Phosphodiesterase (PDE4/PDE IV) (Ki = 1930 nM)

The synthesis and biological evaluation of cAMP-specific phosphodiesterase (PDE IV) inhibitors is described. The PDE IV inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724, 2) was used as a template from which to design a set of rigid oxazolidinones, imidazolidinones, and pyrrolizidinones that mimic Ro 20-1724 but differ in the orientation of the carbonyl group. The endo isomer of each of these heterocycles was more potent than the exo isomer in an enzyme inhibition assay and a cellular assay, which measured TNF alpha secretion from activated human peripheral blood monocytes (HPBM). Imidazolidinone 4a inhibited human PDE IV with a Ki of 27 nM and TNF alpha secretion from HPBM with an IC50 of 290 nM. By comparison, Ro 20-1724 is significantly less active in these assays with activities of 1930 and 1800nM, respectively. [1]

In the albino Wistar phase and 200-250 g adults (3-5 months old), Ro 20-1724 (125, 250, and 500 μg/kg; i.p.; 21 days after first icv streptozotocin) significantly decreased cognitive impairments and oxidative short-circuiting in streptozotocin induction [2].

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Cyclic nucleotides viz cGMP and cAMP are known to play an important role in learning and memory processes. Enhancement of cyclic nucleotide signalling through inhibition of phosphodiesterases (PDEs) has been reported to be beneficial in several neurodegenerative disorders associated with cognitive decline. The present study was undertaken to investigate the effect of RO-20-1724-a PDE4 inhibitor on streptozotocin (STZ) induced experimental sporadic dementia of Alzheimer's type. The STZ was injected twice intracerebroventrically (3 mg/kg i.c.v.) on alternate days (day 1 and day 3) in rats. The STZ injected rats were treated with RO-20-1724 (125, 250 and 500 μg/kgi.p.) for 21 days following first i.c.v. STZ administration. Learning and memory in rats were assessed by passive avoidance [PA (days 14 and 15)] and Morris water maze [MWM (days 17, 18, 19, 20 and 21)] following first i.c.v. STZ administration. On day 22 rat cerebral homogenate was used for all the biochemical estimations. The pharmacological inhibition of PDE4 by RO-20-1724 significantly attenuated STZ induced cognitive deficit and oxidative stress. RO-20-1724 was found to not only improve learning and memory in MWM and PA paradigms but also restore STZ induced elevation in cholinesterase activity. Further, RO-20-1724 significantly reduced malondialdehyde and nitrite levels, and restored the glutathione levels indicating attenuation of oxidative stress. Current data complement previous studies by providing evidence for a subset of cognition enhancing effects after PDE4 inhibition. The observed beneficial effects of RO-20-1724 in spatial memory may be due to its ability to restore cholinergic functions and possibly through its antioxidant mechanisms. [2]

Measurement of acetylcholinesterase activity [2]
The quantitative measurement of acetylcholinesterase activity in brain was performed according to the method described by Ellman et al. (1961). The assay mixture contained 0.05 ml of supernatant, 3 ml of 0.01 M sodium phosphate buffer (pH 8), 0.10 ml of acetylthiocholine iodide and 0.10 ml of DTNB. The change in absorbance was measured immediately at 412 nm spectrophotometrically. The acetylcholinesterase activity in the supernatant was expressed as nmol per mg protein.

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Estimation of malondialdehyde (MDA) [2]
The quantitative measurement of malondialdehyde (MDA) – end product of lipid peroxidation – in brain homogenate was performed according to the method of Wills (1996). The amount of MDA was measured after its reaction with thiobarbituric acid at 532 nm using spectrophotometer. The concentration of MDA was determined from a standard curve and expressed as nmol per mg protein.

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Protein carbonyl assay [2]
Protein carbonyl content was determined by the most common and reliable method based on the reaction of carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenylhydrazone (Levine et al., 1990). In this method, 0.1 ml of supernatant from brain homogenate was incubated with 0.5 ml of 10 mM DNPH for 60 min. Subsequently, the protein was precipitated from the solution using 20% trichloroacetic acid. The pellet was washed after centrifugation (3400 × g) with ethyl acetate:ethanol (1:1 vv− 1) mixture thrice to remove excess of DNPH. The final protein pellet was dissolved in 2.5 ml of 6 M guanidine hydrochloride. Absorbance was recorded at 360 nm using a spectrophotometer. Protein carbonyl level was expressed as nmol carbonyl mg − 1 protein, using a molar extinction coefficient of 22 × 104 M− 1 cm− 1.

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Estimation of nitrite [2]
The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined by a colorimetric assay using Greiss reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) as described by Green et al. (1982). Equal volumes of supernatant and Greiss reagent were mixed, the mixture incubated for 10 min at room temperature in the dark and the absorbance determined at 540 nm spectrophotometrically. The concentration of nitrite in the supernatant was determined from sodium nitrite standard curve and expressed as μmol per mg protein.

