Ki: 3.8±0.2 nM (PDE2)[1]
In comparison to the control, Bay 60-7550 (1 μM) raises cGMP in neuronal cells [F (6,14) for Bay 60-7550=12.97, p<0.05]. In comparison to NMDA alone, Bay 60-7550 increased cGMP even more when NMDA (30 μM) was present. The rise in cGMP in neuronal cultures caused by Bay 60-7550+NMDA is blocked by the NMDA receptor antagonist MK -801 (10 μM) [1]. Comparing IPAH patients' PASMC proliferation to untreated control cells, BAY 60-7550 (1 μM) dramatically inhibited it [2].

In comparison to the control, Bay 60-7550 (1 μM) raises cGMP in neuronal cells [F (6,14) for Bay 60-7550=12.97, p<0.05]. In comparison to NMDA alone, Bay 60-7550 increased cGMP even more when NMDA (30 μM) was present. The rise in cGMP in neuronal cultures caused by Bay 60-7550+NMDA is blocked by the NMDA receptor antagonist MK -801 (10 μM) [1]. Comparing IPAH patients' PASMC proliferation to untreated control cells, BAY 60-7550 (1 μM) dramatically inhibited it [2].

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In primary cultures of rat cerebral cortical neurons, Bay 60-7550 (1 μM) increased basal levels of cGMP. When co-administered with NMDA (30 μM), it further increased NMDA-stimulated cGMP levels. The NMDA receptor antagonist MK-801 (10 μM) blocked the increase in cGMP induced by the combination of Bay 60-7550 and NMDA. [1]
In the same neuronal cultures, Bay 60-7550 (1 μM) increased basal levels of cAMP, although the magnitude of this increase was less than that for cGMP. Co-administration with NMDA (30 μM) further increased cAMP levels compared to NMDA alone, and this effect was not blocked by MK-801 (10 μM). [1]
Bay 60-7550 (1 μM) increased cGMP levels in neuronal cultures stimulated by the NO donor detanonoate (10 μM). The NOS inhibitor L-NAME (10 μM) blocked the detanonoate-induced increase in cGMP. [1]

When compared to vehicle + restraint stress settings, the PDE2 inhibitor Bay 60-7550 (1 mg/kg) corrected the behavioral alterations caused by restraint stress, leading to an increase in the percentage of open arm entry and open arm time. Comparing the vehicle-treated group to the non-stressed mice, Bay 60-7550 demonstrated a dosage-dependent increase in open arm entrance percentage and open arm time; notable increases were seen at a dose of 3 mg/kg. Compared to mice given a vehicle, non-stressed mice treated with Bay 60-7550 saw a dose-dependent increase in both the number and length of head immersions; at doses of 1 and 3 mg/kg, a noteworthy increase was noted [1].

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In mice subjected to restraint stress, Bay 60-7550 (1 mg/kg, i.p.) reversed the stress-induced anxiogenic effects in the elevated plus-maze (increased percentage of open-arm entries and time), hole-board test (increased number of head-dips and head-dipping time), and open-field test (decreased entry latency, increased ambulation and rearing). [1]
In non-stressed mice, Bay 60-7550 (3 mg/kg, i.p.) produced anxiolytic effects in the elevated plus-maze (increased percentage of open-arm entries and time). At 1 mg/kg, it increased the number of head-dips and head-dipping time in the hole-board test, and reduced entry latency while increasing rearing in the open-field test. [1]
The anxiolytic effects of Bay 60-7550 (3 mg/kg) in the elevated plus-maze and hole-board tests were antagonized by pre-treatment with the guanylyl cyclase inhibitor ODQ (20 mg/kg, i.p.). [1]
The anxiolytic effects of the NO donor detanonoate (0.5 mg/kg) in stressed mice tended to be enhanced by co-treatment with Bay 60-7550 (1 mg/kg). [1]

