SAG free base;SAG; SAG (cyclopamine antagonist); SAG (Smo agonist)
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Purity: ≥98%
Smoothened Agonist (SAG), a benzothiophene analog, is a potent and cell-permeable Smoothened (Smo) agonist with EC50 of 3 nM in Shh-LIGHT2 cells. In Shh-light 2 cells, SAG significantly (EC50 ~ 3 nM) activates the Hedgehog signaling pathway. Ptch proteins are not necessary for SAG to cause pathway activation. The hedgehog pathway, which is connected to both normal development and carcinogenesis, uses the Smoothened receptor (SMO) to mediate signal transduction. Certain tumors' growth can be inhibited by SMO antagonists.
| Targets |
Smoothened/Smo ( EC50 = 3 nM )
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| ln Vitro |
In vitro activity: SAG can mitigate the antagonistic effects of glucocorticoids (GCs) on the proliferation of granule neuron precursors (CGNPs), which is driven by Shh.[2]
- In SHH-Light2 cells, SAG (0.1 nM - 100 μM; 30 h) induced firefly luciferase expression with an EC₅₀ of 3 nM, but inhibited expression at higher concentrations. Additionally, SAG (1 - 1000 nM; 1 h) competed for the binding of BODIPY-cyclopamine to SMO-expressing COS-1 cells, yielding an apparent dissociation constant (Kd) of 59 nM for the SAG/SMO complex [2]. - SAG (250 nM; 48 h) significantly increased SMO mRNA and protein expression in MDA-MB-231 cells. It also increased CA XII mRNA expression in MDA-MB-231 cells at 24 h under normoxic and hypoxic conditions, and enhanced cell migration at 24 h [2]. - SAG (10 μM) treatment of primary mouse astrocytes for 24 hours increased Gli1 mRNA levels by 2.3-fold and PTCH1 mRNA levels by 2.5-fold. It also reduced GLT-1 protein levels by 50% and GFAP protein levels by 40% [2]. |
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| ln Vivo |
Smoothened Agonist (SAG) administered systemically inhibits the neurotoxic effects of glucocorticoids. In vivo, Smoothened Agonist can activate Shh transcriptional targets by bridging the blood-brain barrier. Neonatal cerebellar developmental abnormalities induced by GC are effectively prevented by SAG at the treatment dose.[2]
- Systemic administration of SAG (15-20 mg/kg; i.p.) induced pre-axial polydactyly in a dose-dependent manner in mice [2]. - In a mouse model of glucocorticoid-induced neonatal cerebellar injury, SAG (1.0 mM) prevented neurotoxic effects by activating the SHH-SMO pathway. It increased 11β-HSD2 expression and promoted cerebellar granule neuron precursor survival and proliferation. Treatment with SAG did not interfere with glucocorticoid-induced lung maturation and did not promote tumor formation after 1-week treatment [2]. - SAG (1.0 mM) induced more osteogenesis at the defect borders and increased bone volume/tissue volume (BV/TV) at the eight-week time point in CD-1 mice [2]. |
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| Enzyme Assay |
- To determine the binding affinity of SAG to SMO, a competition binding assay was performed. COS-1 cells expressing SMO were incubated with BODIPY-cyclopamine (10 nM) and increasing concentrations of SAG (1 - 1000 nM) for 1 hour at room temperature. Fluorescence polarization was measured to determine the displacement of BODIPY-cyclopamine by SAG, yielding a Kd of 59 nM [2].
- An enzyme reporter assay was conducted using SHH-Light2 cells, which stably express a Gli-responsive luciferase reporter. Cells were treated with SAG (0.1 nM - 100 μM) for 30 hours, and luciferase activity was measured. The EC₅₀ for SAG-induced luciferase expression was 3 nM [2]. |
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| Cell Assay |
- Primary mouse astrocytes were treated with SAG (10 μM) for 24 hours. Total RNA was extracted, and qPCR was performed to measure Gli1, PTCH1, GLT-1, and GFAP mRNA levels. SAG increased Gli1 and PTCH1 mRNA levels and decreased GLT-1 and GFAP mRNA levels. Western blot analysis confirmed the reduction in GLT-1 and GFAP protein levels [2].
- MDA-MB-231 cells were treated with SAG (250 nM) for 24 or 48 hours. Total RNA was extracted, and qPCR was performed to measure SMO and CA XII mRNA levels. SAG increased both SMO and CA XII mRNA levels. Cell migration was assessed using a wound healing assay, where SAG-treated cells showed increased migration compared to controls [2]. CGNP are stimulated by various SAG concentrations (15 to 240 nM) compared with ShhN (3 μg/ml) and vehicle after 24 hours in vitro. With the LightCycler 480, quantitative reverse transcription-PCR is carried out using SYBR Green master mix. |
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| Animal Protocol |
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| ADME/Pharmacokinetics |
After intraperitoneal injection, the half-life of SAG in mouse plasma is about 2 hours. It can be rapidly distributed to various tissues, with the highest concentrations in the liver, kidneys and brain. SAG is mainly metabolized by the cytochrome P450 system, with less than 10% excreted unchanged in the urine [2].
