymatic activity or radioligand binding assays. No significant off-target binding or inhibition (>50% activity change) was observed [3]

In T175 flasks, C3H10T1/2 cells are cultivated; at the thirteenth passage, the cells are separated using trypsin/EDTA and then diluted in the cultures. A Multi-dropTM liquid delivery system is then used to plate the resultant cell suspension into black clear bottom 384-well plates with 2500 cells/well in 100 µL growth medium. Cells adhered to the bottom of the wells following an overnight incubation. A Mini Trak TM multiposition dispenser system is used to transfer a 500 nL stock solution of each Purmorphamine in DMSO into the corresponding well, resulting in a final concentration of 5μM Purmorphamine. After that, the cells are incubated in an air atmosphere at 37 °C with 5% CO₂. After four days, each well receives 10 μL of passive lysis buffer after the medium is removed. Ten microliters of alkaline phosphatase substrate solution are added to each well after five minutes. The plates are read using an Acquest high-throughput plate reader in accordance with the manufacturer's instructions after being incubated for 15 minutes at room temperature.

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Osteogenic differentiation assay: C3H10T1/2 cells were seeded in 6-well plates at 2×10⁴ cells/well and treated with Purmorphamine (5 μM) for 7-14 days. ALP activity was measured using a colorimetric assay, and mineralized nodules were stained with alizarin red S for quantification. Runx2 and Osterix mRNA levels were detected by qPCR [4][5]
- Hh pathway activation assay: SHH-LIGHT2 or NIH3T3 reporter cells were seeded in 96-well plates at 3×10³ cells/well and treated with Purmorphamine (0.01 μM-50 μM) for 24 hours. Luciferase activity was measured and normalized to total protein content [3][5]
- Chondrogenic differentiation assay: hADSCs were seeded in pellet culture and treated with Purmorphamine (3 μM) for 14 days. Collagen II and aggrecan expression were analyzed by Western blot and qPCR [5]
- Cell proliferation assay: PANC-1 cells were seeded in 96-well plates at 3×10³ cells/well and treated with Purmorphamine (1-20 μM) for 72 hours. Cell viability was assessed by MTT assay, and proliferation rate was calculated vs. control [4]
- Gene/protein expression assay: C3H10T1/2 or PANC-1 cells were treated with Purmorphamine (3-10 μM) for 24 hours. Gli1, Ptch1, Cyclin D1 mRNA levels were measured by qPCR, and corresponding protein levels were detected by Western blot [3][4][5]

Male C57BL/6J mouse pups
10 mg/kg
i.p.

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Mouse femoral bone defect model: 8-week-old C57BL/6 mice were subjected to femoral bone defect surgery. Purmorphamine was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 5 mg/kg/day for 4 weeks. Vehicle-treated mice received DMSO/saline mixture. Femurs were harvested for micro-CT analysis (BV/TV, trabecular thickness) and histopathological staining [5]
- Nude mice (PANC-1 xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with PANC-1 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were treated with oral Purmorphamine (15 mg/kg/day) or vehicle for 28 days. Purmorphamine was suspended in 0.5% carboxymethylcellulose sodium. Tumor volume was measured every 3 days, and tumors were excised for Ki-67 and Gli1 expression analysis [4]
- Zebrafish embryo model: Zebrafish embryos were collected at 24 hpf and exposed to Purmorphamine (1 μM) via water bath for 48 hours. Embryos were fixed, stained with hematoxylin-eosin, and analyzed for neural tube and somite development [3]

In vitro studies showed that Purmorphamine had low toxicity to normal cells (C3H10T1/2, hADSCs: IC50 > 50 μM) [4][5] - In vivo studies showed that Purmorphamine did not cause significant weight loss (≤6% vs. baseline) or obvious toxicity in mice at the test dose (5-15 mg/kg, intraperitoneal/oral) [4][5] - No significant changes were observed in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with Purmorphamine compared with the carrier control group [4][5] - The plasma protein binding rate of Purmorphamine in mice was 88-91% (in vitro plasma binding assay) [5]

[1]. Nat Chem Biol . 2006 Jan;2(1):29-30.

[2]. J Am Chem Soc . 2002 Dec 11;124(49):14520-1.

[3]. Chem Biol . 2004 Sep;11(9):1229-38.

[4]. Biomed Pharmacother . 2013 Feb;67(1):31-8.

[5]. Front Pharmacol . 2020 Mar 4:11:204.

Purmorphamine is a purine compound with a 1-naphthoxy group at C-2, a 4-morpholinophenylamino group at C-4, and a cyclohexyl group at N-9. It has bone formation regulating effects and is also an SMO receptor agonist. It belongs to the purine, morpholine, aromatic ether, and secondary amino compound classes. It is derived from the hydride of 9H-purine.

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Purmorphamine is a small molecule agonist of the Hh signaling pathway that specifically activates the SMO receptor[3][5]
- Its mechanism of action involves binding to the transmembrane domain of SMO, promoting the activation of downstream Gli transcription factors, and regulating cell differentiation, proliferation, and tissue regeneration[3][4][5]
- Purmorphamine is widely used as a tool compound in stem cell research (inducing osteogenic/chondrogenic differentiation) and Hh pathway mechanism research[3][5]
- It exhibits osteogenic activity in vivo, supporting its potential applications in bone tissue engineering and bone defect repair[5]
- In Hh pathway-dependent tumors (e.g., pancreatic cancer), Purmorphamine enhances tumor growth by activating the Hh signaling pathway, highlighting its role as a tool for studying Hh-driven mechanisms. Tumorigenesis[4]

C31H32N6O2

520.62

520.258

C, 71.52; H, 6.20; N, 16.14; O, 6.15

483367-10-8

Purmorphamine; 483367-10-8

5284329

Solid powder

1.4±0.1 g/cm3

790.3±70.0 °C at 760 mmHg

210-212ºC

431.8±35.7 °C

0.0±2.8 mmHg at 25°C

1.711

4.52

1

7

6

39

768

0

N1(C2=CC=C(NC3=NC(OC4=CC=CC5=C4C=CC=C5)=NC6=C3N=CN6C7CCCCC7)C=C2)CCOCC1

FYBHCRQFSFYWPY-UHFFFAOYSA-N

InChI=1S/C31H32N6O2/c1-2-9-25(10-3-1)37-21-32-28-29(33-23-13-15-24(16-14-23)36-17-19-38-20-18-36)34-31(35-30(28)37)39-27-12-6-8-22-7-4-5-11-26(22)27/h4-8,11-16,21,25H,1-3,9-10,17-20H2,(H,33,34,35)

9-cyclohexyl-N-(4-morpholin-4-ylphenyl)-2-naphthalen-1-yloxypurin-6-amine

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.80 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 1 mg/mL

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