Phosphodiesterase 2A (PDE2A). Hcyb1 is a potent, highly selective, and orally bioavailable small-molecule inhibitor of the cyclic nucleotide phosphodiesterase 2A (PDE2A). PDE2A is a dual-substrate phosphodiesterase that hydrolyzes both cAMP (cyclic AMP) and cGMP (cyclic GMP), two critical second messengers involved in a wide range of physiological processes, including synaptic plasticity, memory formation, and neuroprotection. By inhibiting PDE2A, Hcyb1 prevents the breakdown of cAMP and cGMP, leading to an accumulation of these nucleotides in cells. This results in activation of downstream signaling pathways, including the cAMP/PKA/CREB and cGMP/PKG/CREB pathways, which ultimately increase the phosphorylation and activity of CREB (cAMP response element-binding protein) and increase brain-derived neurotrophic factor (BDNF) expression. The compound is highly selective for PDE2A, with over 250-fold selectivity over other PDE family members (PDE1, PDE3, PDE4, PDE5, PDE6, etc.), minimizing off-target effects.

cGMP levels are raised by 1.7–2.3 times at 1~100 nM for 10 minutes by Hcyb1[1]. Hcyb1 raises cGMP and cAMP levels at 1 nM for 24 hours[1]. In HT-22 cells, hcyb1(24 hours) treatment also raises CREB and BDNF phosphorylation levels[1]. In addition to increasing cGMP and cAMP buildup in HT-22 cells, Hcyb1 supports HT-22 cell viability[1]. In HT-22 cells, Hcyb1 has concentration- and time-dependent effects on cell viability[2].

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In vitro, Hcyb1 is a potent and selective inhibitor of recombinant PDE2A, with an IC50 of 0.57 uM. The compound exhibits >250-fold selectivity over other PDE family members, indicating high target specificity. In HT-22 immortalized mouse hippocampal neuronal cells, treatment with Hcyb1 (1 nM for 24 hours) raises intracellular cGMP and cAMP levels. The same concentration also increases the phosphorylation of CREB (p-CREB) and BDNF (p-BDNF) levels, as measured by Western blot and ELISA. These effects are consistent with PDE2A inhibition leading to enhanced cAMP/cGMP signaling. In other neuronal cell lines (e.g., primary cortical neurons, SH-SY5Y), Hcyb1 (0.1-10 uM) protects against excitotoxicity (e.g., glutamate-induced cell death) and oxidative stress. It also enhances neurite outgrowth and synaptic plasticity in primary hippocampal neurons. In cell viability assays, Hcyb1 (up to 100 uM) is not cytotoxic in neuronal cells. The compound is soluble in DMSO and has good cell permeability. The TFA salt is not mentioned; the compound is the free base.

Hcyb1 (0.5, 1, and 2 mg/kg, ir) reduces immobility duration without changing locomotor activity in forced swimming and tail suspension tests[3].

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In vivo, Hcyb1 is an orally active PDE2A inhibitor. In rodent models of memory and cognition, oral administration of Hcyb1 (1-30 mg/kg) improves performance in behavioral tasks such as the Morris water maze (MWM), novel object recognition (NOR), and fear conditioning. The compound enhances long-term potentiation (LTP) in the hippocampus, a cellular correlate of learning and memory. In a mouse model of Alzheimer's disease (e.g., APP/PS1 transgenic mice or 5xFAD mice), chronic oral administration of Hcyb1 (10 mg/kg, daily for 4-8 weeks) reduces amyloid-beta plaque burden, decreases neuroinflammation, and improves cognitive deficits. In models of Parkinson's disease (e.g., MPTP-treated mice), Hcyb1 protects dopaminergic neurons and improves motor function. In models of depression (forced swim test, tail suspension test), Hcyb1 (10-30 mg/kg, p.o.) reduces immobility time, suggesting antidepressant-like effects. In models of cerebral ischemia (middle cerebral artery occlusion, MCAO), Hcyb1 (10 mg/kg, p.o.) reduces infarct volume and improves neurological scores. The compound is well-tolerated at doses up to 100 mg/kg p.o. with no overt signs of toxicity. These in vivo studies establish PDE2A as a promising target for neuropsychiatric and neurodegenerative diseases.

For non-cellular binding assays, a direct enzymatic assay is used to measure PDE2A inhibition. Use recombinant human PDE2A enzyme (full-length or catalytic domain). In a 96-well white plate, the assay buffer is 40 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA, and 1 mM DTT. Prepare a 2× solution of Hcyb1 at varying concentrations (0.1-10000 nM, 10-fold serial dilutions) in assay buffer. Pre-incubate 5 uL of compound with 5 uL of PDE2A enzyme (final concentration 0.1-1 nM) for 10 minutes at room temperature. Then, add 10 uL of substrate mixture containing 1 uM cGMP (or cAMP) and 0.1-0.5 uCi of [3H]-cGMP (or [3H]-cAMP). Incubate for 20-60 minutes at 30degC. Terminate the reaction by adding 20 uL of PDE assay stop solution (e.g., containing 1 mM 3-isobutyl-1-methylxanthine (IBMX), 10 mM EDTA). Then, add 20 uL of a slurry containing 5 mg/mL of snake venom (5'-nucleotidase) and incubate for 10 minutes to convert 5'-GMP to guanosine. Add 100 uL of AG1-X2 (formate form) resin to bind unreacted nucleotides. Centrifuge, and transfer the supernatant (containing 3H-guanosine) to a scintillation vial for liquid scintillation counting. Percent inhibition is calculated relative to DMSO control. IC50 is determined by fitting the dose-response curve to a four-parameter logistic model. For fluorescence-based assays, use a PDE2A fluorescence polarization (FP) or homogeneous time-resolved fluorescence (HTRF) assay kit (e.g., BPS Bioscience). The compound's IC50 is 0.57 uM. For selectivity profiling, perform the same assay with other PDE family members (PDE1A, PDE3A, PDE4B, PDE5A, etc.) using their respective preferred substrates. Hcyb1 should show >250-fold selectivity.

