AVN-492 is a highly specific, selective and orally bioavailable antagonist of the 5-HT6R with Ki of 91 pM. AVN-492 exhibits a binding affinity of over three orders of magnitude higher to 5-HT6R (Ki=91 pM) than it does to 5-HT2BR (Ki=170 nM), which is the only other target. Because of this, the compound AVN-492 demonstrated excellent 5-HT6R selectivity against all other subtypes of serotonin receptors and exhibited extreme specificity against all other receptor types, including adrenergic, GABAergic, dopaminergic, histaminergic, and others. In rodents, AVN-492 exhibits a good ADME profile both in vitro and in vivo, along with high oral bioavailability and good brain permeability. An extremely promising method for assessing the possible contribution of the 5-HT6 receptor to cognitive and neurodegenerative impairments is AVN-492. AVN-492 is a promising drug candidate undergoing Phase I trials to be tested for treating these kinds of illnesses.

Physicochemical Properties

Molecular Formula	C17H21N5O2S
Molecular Weight	359.44594168663
Exact Mass	359.14
Elemental Analysis	C, 56.81; H, 5.89; N, 19.48; O, 8.90; S, 8.92
CAS #	1220646-23-0
Related CAS #	1220646-23-0
PubChem CID	56655571
Appearance	Solid powder
LogP	2.9
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	558
Defined Atom Stereocenter Count	0
InChi Key	SNPPEHMSSOEYDH-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H21N5O2S/c1-11-14(21(4)5)12(2)22-17(19-11)15(16(18-3)20-22)25(23,24)13-9-7-6-8-10-13/h6-10H,1-5H3,(H,18,20)
Chemical Name	3-(benzenesulfonyl)-2-N,6-N,6-N,5,7-pentamethylpyrazolo[1,5-a]pyrimidine-2,6-diamine
Synonyms	AVN-492; AVN 492; AVN492
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	5-HT6 Receptor ( Ki = 91 pM )
ln Vitro	In vitro activity: AVN-492's binding affinity to 5-HT6R (Ki=91 pM) is over three times greater than its affinity to bind to 5-HT2BR (Ki=170 nM), which is the only other target. Consequently, AVN-492 exhibits excellent 5-HT6R selectivity against all other subtypes of serotonin receptors and possesses exceptional selectivity against all other receptor types, including adrenergic, GABAergic, dopaminergic, histaminergic, and so forth[1].
ln Vivo	The concentration of AVN-492 in rats' plasma, brain, and CSF varies with dose when given by PO. The plasma and brain drug concentration curves have an atypical form, with the brain-plasma ratio consistently hovering around 11% across all dosages. With 10 mg/kg, the drug concentration in CSF reaches 50% of the plasma level, but its dependence on dosage is basically linear. After administering AVN-492 intravenously (IV) at a dose of 2 mg/kg to mice, the concentrations of the drug in the plasma and brain decreased over time. However, at the 15- and 60-minute mark, the brain/plasma ratio (mean±SEM) remained relatively constant at 13.2±0.7% and 9.0±1.5%, respectively. According to this, AVN-492's steady-state concentration gradient is created at least 15 minutes after the medication is administered[1].
Enzyme Assay	AVN-492 is dissolved to a concentration of 10 mM in 100% DMSO. After that, this DMSO solution is diluted 50 times using either MQ water or a pH-appropriate buffer. The MultiScreen 96-well plates are used for the Caco-2 permeability assay. Simply put, each well with a porous membrane bottom is seeded with Caco-2 cells (ATCC, Cat. No. HTB-37). The cells are grown in a CO2 thermostat at 37°C for 20–23 days, or until they reach total confluence. Every two to three days, the growth medium is swapped. Using Lucifer Yellow CH, a "leak test" is used to determine the physical integrity of the cell monolayer that has been established on the well-porous membrane. The permeability of AVN-492 (200μM) is assessed in both directions: from basal to apical and from apical to basal. The permeabilities of propranolol (which has a high permeability) and ranitidine (which has a low permeability) are only measured in the apical-to-basal direction. Verapamil (100 μM), a Pgp inhibitor, is used with or without Rhodamine 123 (30 μM) to measure its Pgp-dependent permeability. The apical-to-basal and basalto-apical permeabilities are also measured in the presence of verapamil in order to evaluate the Pgp pump's potential involvement in an AVN-492 efflux. LC/MS/MS API2000 is used to determine the concentrations of AVN-322 in donor and acceptor chambers.
Animal Protocol	Mice and Rats: Male CD1 (24–30 g), male SHK (20–25 g), male Wistar rats (220–242 g), and male Balb/C mice (15–20 g) are used for pharmacokinetic, behavioral, and toxicological studies. Male Wistar rats and male CD-1 mice are used for the pharmacokinetic profiling of AVN-492. Three rodents make up each dose-route group. There are two ways to administer AVN-492: orally (PO) or intravenously (IV). The animals are promptly put to death by being placed in a CO2 chamber at various times following the drug administration. Using a cardiopuncture, blood samples are taken. Male Wistar rats are given oral doses of AVN-492 in three different experiments (3 independent groups, 3 animals per group) at concentrations of 1 mg/kg, 3 mg/kg, and 10 mg/kg. The animals are placed in a stereotaxic frame, given 5% halothane anesthesia 60 minutes later, and samples of cerebrospinal fluid (CSF) are extracted from the cisterna magna using a 23G needle. The CSF samples are examined to make sure there is no blood contamination. Following the collection of CSF samples, blood is extracted via cardiopuncture, and the brains are extracted, instantly cleaned in ice-cold saline, and homogenized in a 1:4 brain tissue/water mixture. Using acetonitrile, AVN-492 is extracted from each sample, and the concentrations are calculated.
References	[1]. AVN-492, A Novel Highly Selective 5-HT6R Antagonist: Preclinical Evaluation. J Alzheimers Dis. 2017;58(4):1043-1063.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 100 mg/mL Water: N/A Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.75 mg/mL (10.43 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 37.5 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: ≥ 3.75 mg/mL (10.43 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 37.5 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	2.7820 mL	13.9101 mL	27.8203 mL
	5 mM	0.5564 mL	2.7820 mL	5.5641 mL
	10 mM	0.2782 mL	1.3910 mL	2.7820 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.