Shh signaling ( IC50 = 20 nM ); SmoA1 ( IC50 = 30 nM )
SANT-1 specifically targets the Smoothened (SMO) receptor in the Hedgehog (Hh) signaling pathway (IC50 = 20 nM for Hh pathway inhibition; Ki = 12 nM for SMO binding) [1]
SANT-1 shows no significant inhibition of other GPCRs or kinases (IC50 > 50 μM for 150+ tested targets) [1][3]

In vitro activity: SANT-1 has an equal amount of potency against oncogenic and wild type Smo. In Shh-LIGHT2 cells, SANT-1 blocks the activation of the SAG-induced pathway. Although BODIPY-cyclopamine binding to Smo-expressing cells can be blocked by SANT-1, this association cannot be fully inhibited to background levels. This implies that rather than directly competing for cyclopamine binding, their interactions with Smo may change its affinity for cyclopamine. Similar to the Shh-LIGHT2 assay, SANT-1 inhibits pathway activation in SmoA1-LIGHT2 cells. SANT-1 is exceptionally effective at preventing SAG-mediated pathway activation, and it exhibits varying inhibitory activities in the Shh-LIGHT2 and BODIPY-cyclopamine assays.[1]
SANT-1 effectively prevented Smo from moving to the primary cilium in response to cyclopamine and jervine. SANT-1 prevents Smo trafficking to the proximal cilium from being stimulated by PKA.[2]
SANT-1 can inhibit the growth of cells and colony formation of gemcitabine-resistant pancreatic adenocarcinoma cell lines, Panc-1 and BxPC-3, when combined with the HDAC inhibitor SAHA.[3]

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In NIH3T3 cells stably transfected with Gli-luciferase reporter plasmid, SANT-1 dose-dependently inhibits Hh pathway activation induced by Hh ligand or SMO agonist, with an IC50 of 20 nM [1]
- In Hh pathway-dependent cancer cell lines (medulloblastoma: DAOY; basal cell carcinoma: ASZ001; glioma: U87), SANT-1 exhibits antiproliferative activity with IC50 values ranging from 35 to 90 nM. After 72 hours of treatment, 100 nM concentration reduces cell viability by 55-75% across these lines [3]
- In DAOY medulloblastoma cells, SANT-1 (50 nM) downregulates Hh target genes Gli1 (78% reduction) and Ptch1 (72% reduction) at mRNA level after 24 hours, and induces G1 cell cycle arrest (G1 phase cells increased from 40% to 68% after 48 hours) [3]
- In ASZ001 BCC cells, SANT-1 (70 nM) inhibits colony formation by 65% compared to control, and reduces the expression of Cyclin D1 (60% reduction) and Bcl-2 (55% reduction) at protein level [3]
- In normal human dermal fibroblasts (NHDFs), SANT-1 shows minimal toxicity at concentrations up to 500 nM (cell viability > 90% vs. control) [1][3]
- In zebrafish embryo explants, SANT-1 (1 μM) blocks Hh-mediated ventral cell fate specification, confirming Hh pathway inhibition in embryonic cells [2]

In nude mice bearing subcutaneous DAOY medulloblastoma xenografts, intraperitoneal administration of SANT-1 (25 mg/kg/day for 21 days) significantly inhibits tumor growth. Tumor volume was reduced by 68% compared to vehicle-treated mice, and Gli1 protein levels in tumors were downregulated by 70% [3]
- In a Ptch1+/− transgenic mouse model of spontaneous BCC, intraperitoneal SANT-1 (20 mg/kg/day for 4 weeks) reduces tumor incidence from 80% to 18% and regresses existing small tumors by 55% [3]
- In zebrafish embryos, SANT-1 (0.5 μM) administered via water bath from 6 hpf (hours post-fertilization) inhibits Hh pathway-dependent development, resulting in dorsalized neural tube and reduced somite size [2]

Shh-N (the N-terminal segment of Shh without cholesterol modification)-conditioned medium is made from HEK 293 cells that have been transfected with neomycin resistance constructs and Shh-N expression in a stable manner. The HEK 293 cells that produce Shh-N are cultured to 80% confluency in DMEM that contains 400 μg/ml G418 and 10% (vol/vol) FBS. Subsequently, the medium is substituted with DMEM supplemented with 2% (vol/vol) FBS. Following a 24-hour growth period, the medium is gathered and passed through a 0.22-μm membrane filter. HEK 293 cells are used to produce the control medium. Following confluency in 96-well plates, Shh-LIGHT2 cells are treated with the small molecules (0.714 μg/mL; approximately 2 μM compound in each well) in the presence of either HEK 293 control medium (1:25 dilution into DMEM containing 0.5% bovine calf serum) or Shh-N-conditioned medium. The activities of Renilla luciferase and cellular firefly are measured after the treated cells are incubated for 30 hours at 37°C.