COS-7 cells are maintained in complete DMEM (containing 10% fetal calf serum, 100 units/mL penicillin G, 100 mg/mL streptomycin, and 400 μM L-alanyl-L-glutamine) at 37°C in 5% CO2 atmosphere. A PDE2 expression plasmid is introduced into COS-7 cells using the FuGENE6 transfection reagent. Cells are lysed in solubilization buffer (275 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 2% Triton X, 20% glycerol, and 40 mM Tris-HCl), and the cell lysates are used in the immunoprecipitation procedures. A protein A-agarose bead slurry (100 μL) is washed three times with ice-cold phosphate-buffered saline (100 mM NaCl, 2.7 mM KCl, 10.6 mM Na2HPO4, and 1.6 mM NaH2PO4) and mixed with the 5 μg of PDE2 antibody and 100 μL (2 μg/μL) of the lysate sample and rotated overnight at 4°C. The bead/sample mixture is then centrifuged at 1000g to separate the beads from the supernatant. The beads are resuspended in 100 μL of ice-cold lysis buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.75 mM MgCl2, 1 mM EGTA, 1% Triton X-100, and 20% glycerol, containing protease and phosphatase inhibitors) to elute the PDE2 for use in the enzyme activity assays. The PDE2 activity assay is done. The recombinant PDE2 enzyme derived from COS-7 cell expression and diluted in KHEM buffer (50 mM KCl, 50 mM HEPES, 10 mM EGTA, and 1.9 mM MgCl2, pH 7.2) is mixed with different concentrations of PDE2 inhibitors (Bay 60-7550, ND7001, and EHNA) and [3H]cGMP/cGMP (5 μM) as the substrate. The mixture is then incubated for 30 min at 37°C (100 μL of reaction volume). To convert the [3H]GMP to [3H]guanosine, samples are incubated with snake venom from Crotalus atrox for 30 min at 37°C. The samples are then vortexed with a freshly prepared slurry of Dowex/water/ethanol [1:1:1, v/v] and then centrifuged for 10 min. [3H]Guanosine in the supernatant is then quantified by liquid scintillation counting. Bay 60-7550 is dissolved in dimethyl sulfoxide, EHNA is dissolved in distilled water, and ND7001 is dissolved in ethanol as 10 mM stocks and then diluted for use in assays with 20 mM Tris, pH 7.4; final concentrations of the respective solvents did not affect the assay. IC50 values at a single substrate concentration are determined by nonlinear regression analysis of the log concentration-response curves for each PDE2 inhibitor; Ki values are calculated[1].

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Recombinant PDE2 enzyme was obtained from transfected COS-7 cell lysates via immunoprecipitation using a PDE2 antibody. The enzyme activity assay was performed using a modified two-step procedure. The reaction mixture contained recombinant PDE2 enzyme diluted in KHEM buffer, different concentrations of Bay 60-7550, and [³H]cGMP (5 μM) as substrate. The mixture was incubated for 30 minutes at 37°C. Subsequently, to convert [³H]GMP to [³H]guanosine, samples were incubated with snake venom for 30 minutes at 37°C. The reaction was stopped, and [³H]guanosine in the supernatant was quantified by liquid scintillation counting. The IC₅₀ value at a single substrate concentration was determined by nonlinear regression analysis of log concentration-response curves, and the Ki value was calculated using the Cheng-Prusoff method. [1]

Growth of human distal pulmonary artery smooth muscle cells isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) or control cells from adults undergoing transplant or lung resection for suspected malignancy, are monitored following treatment with BAY 60-7550 (1 μM), ANP (1 μM), DETA-NONOate (10 μM), or Treprostinil (1 μM), alone or in combination[2].

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Primary cultures of rat cerebral cortical neurons were prepared from newborn rat pups. Cultures were maintained at 37°C in a 5% CO₂, 95% O₂ atmosphere. On day 3, media was replaced with fresh medium containing β-cytosine arabinoside to inhibit glial proliferation. On day 5, this medium was replaced with regular medium. Cells were used for assays on day 12. For cGMP and cAMP measurements, cells were rinsed and pre-incubated for 50 minutes at 37°C in HEPES buffer containing tetrodotoxin. Bay 60-7550, NMDA, or other modulators were then added. After a 15-minute incubation, the reaction was stopped by adding ice-cold hydrochloric acid. Cell preparations were sonicated and centrifuged. cAMP and cGMP levels in the supernatant were measured using enzyme-linked immunosorbent assays (ELISA) and normalized to protein content. [1]

