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-imidazolidinone is a member of methoxybenzenes.
Inhibitor of phosphodiesterases.

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In comparison with rolipram, there has been little research on RO-20-1724 for central nervous system diseases. Few studies indicate antidepressant-like effects for RO-20-1724, albeit at lower potency than rolipram (Wachtel, 1983). Potential effects of RO-20-1724 on cognitive functions have not been addressed before. The present study reveals important cognition-enhancing effects of RO-20-1724 against i.c.v. STZ induced cognitive deficit and oxidative stress. Consistent with our previous reports (Deshmukh et al., 2009, Sharma et al., 2010), bilateral i.c.v. STZ administration in rats was found to cause significant deficits in spatial learning and memory as indicated by impaired acquisition and retention in Morris water maze and passive avoidance tasks in the present study. The changes in locomotor activity have been suggested to modulate the learning and memory in passive avoidance and Morris water maze paradigms (Sharma and Gupta, 2003, Deshmukh et al., 2009, Sharma et al., 2010). However no significant difference in spontaneous locomotor activity was observed in any of the experimental groups in the present study. This excludes the possibility that the locomotor activity per se may have contributed to the changes in passive avoidance and Morris water maze in vehicle treated and RO-20-1724 treated STZ rats. During acquisition trials, STZ rats showed gradual decrease in escape latency. However, STZ injected rats took significantly larger time to reach the submerged platform as compared to vehicle control on days 25, 26 and 27 (Fig. 3A). Similar results were also obtained in passive avoidance learning, wherein STZ injected rats showed decrease in retention latency on day 15 (Fig. 2).[2]
Conversely, the pharmacological inhibition of PDE4 by RO-20-1724 in STZ rats significantly attenuated STZ induced cognitive deficit. Chronic administration of RO-20-1724 in STZ rats showed significant dose dependent improvement in cognitive performance in both the tasks. In the probe test conducted on day 28 in water maze (Fig. 3B), STZ rats spent less time in the target quadrant and in passive avoidance during the retention trial the mean retention latency was decreased in STZ injected rats indicating poorer consolidation of memory. Whereas, RO-20-1724 treatment in STZ injected rats produced significant improvement in memory consolidation, as evidenced by increased time spent in target quadrant. It has been reported that the sub-chronic administration of rolipram (per se) in normal rats improves learning and memory (Rutten et al., 2008). However, such an effect for RO-20-1724 per se was not observed in the present study. Lack of effect for RO-20-1724 per se observed in the present study may be dose related. Morris water maze (MWM) is employed in the present study as an exteroceptive model to evaluate spatial learning and memory (Morris, 1984). Most importantly, spatial learning in general and MWM performance in particular appear to depend upon the coordinated action of different brain regions constituting a functionally integrated neural network (Hooge and Deyn, 2001). Whereas, passive avoidance learning (PAL) refers to the learned inhibition of behavior in order to avoid punishment. Both hippocampus and amygdala are thought to be involved in fear conditioning (passive avoidance). All these regions of the brain are mainly involved in cholinergic transmission and play vital role in learning and memory processing, and seem to be more prone to oxidative damage (Arendt, 2001, Hartman et al., 2005, Pratico and Delanty, 2000).[2]
In the present study, acetyl cholinesterase activity was found to be increased following STZ injection in rat brain which is in accord with earlier report (Deshmukh et al., 2009). Whereas, RO-20-1724 treatment in STZ rats was able to significantly restore the acetyl cholinesterase activity (Fig. 5). These results suggest that the pharmacological inhibition of PDE4 by RO-20-1724 may improve memory performance of STZ injected rats by improving cholinergic functions. Although, there are no reports on RO-20-1724 in relation to its effects on cholinergic functions, however, PDE4 inhibitors (PDE4-Is) has been reported to promote cholinergic activity (Egawa et al., 1997). RO-20-1724 inhibits PDE4 enzyme, like rolipram, an extensively studied PDE4-I; it increases intensity and duration of cAMP-mediated signaling (Scuvee-Moreau et al., 1987). Indeed, administration of analogs of cAMP has also been reported to promote the activity of cholinergic neurons and potentiate acetylcholine responses (Fu, 1993, Nakamura et al., 1994). Moreover, in several spatial memory tasks (e.g., water escape task and radial arm maze) PDE4-Is has shown to improve spatial memory not only in unimpaired rats and mice (Bach et al., 1999), but also in rats of which spatial memory was impaired by age or microsphere embolism-induced cerebral ischemia (Nagakura et al., 2002). In addition, PDE4-I has also been shown to improve passive avoidance learning in scopolamine treated rats (Egawa et al., 1997). Moreover, PDE inhibitors have been reported to mediate cellular signaling processes by elevating the levels of cAMP and/or cGMP, which ultimately can lead to gene transcription through activation of cAMP response element binding protein (CREB) signaling pathway (Impey et al., 1996, Lu et al., 1999). Further, both cGMP/PKG/CREB and cAMP/PKA/CREB pathways have been suggested to play a major role in cognition enhancing effects of PDE inhibitors (Prickaerts et al., 2004, Blokland et al., 2006, Rutten et al., 2007). Accumulating evidence supports the concept of reactive oxygen species and its involvement in oxidative pathway of memory impairment (Bruce-Keller et al., 1998). In line with previous studies STZ induced cognitive deficit was also found to be associated with oxidative–nitritive stress and cholinergic deficit (Sharma and Gupta, 2001, Deshmukh et al., 2009, Sharma et al., 2010). Oxidative damage to macromolecules (lipid, protein and nucleic acids, etc.) has been considered as an important factor in the acceleration of aging and age-related neurodegenerative disorders (Wickens, 2001). In the present study, STZ injection in rats has shown to cause peroxidation of membrane lipids and proteins as evidenced by significant increase in malondialdehyde levels and protein carbonyl formation. Moreover, STZ has also caused a significant increase in nitrite and decrease in glutathione levels indicating increased oxidative–nitritive stress (Fig. 6, Fig. 7, Fig. 8, Fig. 9). In the present study, pharmacological inhibition of PDE4 by RO-20-1724 in STZ rats showed significant dose dependent reduction in the levels of malondialdehyde, protein carbonyl formation and nitrite and restored glutathione levels.[2]
In summary, 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. Although, the molecular mechanisms mediating the memory-impairing effects of STZ remain to be elucidated, it is possible that STZ may interfere with protein kinase intracellular signaling pathways crucially involved in synaptic plasticity, including the cAMP/PKA/CREB pathway. Therefore, favorable modulation of cyclic nucleotide signaling by RO-20-1724 may also play a role for the 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.)