AT-533 is a novel inhibitor of Hsp90 and HSV. AT-533 inhibits the growth of tumors and angiogenesis by obstructing the signaling pathway of HIF-1α, VEGF, and VEGFR-2. Additionally, AT-533 prevents the downstream pathways—Akt/mTOR/p70S6K, Erk1/2, and FAK—from being activated. Human umbilical vein endothelial cells (HUVECs) are inhibited by AT-533 in terms of tube formation, cell migration, and invasion.

Physicochemical Properties

Molecular Formula	C23H30N4O3
Molecular Weight	410.509305477142
Exact Mass	410.231
CAS #	908112-37-8
Related CAS #	908112-37-8
PubChem CID	24820800
Appearance	Off-white to light yellow solid
LogP	3.3
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	661
Defined Atom Stereocenter Count	0
InChi Key	FMOSGTTYAFQMSD-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H30N4O3/c1-13-21-19(11-23(2,3)12-20(21)29)27(26-13)15-6-9-17(22(24)30)18(10-15)25-14-4-7-16(28)8-5-14/h6,9-10,14,16,25,28H,4-5,7-8,11-12H2,1-3H3,(H2,24,30)
Chemical Name	2-[(4-hydroxycyclohexyl)amino]-4-(3,6,6-trimethyl-4-oxo-5,7-dihydroindazol-1-yl)benzamide
Synonyms	AT-533; AT533; AT 533
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	HSP90; HSV-1; ERK1; ERK2; NF-κB; Akt; HIF-1α; VEGF/VEGFR-2
ln Vitro	Twenty ng/mL VEGF-induced tube, cell migration, and HUVEC templates are inhibited by AT-533 (0-1350 nM; 24 h or 48 h) [1]. Under hypoxic conditions, AT-533 (2 μM or 75 μM; 24 h) suppresses the coupling of HIF-1α/VEGF signaling in breast cancer cells and phosphorylates FAK, Akt/mTOR/p70S6K, and Erk1/2 [1]. AT-533 (0.5 μM; 2 h, 4 h) on chorion membrane (CAM) decreases the HSV-1-induced TNF-α, IL- Production of 1β and IL-6. AT-533 (10 nM, 50 nM; 48 h).
ln Vivo	AT-533 (10 mg/kg; intraperitoneal injection; once daily for 21 days) Fluorescent dye MDA-MB-231 Expression of HIF-1α/VEGF coupling signaling-related proteins in breast cancer xenograft tumor transplant model [1]. AT-533 (1, 2, 4 mg/kg; i.p.; once daily for 30 days) did not produce death, recovery, weight loss, or adverse effects in the Sprague-Dawley subacute poisoning study [
Cell Assay	Cell Viability Assay [1] Cell Types: Human Umbilical Vein Endothelial Cells (HUVEC): MCF-7 and MDA-MB-231 Tested Concentrations: 0, 5.6, 16.7, 50, 150, 450 and 1350nM Incubation Duration[2]: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours). Experimental Results: Cell viability was inhibited at 48 hrs (hours) with an IC50 value of 50.1 nM. Western Blot Analysis[1] Cell Types: MCF-7 cells and MDA- MB-231 Cell Tested Concentrations: 5 nM, 10 nM, 50 nM and 75 nM Incubation Duration: 24 hrs (hours) Experimental Results: Inhibition of VEGF-2, phosphorylation of Akt, mTOR, Erk1/2, FAK.
Animal Protocol	Animal/Disease Models: MDA-MB-231 breast cancer xenograft male C57BL/6 mice [1] Doses: 10 mg/kg; 3]. Route of Administration: intraperitoneal (ip) injection; one time/day for 21 days. Experimental Results: The expression of HIF-1α and VEGF was Dramatically down-regulated.
References	[1]. AT-533, a novel Hsp90 inhibitor, inhibits breast cancer growth and HIF-1α/VEGF/VEGFR-2-mediated angiogenesis in vitro and in vivo. Biochem Pharmacol. 2020 Feb;172:113771. [2]. AT-533, a Hsp90 inhibitor, attenuates HSV-1-induced inflammation. Biochem Pharmacol. 2019 Aug;166:82-92. [3]. Subacute toxicological evaluation of AT-533 and AT-533 gel in Sprague-Dawley rats. Exp Ther Med. 2021 Jun;21(6):632.

Solubility Data

Solubility (In Vitro)

DMSO: ~33.3 mg/mL (81.2 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.09 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.09 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 25.0 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: ≥ 2.5 mg/mL (6.09 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 25.0 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	2.4360 mL	12.1800 mL	24.3599 mL
	5 mM	0.4872 mL	2.4360 mL	4.8720 mL
	10 mM	0.2436 mL	1.2180 mL	2.4360 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.