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Estimation of glutathione [2]
Reduced glutathione in brain was estimated according to the method described by Ellman (1959). One milliliter supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 °C for 1 h. The samples were centrifuged at 1200 × g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer. The concentration of glutathione in the supernatant was determined from a standard curve and expressed as μmol per mg protein.

Experimental procedure and drug administration [2]
The rats were anesthetized with ketamine (100 mg/kg, ip) and xylazine (5 mg/kg, ip). The head of the anesthetized rat was placed in position in the stereotaxic apparatus and a midline sagittal incision was made in the scalp. Two holes were drilled through the skull for placement of injection cannulae into the lateral cerebral ventricles using following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to sagittal suture; 3.6 mm ventral from the surface of the brain (Paxinos and Watson, 1986). The cannulated rats were randomly divided into different groups consisting n = 8 in each group. The STZ [3 mg/kg i.c.v. (1 μl/min)] was dissolved in citrate buffer [3 mg/ml (pH 4.4)] just prior to administration and injected twice intracerebroventrically on alternate days (day 1 and day 3) in rats through a cannula using Hamilton microsyringe in a volume of 5 μl into each lateral cerebral ventricle (bilateral) (Deshmukh et al., 2009, Sharma et al., 2010). Starting from day 1, STZ injected rats were treated with either vehicle [DMSO:saline 10:90/2 ml/kg ip and citrate buffer for i.c.v., n = 8 in each group) or Ro 20-1724 (125, 250 and 500 μg/kg i.p.) for 21 days following first i.c.v. STZ administration. The vehicle for STZ and Ro 20-1724 both were administered to the same animals and served as double vehicle control. The doses used in the present study were selected based on earlier report (Halene and Siegel, 2008). Further, Ro 20-1724 [500 μg/kg i.p, (per se)] has also administered to normal (cannulated) rats for 21 days without STZ injection.

[1]. Design and synthesis of conformationally constrained analogues of 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) as potent inhibitors of cAMP-specific phosphodiesterase. J Med Chem. 1995;38(24):4848-4854.

[2]. Neuroprotective effect of RO-20-1724-a phosphodiesterase4 inhibitor against intracerebroventricular streptozotocin induced cognitive deficit and oxidative stress in rats. Pharmacol Biochem Behav. 2012;101(2):239-245.