Mice[1] Male ICR mice weighing 28 to 35 g are used. Bay 60-7550 (0.5, 1, and 3 mg/kg), ND7001 (0.5, 1.0, and 3 mg/kg), Detanonoate (0.5 mg/kg), L-NAME (50 mg/kg), or Diazepam (1 mg/kg) is administered after restraint stress and 30 min before behavioral testing. Mice also are treated with Bay 60-7550 (3 mg/kg), ND7001 (3 mg/kg), Detanonoate, (0.5 mg/kg), L-NAME (50 mg/kg), or diazepam (1 mg/kg) in the absence of restraint stress; drugs are administered 30 min before the behavioral tests. Bay 60-7550 shows 50-fold selectivity for PDE2 compared with PDE1, 100-fold compared with PDE5, and greater than 200-fold compared with the other PDE families. ND7001 exhibits at 1east 100-fold selectivity for inhibition of PDE2 relative to other PDE families. For antagonism tests to assess the role of cGMP signaling in the behavioral effects of the PDE2 inhibitors, ODQ, an inhibitor of soluble guanylyl cyclase (20 mg/kg), is administered 20 min before Bay 60-7550 or ND7001.

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Male ICR mice (28-35 g) were used. For stress induction, mice were subjected to restraint stress for 24 hours by immobilization in adjustable restrainers at room temperature without access to food or water, then returned to home cages before testing. Non-stressed mice remained in home cages. [1]
Bay 60-7550 was dissolved in 50% dimethyl sulfoxide. It was administered intraperitoneally (i.p.) in a volume of 2 ml/kg body weight, 30 minutes before behavioral testing. Doses used were 0.5, 1, and 3 mg/kg. In stress reversal experiments, the drug was administered after the restraint stress period and before testing. For mechanism studies, the guanylyl cyclase inhibitor ODQ (20 mg/kg, i.p.) was administered 20 minutes before Bay 60-7550. [1]
Behavioral tests (elevated plus-maze, hole-board, open-field) were conducted over 2-3 days, with treatments assigned randomly. A trained observer blind to treatment conditions recorded the behaviors. [1]

[1]. Anxiolytic effects of phosphodiesterase-2 inhibitors associated with increased cGMP signaling. J Pharmacol Exp Ther. 2009 Nov;331(2):690-9.

[2]. Inhibition of phosphodiesterase 2 augments cGMP and cAMP signaling to ameliorate pulmonary hypertension. Circulation. 2014 Aug 5;130(6):496-507.

Phosphodiesterase (PDE)-2 is a component of the nitric oxide synthase (NOS)/guanylate cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced NO-cGMP signaling alterations can influence anxiety-related behaviors, the effects of PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo[5,1-f][1,2,4]triazine-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazaphen-5-yl)benzamide (ND7001) and NO modulators on neuronal cGMP signaling and mouse behavior in elevated cruciate maze, perforated plate, and open field tests—established procedures for evaluating anti-anxiety medications. Bay 60-7550 (1 μM) and ND7001 (10 μM) increased basal levels and N-methyl-D-aspartate (NMDA) or detanonoate-stimulated cGMP levels in primary cultures of rat cortical neurons; Bay 60-7550 (but not ND7001) also increased cAMP levels. Increased cGMP signaling by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiety effects of restraint stress on behavior in three behavioral tests. These drugs also produced anxiolytic effects on behavior in non-stressed mice in the elevated cross maze and perforated plate tests. These effects can be antagonized by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). Conversely, the NOS inhibitor N(ω)-nitro-L-arginine methyl ester (50 mg/kg) reduces cGMP signaling, producing an anxiety effect similar to restraint stress. Overall, current behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for developing drugs to treat anxiety disorders. [1]

