-SAG Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C28H28CLN3OS |
| Molecular Weight | 490.06 |
| Exact Mass | 489.1642 |
| Elemental Analysis | C, 68.63; H, 5.76; Cl, 7.23; N, 8.57; O, 3.26; S, 6.54 |
| CAS # | 364590-63-6 |
| Related CAS # | SAG;912545-86-9;SAG hydrochloride;2095432-58-7;SAG dihydrochloride;2702366-44-5 |
| PubChem CID | 71433770 |
| Appearance | Typically exists as solid at room temperature |
| LogP | 8.72 |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 5 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 37 |
| Complexity | 666 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | ClC1=C(C(N(C2CCC(NC)CC2)CC2C=CC=C(C3C=CN=CC=3)C=2)=O)SC2C=CC=CC1=2 |
| InChi Key | IYXUQUYRWPGIQL-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H28ClN3OS.2ClH.H2O/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;2*1H;1H2 |
| Chemical Name | 3-chloro-N-[4-(methylamino)cyclohexyl]-N-[(3-pyridin-4-ylphenyl)methyl]-1-benzothiophene-2-carboxamide;hydrate;dihydrochloride |
| Synonyms | (Rac)-SAG; (Rac)-SAG |
| 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 |
| 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 |
At an EC50 of 3 nM, SAG (0.1 nM-100 μM; 30 h) stimulates firefly luciferase expression in Shh-LIGHT2 cells, which is subsequently inhibited at higher dosages [1]. In Cos-1 cells expressing Smo, SAG (1-1000 nM; 1 h) competes for the binding of BODIPY-cyclopamine to the SAG/Smo complex, leading to an apparent dissociation constant (Kd) of 59 nM [1]. The inhibitory impact of ShhN-induced activation of the Robotnikinin pathway is inhibited by SAG (100 nM) [2]. In MDAMB231 cells, SAG (250 nM; 48 h) dramatically raised the expression of SMO mRNA and protein [3]. Under normoxic and hypoxic conditions, SAG (250 nM; 24 and 48 h) enhances mRNA expression in CAXII MDAMB231 cells for 24 h [3]. MDAMB231 cell migration is increased by SAG (250 nM; 24 h) [3].
- 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 [3]. - 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 |
near the eight-week mark, SAG (1.0 mM) dramatically raised BV/TV and largely increased osteogenesis near the defect border in CD-1 mice [4]. In mice, SAG (15–20 mg/kg; ip) consistently and dose-dependently causes preaxial polydactyly [5].
Systemic administration of SAG (15-20 mg/kg; i.p.) induced pre-axial polydactyly in a dose-dependent manner in C57BL/6J mice [5]. - 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]. - Combining SAG (1.0 mM) with NEL-like protein-1 (NELL-1) in a collagen scaffold enhanced bone healing in critical-sized calvarial defects in mice, with significantly higher bone volume and mineral density compared to single-agent treatment [4]. |
| 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 [3]. |
| 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 C57BL/6J mice (gestational day 10.5) received a single intraperitoneal injection of SAG (20 mg/kg). Offspring were evaluated for limb malformations at birth [5]. - 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]. - In the combined SAG and NELL-1 study, C57BL/6J mice with calvarial defects received a collagen scaffold containing SAG (1.0 mM) and NELL-1 (10 μg/mL). Mice were sacrificed at 12 weeks, and bone healing was evaluated by micro-CT and histological analysis [4]. |
| 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 hepatic or renal toxicity (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]. Small molecule modulation of Smoothened activity. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14071-6. [2]. A small molecule that binds Hedgehog and blocks its signaling in human cells. Nat Chem Biol. 2009 Mar;5(3):154-6. [3]. Inhibition of smoothened in breast cancer cells reduces CAXII expression and cell migration. J Cell Physiol. 2018 Dec; 233(12): 9799-9811. [4]. Combining Smoothened Agonist (SAG) and NEL-like protein-1 (NELL-1) Enhances Bone Healing. Plast Reconstr Surg. 2017 Jun;139(6):1385-1396. [5]. Preaxial polydactyly following early gestational exposure to the smoothened agonist, SAG, in C57BL/6J mice. 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 [2,4]. - Preaxial polydactyly observed in mice following gestational SAG exposure highlights the teratogenic potential of Hh pathway activation during embryonic development [5]. |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (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 |