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| Toxicity/Toxicokinetics |
In acute toxicity studies, intraperitoneal injection of SAG at doses up to 500 mg/kg did not cause death or significant adverse reactions in mice. Subchronic toxicity studies (14 days) showed no hepatotoxicity or nephrotoxicity (normal liver and kidney function indicators) or hematological abnormalities [2]. - SAG did not show significant plasma protein binding in mouse plasma (less than 20%) [2].
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| Additional Infomation |
SAG is a small molecule agonist of the smoothed receptor that activates the Hedgehog (Hh) signaling pathway. It has been investigated for use in regenerative medicine, particularly in cartilage and bone repair and neuroprotection against glucocorticoid-induced damage [2]. SAG activation of the Hh signaling pathway promotes the proliferation and survival of various cell types, including chondrocytes, neuronal progenitor cells, and astrocytes. However, chronic activation of the Hh signaling pathway is associated with tumorigenesis, but short-term treatment with SAG did not promote tumor formation in animal models [2]. Studies have shown that SAG can enhance osteogenic activity in vivo, making it a potential drug for treating bone defects. Its ability to activate the Hh signaling pathway in stem cells/progenitor cells contributes to its regenerative effects [2]. 3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[3-(pyridin-4-yl)benzyl]-1-benzothiophene-2-carboxamide belongs to the 1-benzothiophene class of compounds, in which the amide nitrogen atom is substituted with trans-4-(methylamino)cyclohexyl and 3-(pyridin-4-yl)benzyl. It is a smoothed (Smo) receptor agonist that antagonizes the action of cyclopamine on Smo receptors. It activates the Hedgehog signaling pathway in a patched-independent manner and functions as an Smo receptor agonist. It is a member of the 1-benzothiophene class, organochlorine compounds, biaryl compounds, phenylpyridine compounds, tertiary carboxamides, and secondary amino compounds.
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| Molecular Formula |
C28H28CLN3OS
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| Molecular Weight |
490.05942440033
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| Exact Mass |
489.164
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| Elemental Analysis |
C, 68.63; H, 5.76; Cl, 7.23; N, 8.57; O, 3.26; S, 6.54
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| CAS # |
912545-86-9
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| Related CAS # |
SAG hydrochloride; 2095432-58-7; SAG dihydrochloride; 2702366-44-5; SAG-d3; (Rac)-SAG; 364590-63-6
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| PubChem CID |
5284330
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| Appearance |
White to yellow solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
688.6±55.0 °C at 760 mmHg
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| Flash Point |
370.3±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.678
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| LogP |
6.42
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
34
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| Complexity |
666
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N([C@H]1CC[C@H](NC)CC1)(CC1C=CC=C(C2C=CN=CC=2)C=1)C(C1SC2C=CC=CC=2C=1Cl)=O
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| InChi Key |
VFSUUTYAEQOIMW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C28H28ClN3OS/c1-30-22-9-11-23(12-10-22)32(28(33)27-26(29)24-7-2-3-8-25(24)34-27)18-19-5-4-6-21(17-19)20-13-15-31-16-14-20/h2-8,13-17,22-23,30H,9-12,18H2,1H3
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| Chemical Name |
3-chloro-N-[4-(methylamino)cyclohexyl]-N-[(3-pyridin-4-ylphenyl)methyl]-1-benzothiophene-2-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 10 mg/mL (20.41 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 100.0 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. Solubility in Formulation 2: ≥ 10 mg/mL (20.41 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 100.0 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. View More
Solubility in Formulation 3: ≥ 10 mg/mL (20.41 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0406 mL | 10.2028 mL | 20.4057 mL | |
| 5 mM | 0.4081 mL | 2.0406 mL | 4.0811 mL | |
| 10 mM | 0.2041 mL | 1.0203 mL | 2.0406 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05823207 | Not yet recruiting | Other: sage essential oil | Premenstrual Syndrome Anxiety |
Cansu Mine Aydin | May 2023 | Not Applicable |
| NCT04735419 | Recruiting | Other: No intervention | Streptococcus Agalactiae Infection | St George's, University of London | December 1, 2021 | N/A |
| NCT01888471 | NCT01888471 | Procedure: Blood sample Other: Vaginal swab sample |
Streptococcus Agalactiae | GlaxoSmithKline | December 11, 2013 | Not Applicable |
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