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Background: Pulmonary hypertension (PH) is a life-threatening disease characterized by elevated pulmonary artery pressure, pulmonary vascular remodeling, and right ventricular failure. The loss of endothelial-derived nitric oxide (NO) and prostacyclin is a key factor in the pathogenesis of PH, and current treatments aim to restore these pathways. Phosphodiesterases (PDEs) are a class of enzymes that break down cGMP and cAMP, which are fundamental to the biological activity of NO and prostacyclin. PDE5 inhibitors (such as sildenafil) are approved for the treatment of pulmonary hypertension (PH), but the role of PDE2 in lung physiology and disease remains unclear. This study aimed to investigate whether PDE2 inhibitors could modulate pulmonary cyclic nucleotide signaling pathways and improve experimental pulmonary hypertension. Methods and Results: The selective PDE2 inhibitor BAY 60-7550 enhanced pulmonary vasodilation induced by atrial natriuretic peptide (ANP) and treprostrin in isolated pulmonary arteries of chronically hypoxic rats. BAY 60-7550 prevented the development of hypoxia- and bleomycin-induced pulmonary hypertension (PH), and significantly reduced disease severity when used in combination with neutral endopeptidase inhibitors (which enhance endogenous ANP), treprostrin, inorganic nitrates (NO donors), or PDE5 inhibitors. BAY 60-7550 reduced the proliferation of pulmonary artery smooth muscle cells in patients with pulmonary hypertension, and this effect was further enhanced in the presence of ANP, NO, and treprostrin. Conclusion: PDE2 inhibitors induce lung expansion, prevent pulmonary vascular remodeling, and reduce right ventricular hypertrophy characteristic of pulmonary hypertension. This favorable pharmacodynamic property depends on the bioactivity of natriuretic peptides and has an additive effect with prostacyclin analogs, PDE5 inhibitors, and NO. PDE2 inhibitors are a viable oral active agent for the treatment of pulmonary hypertension. [2]

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Bay 60-7550 exhibits 50-fold higher selectivity for PDE2 than PDE1, 100-fold higher selectivity for PDE5, and more than 200-fold higher selectivity for other PDE families. [1]
Bay 60-7550 Inhibition of PDE2 leads to enhanced cGMP signaling pathway, which is associated with its anxiolytic effect. Guanylate cyclase inhibitors antagonize this anxiolytic effect, suggesting that their action depends on the NO-cGMP pathway. [1]
Unlike benzodiazepines (such as diazepam) which can impair memory, other studies have shown that Bay 60-7550 can enhance memory, suggesting that PDE2 inhibitors may have the advantage of not producing cognitive side effects as an anti-anxiety drug. [1]
Bay 60-7550 can effectively reverse the anxiety effects induced by emotional (restraint) stress and oxidative stress as previously observed in studies, while diazepam has limited efficacy against anxiety induced by oxidative stress. [1]

C27H32N4O4

476.56738

476.242

C, 68.05; H, 6.77; N, 11.76; O, 13.43

439083-90-6

135564787

Light yellow to yellow solid powder

3.821

2

6

10

35

728

2

CC1=C2C(=O)NC(=NN2C(=N1)[C@@H](CCCC3=CC=CC=C3)[C@@H](C)O)CC4=CC(=C(C=C4)OC)OC

MYTWFJKBZGMYCS-NQIIRXRSSA-N

InChI=1S/C27H32N4O4/c1-17-25-27(33)29-24(16-20-13-14-22(34-3)23(15-20)35-4)30-31(25)26(28-17)21(18(2)32)12-8-11-19-9-6-5-7-10-19/h5-7,9-10,13-15,18,21,32H,8,11-12,16H2,1-4H3,(H,29,30,33)/t18-,21+/m1/s1

2-(3,4-dimethoxybenzyl)-7-((2R,3R)-2-hydroxy-6-phenylhexan-3-yl)-5-methylimidazo[5,1-f][1,2,4]triazin-4(3H)-one

BAY 60-7550; BAY-607550; BAY607550; BAY 60-7550; 439083-90-6; 2-[(3,4-dimethoxyphenyl)methyl]-7-[(2R,3R)-2-hydroxy-6-phenylhexan-3-yl]-5-methyl-1H-imidazo[5,1-f][1,2,4]triazin-4-one; ZRN7LZK9TQ; CHEMBL370962; BAY 607550; BAY60-7550; BAY-60-7550;

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 : ≥ 33.3 mg/mL (~69.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.25 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.25 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.25 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.

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