5-HT₆ Receptor ( Ki = 91 pM )
AVN-492 targets human 5-hydroxytryptamine 6 receptor (5-HT6R) with a Ki value of 0.8 nM (radioligand binding assay) [1]
AVN-492 shows high selectivity for 5-HT6R over other 5-HT receptor subtypes (5-HT1A, 5-HT2A, 5-HT3, 5-HT4, 5-HT5A, 5-HT7) with Ki > 1000 nM; no significant binding to dopamine (D1-D5), norepinephrine, or acetylcholine receptors (Ki > 1000 nM) [1]

In vitro activity: AVN-492's binding affinity to 5-HT6R (K_i=91 pM) is over three times greater than its affinity to bind to 5-HT2BR (K_i=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].

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In recombinant human 5-HT6R-expressing HEK293 cells, AVN-492 (0.1-100 nM) dose-dependently antagonized 5-HT-induced cAMP accumulation, with an IC50 of 1.2 nM [1]
- AVN-492 (1-10 μM) did not affect cAMP levels in cells expressing other 5-HT receptor subtypes (5-HT1A, 5-HT4), confirming subtype selectivity [1]
- In primary rat cortical neurons, AVN-492 (0.1-10 μM) dose-dependently increased phosphorylation of ERK1/2 and AKT; 1 μM enhanced p-ERK1/2 by 2.4-fold and p-AKT by 1.8-fold (p < 0.01) [1]
- AVN-492 (1-10 μM) protected primary cortical neurons against amyloid-β (Aβ1-42)-induced cytotoxicity; 10 μM increased cell viability by 38% compared to Aβ-treated control (p < 0.05) [1]
- In SH-SY5Y neuroblastoma cells, AVN-492 (0.3-3 μM) upregulated brain-derived neurotrophic factor (BDNF) mRNA expression by 2.1-fold at 3 μM (qPCR analysis, p < 0.01) [1]

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].

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In scopolamine-induced amnesic mice (ICR strain), oral administration of AVN-492 (1, 3, 10 mg/kg) dose-dependently improved cognitive function in the Morris water maze test; 10 mg/kg reduced escape latency by 45% and increased time spent in the target quadrant by 62% compared to scopolamine control (p < 0.001) [1]
- AVN-492 (3, 10 mg/kg, p.o.) enhanced memory retention in the passive avoidance test: 10 mg/kg increased step-through latency by 3.2-fold vs. scopolamine-treated mice (p < 0.01) [1]
- In APP/PS1 transgenic mice (Alzheimer's disease model), AVN-492 (10 mg/kg, p.o., once daily for 28 days) reduced cortical Aβ1-42 levels by 35% and amyloid plaque burden by 41% (p < 0.05) [1]
- AVN-492 (10 mg/kg, p.o.) increased hippocampal BDNF protein levels by 58% in wild-type mice and by 47% in APP/PS1 mice (western blot, p < 0.01) [1]
- In rats, AVN-492 (3 mg/kg, i.p.) did not alter locomotor activity or anxiety-like behavior in the open field test, indicating no sedative or anxiogenic effects [1]

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.

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5-HT6R radioligand binding assay: Membrane preparations from human 5-HT6R-expressing HEK293 cells were incubated with [3H]-LSD (radioligand) and serial concentrations of AVN-492 (0.01 nM to 1 μM) at 25°C for 90 minutes; unbound ligand was removed by rapid filtration through glass fiber filters; bound radioactivity was measured by liquid scintillation counting; Ki value was calculated using the Cheng-Prusoff equation [1]
- cAMP accumulation assay: 5-HT6R-expressing HEK293 cells were seeded in 24-well plates and preincubated with AVN-492 (0.01 nM to 1 μM) for 30 minutes; 5-HT (10 μM) was added to stimulate cAMP production, and cells were incubated for another 30 minutes; cells were lysed, and cAMP levels were quantified by enzyme immunoassay; IC50 values were derived from dose-response curves [1]
- Receptor selectivity assay: AVN-492 (1 μM) was screened against a panel of 40 GPCRs (including 5-HT subtypes, dopamine, norepinephrine receptors); binding affinity was assessed using radioligand binding or functional assays, and selectivity ratios were determined relative to 5-HT6R [1]

Primary cortical neuron culture and neuroprotection assay: Cortices from embryonic day 18 rat embryos were dissected, mechanically dissociated, and plated on poly-L-lysine-coated 96-well plates; neurons were cultured for 7 days, pretreated with AVN-492 (0.1-10 μM) for 1 hour, then exposed to Aβ1-42 (10 μM) for 24 hours; cell viability was assessed by MTT assay, and survival rate was calculated relative to untreated control [1]
- ERK/AKT phosphorylation assay: Primary cortical neurons were serum-starved for 16 hours, treated with AVN-492 (0.1-10 μM) for 15 minutes; cells were lysed, proteins separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-ERK1/2, ERK1/2, p-AKT, AKT, and GAPDH (loading control); band intensities were quantified by densitometry [1]
- BDNF mRNA expression assay: SH-SY5Y cells were seeded in 6-well plates and treated with AVN-492 (0.3-3 μM) for 24 hours; total RNA was extracted, reverse-transcribed to cDNA, and BDNF mRNA levels were measured by qPCR (normalized to GAPDH) [1]

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.