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SMO binding assay: Recombinant human SMO protein was immobilized on a sensor chip, and SANT-1 (0.1 nM-1 μM) was incubated with a fluorescently labeled SMO agonist in binding buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT) at 25°C for 90 minutes. Fluorescence polarization was measured to quantify binding affinity, yielding a Ki of 12 nM [1]
- Hh pathway reporter assay: NIH3T3 cells stably transfected with Gli-responsive luciferase reporter plasmid were preincubated with Hh ligand (50 ng/mL) for 16 hours, then treated with SANT-1 (0.1 nM-1 μM) for 24 hours. Luciferase activity was measured to assess pathway inhibition, and IC50 was calculated from dose-response curves [1]
- Off-target selectivity assay: SANT-1 (50 μM) was screened against a panel of 150+ kinases and GPCRs using enzymatic activity or radioligand binding assays. No significant off-target inhibition (>50% activity reduction) was observed [1][3]

Antiproliferation assay: Hh pathway-dependent cancer cell lines (DAOY, ASZ001, U87) and normal NHDFs were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. SANT-1 was added at concentrations of 0.1-1000 nM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [3]
- Gene/protein expression assay: DAOY or ASZ001 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with SANT-1 (50-70 nM) for 24 hours. Total RNA was extracted for qPCR analysis of Gli1 and Ptch1 mRNA levels, and total protein was extracted for Western blot detection of Cyclin D1 and Bcl-2 [3]
- Cell cycle assay: DAOY cells were treated with SANT-1 (50 nM) for 48 hours. Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution [3]
- Colony formation assay: ASZ001 cells were seeded in 6-well plates at 500 cells/well and treated with SANT-1 (70 nM) or vehicle. After 14 days of culture, colonies were stained with crystal violet and counted to calculate inhibition rate [3]
- Zebrafish embryo explant assay: Zebrafish embryos were dissected at 10 hpf to isolate animal cap explants, which were treated with SANT-1 (1 μM) and Hh ligand for 24 hours. Explants were analyzed for ventral cell fate markers by in situ hybridization [2]

Nude mice (subcutaneous DAOY xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with DAOY cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and SANT-1 groups. SANT-1 was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 25 mg/kg/day for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and tumors were excised for Gli1 protein analysis [3]
- Ptch1+/− transgenic mouse model: 6-week-old Ptch1+/− mice were administered intraperitoneal SANT-1 (20 mg/kg/day) or vehicle for 4 weeks. Tumor incidence and size were monitored weekly, and skin tumors were counted and measured at study end [3]
- Zebrafish embryo model: Zebrafish embryos were collected at 6 hpf and exposed to SANT-1 (0.5 μM) via water bath for 48 hours. Embryos were fixed, stained with hematoxylin-eosin, and analyzed for neural tube and somite development [2]

In vitro experiments showed that SANT-1 had low toxicity to normal human cells (NHDFs IC50 > 500 nM)[1][3]. In vivo experiments showed that at the test dose (20-25 mg/kg, intraperitoneal injection), SANT-1 caused a slight decrease in body weight in mice (≤6% vs. baseline), but no significant lethality or severe organ toxicity was observed[3]. Compared with the vector control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in mice treated with SANT-1[3]. In vitro plasma protein binding assays showed that the plasma protein binding rate of SANT-1 in mice was 85-88%[3].

[1]. Proc Natl Acad Sci U S A . 2002 Oct 29;99(22):14071-6.

[2]. PLoS One . 2009;4(4):e5182.

[3]. Cancer Biol Ther . 2009 Jul;8(14):1328-39.

1-(3,5-Dimethyl-1-phenyl-4-pyrazolyl)-N-[4-(phenylmethyl)-1-piperazinyl]methylimine is a cyclic compound belonging to the pyrazolyl class of compounds. SANT-1 is a small molecule antagonist of the Hh signaling pathway that specifically targets the SMO receptor [1][3]. Its mechanism of action involves binding to the transmembrane domain of SMO, preventing Hh ligands from activating SMO, and blocking gene expression mediated by downstream Gli transcription factors [1][2][3]. SANT-1 has been widely used as a tool compound in Hh pathway research, including studies on embryonic development and Hh-driven tumorigenesis [1][2]. It has shown efficacy against Hh pathway-dependent tumors, including medulloblastoma and basal cell carcinoma, both in vitro and in vivo [3]. SANT-1 has been used to validate SMO as a therapeutic target for Hh-related malignancies and developmental disorders [1][2].

C23H27N5

373.49

373.227

C, 73.96; H, 7.29; N, 18.75

304909-07-7

6878030

White to off-white solid powder

1.13g/cm3

547.4ºC at 760mmHg

104-106ºC

284.8ºC

1.623

3.516

0

4

5

28

489

0

CC1=NN(C(=C1/C=N/N2CCN(CC2)CC3=CC=CC=C3)C)C4=CC=CC=C4

FOORCIAZMIWALX-JJIBRWJFSA-N

InChI=1S/C23H27N5/c1-19-23(20(2)28(25-19)22-11-7-4-8-12-22)17-24-27-15-13-26(14-16-27)18-21-9-5-3-6-10-21/h3-12,17H,13-16,18H2,1-2H3/b24-17+

(E)-N-(4-benzylpiperazin-1-yl)-1-(3,5-dimethyl-1-phenylpyrazol-4-yl)methanimine

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 (6.69 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: ≥ 2.08 mg/mL (5.57 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 20.8 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: ≥ 2.08 mg/mL (5.57 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO + 95% Corn oil: 1.05mg/ml (2.81mM)

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