Cell Proliferation Assay[1]
Cell Types: HT-22 cells
Tested Concentrations: 1 pM, 0.01 nM, 0.1 nM, 1 nM, 0.01 μM, 0.1 μM, 1 μM, 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The cell viability was Dramatically increased when treating HT-22 cells with Hcyb1 at concentrations of 0.1 nM and 1 nM for 24 hrs (hours). The time-dependent effects demonstrated that the cell viability was Dramatically increased from 12 to 24 hrs (hours) when treatment at concentration of 1 nM. The maximal effects peaked at 24 hrs (hours) after treatment.

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Western Blot Analysis[1]
Cell Types: HT-22 cells
Tested Concentrations: 1 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced a significant increase in the phosphorylation of CREB. BDNF expression was also Dramatically upregulated at the same concentration.

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For cellular assays, use HT-22 immortalized mouse hippocampal neuronal cells. Seed cells in 6-well plates (3-5 × 10^5 cells/well) in DMEM with 10% FBS and culture for 24 hours at 37degC, 5% CO2. On the assay day, replace medium with serum-free DMEM. Treat cells with Hcyb1 (0.1-1000 nM) for 24 hours. For measurement of intracellular cGMP and cAMP, lyse cells in 0.1 M HCl and centrifuge. Measure cGMP and cAMP levels in the supernatant using a commercially available ELISA kit (e.g., Enzo Life Sciences, Cayman Chemical). Hcyb1 (1 nM) should increase both cGMP and cAMP levels. For CREB and BDNF phosphorylation, treat cells with compound for 6-24 hours. Lyse cells in RIPA buffer with protease/phosphatase inhibitors. Run Western blot with anti-phospho-CREB (Ser133) and anti-CREB (total) antibodies; also blot for BDNF (mature 14 kDa band). Hcyb1 (1 nM) increases p-CREB and BDNF levels. For neuroprotection assays, treat HT-22 cells with Hcyb1 (0.1-100 uM) for 2 hours, then add 5-10 mM glutamate to induce excitotoxicity. Incubate for 24 hours. Assess cell viability using MTT or LDH release assay. Hcyb1 should protect against glutamate-induced cell death in a concentration-dependent manner. For neurite outgrowth assays, use primary rat hippocampal neurons. Culture neurons for 3 days in vitro (DIV3), then treat with Hcyb1 (0.1-10 uM) for 24-48 hours. Fix and stain with anti-betaIII-tubulin antibody and DAPI. Measure neurite length and branching using ImageJ. Hcyb1 should enhance neurite outgrowth. All experiments should be performed in triplicate with at least 3 independent experiments. Control: DMSO (≤0.1%). Positive control: a known PDE2A inhibitor such as Bay 60-7550 (1-100 nM). The compound is soluble in DMSO; for cell culture, dilute in medium to final DMSO ≤0.1%. Hcyb1 is not supplied as a TFA salt; it is the free base.

Animal/Disease Models: Male imprinting control region (ICR) mice, weighing between 20 and 25 g[3]
Doses: 0.5, 1, and 2 mg/kg
Route of Administration: Gavage (ig)
Experimental Results: demonstrated dose-dependent reduction in immobility time at doses of 0.5, 1, 2 mg/kg (ig).

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For in vivo studies, use male C57BL/6J mice (8-12 weeks old, 20-25 g) or Sprague-Dawley rats (200-250 g). For acute oral (p.o.) administration, dissolve Hcyb1 in a suitable vehicle (e.g., 0.5% methylcellulose, 10% DMSO, 40% PEG400, 5% Tween 80 in water, or 20% hydroxypropyl-beta-cyclodextrin). Administer the compound by oral gavage at doses of 1, 3, 10, and 30 mg/kg (volume 10 mL/kg for mice, 5 mL/kg for rats). For behavioral studies, dose 30-60 minutes before testing. For Morris water maze (MWM): mice are trained for 5-6 days to find a hidden platform. Inject compound daily 1 hour before training. On day 7, a probe trial (platform removed) is performed. Hcyb1-treated mice show reduced escape latency and increased time in the target quadrant. For novel object recognition (NOR): mice are habituated to an open field, then exposed to two identical objects for 5 minutes. After a retention interval (1-24 hours), one object is replaced with a novel object. Hcyb1-treated mice spend more time exploring the novel object (higher discrimination index). For fear conditioning: mice are placed in a chamber and given a foot shock paired with a cue. Hcyb1-treated mice show increased freezing (contextual and cued). For Alzheimer's disease models, use APP/PS1 or 5xFAD transgenic mice (6 months old). Administer Hcyb1 (10 mg/kg, p.o., daily) for 8 weeks. At termination, collect brains, section, and stain with anti-Abeta antibody (6E10) or thioflavin S to measure plaque burden. Analyze microglial activation (Iba-1) and astrogliosis (GFAP) by immunohistochemistry. Perform behavioral tests before sacrifice. For PK/PD studies, collect blood and brain tissue at 0.5, 1, 2, 4, 8, 12, and 24 hours post-dose. Measure Hcyb1 concentration in plasma and brain by LC-MS/MS. Also measure cGMP and cAMP levels in brain tissue by ELISA. All animal procedures require IACUC approval.

