GANT 58 (NSC 75503), a tetrapyridinylthiophene compound, is a novel, cell-permeable and potent Gli antagonist that inhibits GLI1-induced transcription with IC50 of 5 μM. GANT58 acts as downstream Hedgehog (Hh) pathway blocker and targets Gli-mediated gene transactivation (IC50 ~ 5 µM in SAG-stimulated Shh-L2 cells).

Physicochemical Properties

Molecular Formula	C24H16N4S
Molecular Weight	392.47564
Exact Mass	392.109
Elemental Analysis	C, 73.45; H, 4.11; N, 14.28; S, 8.17
CAS #	64048-12-0
PubChem CID	253078
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	414.1±40.0 °C at 760 mmHg
Flash Point	169.0±17.7 °C
Vapour Pressure	0.0±0.9 mmHg at 25°C
Index of Refraction	1.659
LogP	3.12
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	29
Complexity	449
Defined Atom Stereocenter Count	0
SMILES	C1(C2=C(C(C3=CC=NC=C3)=C(S2)C4=CC=NC=C4)C5=CC=NC=C5)=CC=NC=C1
InChi Key	USWLOKMMUTWFMD-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H16N4S/c1-9-25-10-2-17(1)21-22(18-3-11-26-12-4-18)24(20-7-15-28-16-8-20)29-23(21)19-5-13-27-14-6-19/h1-16H
Chemical Name	2,3,4,5-Tetra(4-pyridyl)thiophene
Synonyms	NSC75503; NSC-75503; NSC 75503; GANT 58; GANT-58.
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	Gli(IC50= 5 μM)
ln Vitro	Hh signaling is inhibited downstream by GANT58. Indeed, GANT58 inhibits Hh signaling by blocking Smo and Sufu. Because transcription induced by GLI1 with a mutated nuclear export signal is still blocked, GANT58 primarily functions at the nuclear level. GANT58 successfully inhibits the growth of tumor cells in vitro in a manner that is dependent on GLI. This is achieved by using human prostate cancer cells that have downstream activation of the Hh pathway to block cell growth[1]. It has been demonstrated that GANT58 (NSC75503) prevents GLI1 (as well as the other GLI species) from activating transcription. It has been demonstrated that GANT58 inhibits GLI transactivation[2].
ln Vivo	Tumors (median size ≈250 mm3) are created by injecting GLI1-positive 22Rv1 prostate cancer cells subcutaneously into nude mice. Every day, n = 4–5 injections of solvent alone, GANT61, GANT58, or cyclopamine at a dose of 50 mg/kg are given to naked mice. But after three days, the animals receiving cyclopamine showed signs of severe ulcerations at the injection sites. Consequently, the treatment plan was modified to include injections only once every two days.This protocol is also introduced for the GANTs so that all compounds can be compared, even though the mice treated with these compounds did not exhibit any toxicity. Every injection is made two to three centimeters away from the tumors. For all compounds, there is suppression of tumor cell growth during the course of an 18-day treatment period. The administration of GANT58 or cyclopamine inhibits the growth of additional xenografts and restricts the growth of tumors[1].
References	[1]. Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 2007 May 15;104(20):8455-60. [2]. GLI1 is a central mediator of EWS/FLI1 signaling in Ewing tumors. PLoS One. 2009 Oct 27;4(10):e7608.
Additional Infomation	4-(2,4,5-tripyridin-4-yl-3-thiophenyl)pyridine is a member of pyridines.

Solubility Data

Solubility (In Vitro)

Ethanol : ~20 mg/mL (~50.96 mM) DMSO : ~9.09 mg/mL (~23.16 mM)

Solubility (In Vivo)

Solubility in Formulation 1: 2 mg/mL (5.10 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear EtOH 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: 2 mg/mL (5.10 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear EtOH 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: ≥ 0.91 mg/mL (2.32 mM) 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 9.1 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 4: ≥ 0.91 mg/mL (2.32 mM) 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 9.1 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 5: ≥ 0.91 mg/mL (2.32 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 9.1 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 6: 10% EtOH+40% PEG300+5% Tween-80+45% Saline: 2 mg/mL (5.10 mM) Solubility in Formulation 7: 5 mg/mL (12.74 mM) in 45% PEG300 5% Tween-80 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.5479 mL	12.7395 mL	25.4790 mL
	5 mM	0.5096 mL	2.5479 mL	5.0958 mL
	10 mM	0.2548 mL	1.2740 mL	2.5479 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.