4-[(3-Butoxy-4-methoxyphenyl)methyl]-2-imidazolidineone belongs to the methoxybenzene class of compounds. It is a phosphodiesterase inhibitor. Compared to rolipran, there is less research on RO-20-1724 in relation to central nervous system disorders. A few studies have shown that RO-20-1724 has antidepressant-like effects, but its potency is lower than that of rolipran (Wachtel, 1983). The potential effects of RO-20-1724 on cognitive function have not been previously investigated. This study reveals that RO-20-1724 has a significant cognitive-enhancing effect on intraventricular streptozotocin (STZ)-induced cognitive impairment and oxidative stress. Consistent with our previous reports (Deshmukh et al., 2009; Sharma et al., 2010), this study found that bilateral intraventricular streptozotocin (STZ) injection led to a significant decline in spatial learning and memory abilities in rats, manifested as impaired acquisition and retention of Morris water maze and passive avoidance tasks. Changes in motor activity are thought to modulate learning and memory in the passive avoidance and Morris water maze paradigms (Sharma and Gupta, 2003; Deshmukh et al., 2009; Sharma et al., 2010). However, no significant differences in spontaneous motor activity were observed in any of the experimental groups in this study. This excludes the possibility that motor activity itself may have caused changes in passive avoidance and Morris water maze task performance in the carrier-treated and RO-20-1724-treated STZ rats. During the learning phase, the escape latency of STZ rats gradually shortened. However, compared with the carrier control group, the time required for STZ-injected rats to reach the underwater platform was significantly prolonged on days 25, 26, and 27 (Fig. 3A). Similar results were observed in passive avoidance learning, with a shortened memory retention latency on day 15 in STZ-injected rats (Fig. 2) [2]. Conversely, in STZ rats, pharmacological inhibition of PDE4 with RO-20-1724 significantly reduced STZ-induced cognitive deficits. Long-term administration of RO-20-1724 to STZ rats significantly improved cognitive performance on two tasks in a dose-dependent manner. In the water maze probe test performed on day 28 (Figure 3B), STZ rats spent less time in the target quadrant; in the passive avoidance memory retention test, the average memory retention latency of STZ-injected rats was shorter, indicating poorer memory consolidation. However, in streptozotocin (STZ)-injected rats, RO-20-1724 treatment significantly improved memory consolidation, manifested as an increase in time spent in the target quadrant. Subchronic administration of rolipram to normal rats has been reported to improve learning and memory (Rutten et al., 2008). However, this effect of RO-20-1724 itself was not observed in this study. The lack of observed effect of RO-20-1724 itself in this study may be dose-related. This study used the Morris water maze (MWM) as an exosensory model to assess spatial learning and memory (Morris, 1984). Most importantly, spatial learning (especially MWM performance) seems to depend on the coordinated action of different brain regions that constitute functionally integrated neural networks (Hooge and Deyn, 2001). Passive avoidance learning (PAL) refers to learning to inhibit a behavior in order to avoid punishment. The hippocampus and amygdala are both thought to be involved in fear conditioning (passive avoidance). These brain regions are mainly involved in cholinergic transmission, play a crucial role in learning and memory processing, and seem to be more susceptible to oxidative damage (Arendt, 2001; Hartman et al., 2005; Pratico and Delanty, 2000) [2]. In this study, we found that acetylcholinesterase activity increased after intracerebral injection of streptozotocin (STZ) in rats, which is consistent with previous reports (Deshmukh et al., 2009). However, treatment of STZ rats with RO-20-1724 significantly restored acetylcholinesterase activity (Figure 5). These results suggest that the pharmacological inhibition of PDE4 by RO-20-1724 may improve memory in STZ-injected rats by improving cholinergic function. Although there are currently no reports on the effects of RO-20-1724 on cholinergic function, studies have reported that PDE4 inhibitors (PDE4-Is) can promote cholinergic activity (Egawa et al., 1997). RO-20-1724, similar to the widely studied PDE4-I rolipram, inhibits the PDE4 enzyme; it enhances the strength and duration of cAMP-mediated signal transduction (Scuvee-Moreau et al., 1987). In fact, studies have reported that administration of cAMP analogs can also promote the activity of cholinergic neurons and enhance the acetylcholine response (Fu, 1993; Nakamura et al., 1994). Furthermore, in multiple spatial memory tasks (e.g., the water maze and the radial arm maze), PDE4 inhibitors not only improved spatial memory in normal rats and mice (Bach et al., 1999), but also improved spatial memory in rats with impaired spatial memory due to age-related or microsphere-induced cerebral ischemia (Nagakura et al., 2002). In addition, PDE4 inhibitors improved passive avoidance learning in scopolamine-treated rats (Egawa et al., 1997). Moreover, PDE4 inhibitors have been reported to modulate cellular signaling processes by increasing cAMP and/or cGMP levels, ultimately promoting gene transcription by activating the cAMP response element-binding protein (CREB) signaling pathway (Impey et al., 1996; Lu et al., 1999). Furthermore, both the cGMP/PKG/CREB and cAMP/PKA/CREB pathways are considered to play important roles in the cognitive-enhancing effects of PDE inhibitors (Prickaerts et al., 2004; Blokland et al., 2006; Rutten et al., 2007). Increasing evidence supports the view that reactive oxygen species (ROS) and their involvement in oxidative pathways of memory impairment (Bruce-Keller et al., 1998). Consistent with previous studies, streptozotocin (STZ)-induced cognitive deficits have also been found to be associated with oxidative-nitrifying stress and cholinergic deficiencies (Sharma and Gupta, 2001; Deshmukh et al., 2009; Sharma et al., 2010). Oxidative damage to macromolecules (lipids, proteins, and nucleic acids, etc.) is considered a significant factor accelerating aging and age-related neurodegenerative diseases (Wickens, 2001). In this study, streptozotocin (STZ) injection into rats caused membrane lipid and protein peroxidation, manifested as a significant increase in malondialdehyde levels and protein carbonylation. In addition, STZ also caused a significant increase in nitrite levels and a significant decrease in glutathione levels, indicating enhanced oxidative-nitrifying stress (Figures 6, 7, 8, and 9). In this study, in STZ rats, RO-20-1724 was used to pharmacologically inhibit PDE4, and the results showed a significant decrease in malondialdehyde, protein carbonylation, and nitrite levels in a dose-dependent manner, while glutathione levels returned to normal. [2] In summary, the data from this study supplement previous studies and provide evidence for the cognitive enhancement effect after PDE4 inhibition. The beneficial effects of RO-20-1724 on spatial memory may be attributed to its ability to restore cholinergic function or may be related to its antioxidant mechanism. Although the molecular mechanisms by which STZ impairs memory remain to be elucidated, STZ may interfere with intracellular signaling pathways of key protein kinases involved in synaptic plasticity, including the cAMP/PKA/CREB pathway. Therefore, the beneficial regulation of cyclic nucleotide signaling by RO-20-1724 may also contribute to its observed beneficial effects.

C15H22N2O3

278.35

278.163

C, 64.73; H, 7.97; N, 10.06; O, 17.24

29925-17-5

5087

White to off-white solid powder

1.099 g/cm3

483.8ºC at 760 mmHg

246.4ºC

1.62E-09mmHg at 25°C

1.521

2.755

2

3

7

20

312

0

PDMUULPVBYQBBK-UHFFFAOYSA-N

InChI=1S/C15H22N2O3/c1-3-4-7-20-14-9-11(5-6-13(14)19-2)8-12-10-16-15(18)17-12/h5-6,9,12H,3-4,7-8,10H2,1-2H3,(H2,16,17,18)

4-[(3-butoxy-4-methoxyphenyl)methyl]imidazolidin-2-one

Ro-20-1724 Ro 20-1724 Ro20-1724

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~179.63 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.98 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 (8.98 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 (8.98 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.)