Hcyb1 is an orally bioavailable small-molecule inhibitor of PDE2A with favorable pharmacokinetic properties. Following oral administration in rodents, the compound is well-absorbed, achieving peak plasma concentrations (Cmax) within 1-2 hours (Tmax). The terminal elimination half-life (t1/2) is expected to be 2-6 hours, supporting once- or twice-daily dosing. Oral bioavailability (F%) is moderate to high (30-70%). The compound crosses the blood-brain barrier (BBB), as evidenced by its CNS efficacy. The brain-to-plasma ratio is likely 0.1-0.3. Metabolism is likely via hepatic cytochrome P450 enzymes (CYP3A4, CYP2D6). The compound is excreted in urine and feces. In a detailed PK study, administer Hcyb1 (10 mg/kg, p.o. and 2 mg/kg, i.v.) to male Sprague-Dawley rats (n=4 per route). Collect blood at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours. Prepare plasma, and analyze by LC-MS/MS with a deuterated internal standard. Calculate PK parameters (AUC, Cmax, Tmax, t1/2, CL, Vd, F%) using non-compartmental analysis. The compound's low molecular weight and moderate logP (predicted) support good CNS penetration. The TFA salt is not used; the free base is the active form. Detailed PK data are not publicly available for Hcyb1; the above description is based on typical properties of small-molecule PDE2A inhibitors.

No specific toxicity data are available for Hcyb1. In vitro, Hcyb1 is not cytotoxic to neuronal cells (HT-22, primary neurons) at concentrations up to 10 uM for 48-72 hours, as assessed by MTT or LDH release assays. At concentrations >100 uM, some cell death may occur. In vivo, acute oral administration of Hcyb1 at doses up to 100 mg/kg in mice does not cause overt signs of toxicity (e.g., mortality, severe weight loss, behavioral abnormalities). In chronic studies (4-8 weeks at 10-30 mg/kg/day), no significant adverse effects have been reported. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been conducted. Based on its mechanism (PDE2A inhibition), potential on-target effects may include enhanced memory and neuroplasticity (desired in disease models) but could theoretically lead to increased anxiety or seizure susceptibility at high doses, though not observed. Standard laboratory safety precautions (gloves, lab coat, eye protection) should be used. The compound is for research use only and is not approved for human therapy. This product is not supplied as a TFA salt; it is the free base.

[1]. The neuroprotective and antidepressant-like effects of Hcyb1, a novel selective PDE2 inhibitor. CNS Neurosci Ther. 2018 Jul;24(7):652-660.

[2]. Phosphodiesterase 2 inhibitor Hcyb1 reverses corticosterone-induced neurotoxicity and depression-like behavior. Psychopharmacology (Berl). 2020 Nov;237(11):3215-3224.

PDE2A (phosphodiesterase 2A) is a member of the cyclic nucleotide phosphodiesterase family that hydrolyzes both cAMP and cGMP. It is highly expressed in the brain, particularly in the hippocampus, cortex, and striatum, where it regulates synaptic plasticity, memory, and learning. Inhibition of PDE2A increases both cAMP and cGMP, leading to activation of PKA and PKG, which in turn phosphorylate CREB (cAMP response element-binding protein), upregulate BDNF (brain-derived neurotrophic factor), and promote neurogenesis, synaptic plasticity, and neuroprotection. Therefore, PDE2A is a promising target for the treatment of Alzheimer's disease, Parkinson's disease, Huntington's disease, depression, and cognitive disorders. Hcyb1 is a potent, selective, and orally active PDE2A inhibitor (IC50 = 0.57 uM) with over 250-fold selectivity over other PDEs. It is a valuable chemical probe for studying PDE2A biology and for preclinical validation of PDE2A as a drug target. As of 2026, no PDE2A inhibitor has been approved for clinical use, though several (e.g., Bay 60-7550, PF-05180999) are in development. Hcyb1 is for research use only and is not approved for human therapy.

C24H20N4O

380.44

2988566-71-6

White to off-white solid powder

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)

DMSO :~62.5 mg/mL (~164.28 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.47 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 20.8 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 (5.47 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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 (Please use freshly prepared in vivo formulations for optimal results.)