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.

Physicochemical Properties

Molecular Formula	C28H28CLN3OS
Molecular Weight	490.05942440033
Exact Mass	489.164
Elemental Analysis	C, 68.63; H, 5.76; Cl, 7.23; N, 8.57; O, 3.26; S, 6.54
CAS #	912545-86-9
Related CAS #	SAG hydrochloride; 2095432-58-7; SAG dihydrochloride; 2702366-44-5; SAG-d3; (Rac)-SAG; 364590-63-6
PubChem CID	5284330
Appearance	White to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	688.6±55.0 °C at 760 mmHg
Flash Point	370.3±31.5 °C
Vapour Pressure	0.0±2.1 mmHg at 25°C
Index of Refraction	1.678
LogP	6.42
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	666
Defined Atom Stereocenter Count	0
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
InChi Key	VFSUUTYAEQOIMW-UHFFFAOYSA-N
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
Chemical Name	3-chloro-N-[4-(methylamino)cyclohexyl]-N-[(3-pyridin-4-ylphenyl)methyl]-1-benzothiophene-2-carboxamide
Synonyms	SAG free base;SAG; SAG (cyclopamine antagonist); SAG (Smo agonist)
HS Tariff Code	2934.99.9001
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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Smoothened/Smo ( EC50 = 3 nM )
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].
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].
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].
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.
Animal Protocol	- For the glucocorticoid-induced cerebellar injury model, neonatal mice (postnatal day 0) received daily intraperitoneal injections of SAG (15 mg/kg) or vehicle for 7 days. Glucocorticoids (dexamethasone, 0.5 mg/kg) were administered subcutaneously daily for 7 days starting on postnatal day 3. Mice were sacrificed on postnatal day 10, and cerebella were analyzed for histological changes, cell proliferation (Ki-67 staining), and apoptosis (TUNEL assay) [2]. - For the polydactyly induction study, pregnant mice (gestational day 10.5) received a single intraperitoneal injection of SAG (20 mg/kg). Offspring were evaluated for limb malformations at birth [2]. - For the bone regeneration study, CD-1 mice with critical-sized calvarial defects were treated with SAG (1.0 mM) in a collagen scaffold implanted into the defect site. Mice were sacrificed at 8 weeks, and micro-CT analysis was performed to assess bone volume and mineral density [2]. Rats: 2.5 nM, intracerebroventricular administration; Mice: 20 μg/g, i.p. Rats and mice
ADME/Pharmacokinetics	- SAG has a plasma half-life of approximately 2 hours in mice after intraperitoneal administration. It is rapidly distributed to tissues, with highest concentrations in the liver, kidney, and brain. SAG is metabolized primarily by the cytochrome P450 system, with less than 10% excreted unchanged in urine [2].
Toxicity/Toxicokinetics	- In acute toxicity studies, SAG administered intraperitoneally to mice at doses up to 500 mg/kg did not cause mortality or significant adverse effects. Subchronic toxicity studies (14 days) showed no evidence of肝肾毒性 (normal liver and kidney function markers) or hematological abnormalities [2]. - SAG did not show significant plasma protein binding (less than 20%) in mouse plasma [2].
References	[1]. Proc Natl Acad Sci U S A . 2002 Oct 29;99(22):14071-6. [2]. Sci Transl Med . 2011 Oct 19;3(105):105ra104. [3]. Birth Defects Res . 2017 Jan 20;109(1):49-54.
Additional Infomation	- SAG is a small-molecule agonist of the Smoothened receptor, activating the Hedgehog (Hh) signaling pathway. It has been studied for its potential in regenerative medicine, particularly in cartilage and bone repair, as well as in neuroprotection against glucocorticoid-induced injury [2]. - The activation of Hh signaling by SAG promotes cell proliferation and survival in various cell types, including chondrocytes, neuronal precursors, and astrocytes. However, chronic activation of Hh signaling is associated with tumorigenesis, but transient treatment with SAG in animal models did not promote tumor formation [2]. - SAG has been shown to enhance osteogenesis in vivo, making it a potential therapeutic agent for bone defects. Its ability to activate Hh signaling in stem/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 is a member of the class of 1-benzothiophenes that is 3-chloro-1-benzothiophene-2-carboxamide in which the amide nitrogen is substituted by trans-4-(methylamino)cyclohexyl and 3-(pyridin-4-yl)benzyl groups. A smoothened (Smo) receptor agonist that antagonizes cyclopamine action at the Smo receptor. Activates the Hedgehog signaling pathway in a Patched independent manner. It has a role as a SMO receptor agonist. It is a member of 1-benzothiophenes, an organochlorine compound, a biaryl, a phenylpyridine, a tertiary carboxamide and a secondary amino compound.

Solubility Data

Solubility (In Vitro)

DMSO: 71~98 mg/mL (134.8~200 mM) Water: N/A Ethanol: ~40 mg/mL (~75.9 mM)

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. 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. 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 corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

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.