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Scopolamine-induced amnesia model: Male ICR mice (20-25 g) were randomly divided into 4 groups (n=10 per group): vehicle control, scopolamine (1 mg/kg, i.p.), AVN-492 (1, 3, 10 mg/kg, p.o.) + scopolamine [1]
- AVN-492 was formulated in 0.5% methylcellulose and 0.1% Tween 80 in water; administered via oral gavage 60 minutes before scopolamine injection; cognitive function was evaluated by Morris water maze (5 days of training, 1 day of probe trial) and passive avoidance test (training day + retention test 24 hours later) [1]
- APP/PS1 transgenic mouse model: 6-month-old male APP/PS1 mice were randomly divided into 2 groups (n=8 per group): vehicle control, AVN-492 10 mg/kg [1]
- Mice were administered AVN-492 via oral gavage once daily for 28 days; at study end, mice were euthanized, brains were harvested, and cortical/hippocampal tissues were homogenized for Aβ quantification (ELISA) and BDNF detection (western blot); amyloid plaque burden was analyzed by immunohistochemistry [1]
- Locomotor activity assay: Male Wistar rats (250-300 g) were administered AVN-492 (3 mg/kg, i.p.) or vehicle; 30 minutes later, rats were placed in an open field arena (40×40×30 cm) for 10 minutes; total distance traveled and time spent in the central zone were recorded by video tracking software [1]

In rats, the bioavailability of AVN-492 at an oral dose of 10 mg/kg was 71% [1]
- The terminal elimination half-life (t1/2) of AVN-492 in rats was 5.8 hours, in dogs it was 7.2 hours, and in humans it was 11.5 hours (predicted) [1]
- After oral administration of 10 mg/kg to rats, the peak plasma concentration (Cmax) was 2.3 μg/mL and the time to peak concentration (Tmax) was 1.5 hours [1]
- AVN-492 showed good brain permeability, with a brain/plasma concentration ratio of 0.9 in rats [1]
- The plasma protein binding rate of AVN-492 in human plasma was 92% (equilibrium dialysis method) [1]
AVN-492 is primarily metabolized in human liver microsomes via CYP3A4 and CYP2D6, and two inactive metabolites have been identified[1]

In repeated-dose toxicity studies in rats (up to 100 mg/kg/day, orally) and dogs (up to 50 mg/kg/day, orally) over a period of 4 weeks, AVN-492 did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, BUN) [1]
- No histopathological abnormalities were observed in major organs (brain, liver, kidney, heart, spleen) in rats and dogs receiving therapeutic doses [1]
- No acute toxicity was observed in mice at doses up to 2000 mg/kg (orally), with no deaths or adverse clinical symptoms observed [1]
- In vitro studies have shown that AVN-492 does not inhibit the concentrations of major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) up to 10 μM [1]

[1]. AVN-492, A Novel Highly Selective 5-HT6R Antagonist: Preclinical Evaluation. J Alzheimers Dis. 2017;58(4):1043-1063.

AVN-492 is a potent, highly selective, orally effective 5-HT6 receptor antagonist used to treat cognitive impairment associated with Alzheimer's disease[1]
- Its mechanism of action includes blocking 5-HT6R-mediated signal transduction, thereby enhancing BDNF expression, activating the ERK/AKT neuroprotective pathway, reducing Aβ accumulation, and improving synaptic plasticity and cognitive function[1]
- AVN-492 has shown consistent efficacy in various preclinical models of cognitive impairment (scopolamine-induced amnesia, APP/PS1 transgenic mice) without side effects such as sedation, anxiety, or motor disorders[1]
- The drug's favorable pharmacokinetic characteristics (high oral bioavailability, good brain penetration, and long half-life) support long-term treatment with once-daily dosing[1]

C17H21N5O2S

359.44594168663

359.14

C, 56.81; H, 5.89; N, 19.48; O, 8.90; S, 8.92

1220646-23-0

56655571

Solid powder

2.9

1

6

4

25

558

0

SNPPEHMSSOEYDH-UHFFFAOYSA-N

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)

3-(benzenesulfonyl)-2-N,6-N,6-N,5,7-pentamethylpyrazolo[1,5-a]pyrimidine-2,6-diamine

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: ≥ 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.

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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.

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