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Keywords:

  • antiinflammatory;
  • phosphodiesterase;
  • phosphodiesterase4B2 inhibitor;
  • 1,2,4-oxadiazole

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

A series of 3,5-disubstituted-1,2,4-oxadiazoles has been prepared and evaluated for phosphodiesterase inhibition (PDE4B2). Among the prepared 3,5-disubstituted-1,2,4-oxadiazoles, compound 9a is the most potent inhibitor (PDE4B2 IC50 = 5.28 μm). Structure–activity relationship studies of 3,5-disubstituted-1,2,4-oxadiazoles revealed that substituents 3-cyclopentyloxy-4-methoxyphenyl group at 3-position and cyclic ring bearing heteroatoms at 5-position are important for activity. Molecular modeling study of the 3,5-disubstituted-1,2,4-oxadiazoles with PDE4B has shown similar interactions of 3-cyclopentyloxy-4-methoxyphenyl group; however, heteroatom ring is slightly deviating when compared to Piclamilast. 3-(3-Cyclopentyloxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9a) exhibited good analgesic and antiinflammatory activities in formalin-induced pain in mice and carrageenan-induced paw edema model in rat.

Secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), are of immense interest among chemists and biologists since their discovery (1). Phosphodiesterase4 (PDE4) is a hydrolytic enzyme responsible for the degradation of cAMP in various cell types. Inhibition of PDE4 enzyme can reduce cAMP degradation that is proven to be beneficial in controlling the proinflammatory cytokines like tumor necrosis factor-α, interleukins, leukotrienes, and interferons, and it also reduces the recruitment and activation of inflammatory cells like neutrophils, monocytes, and macrophages (2–9). Inhibitors of PDE4 enzyme exhibit broad range of antiinflammatory and antibronchoconstrictor effects in animal models (10). The diverse biological effects have implicated PDE4 as a potential target for a range of inflammatory diseases including asthma and chronic obstructive pulmonary disease (2). However, the therapeutic window of orally administered PDE4 inhibitors in clinical trials is limited by gastrointestinal side-effects such as nausea, vomiting, abdominal pain, diarrhea, and dyspepsia (10–13). Of the PDE4 isoforms (PDE4A, B, C, D), PDE4B is the predominant isoform present in human monocytes and neutrophils that play vital role in inflammation (14). First generation PDE4 inhibitor, Rolipram (1), caused significant side-effects such as nausea and emesis in clinical trials (15). Second generation PDE4 inhibitor, Piclamilast (2), was discontinued from clinical trials because of undesired side-effects and poor pharmacokinetics (16). Tetomilast (3) from Otsuka Pharmaceuticals has reached third phase in clinical trials (17).

Recently, there has been growing interest in compounds containing the 1,2,4-oxadiazole scaffold because of their unique chemical structure and broad spectrum of biological properties including tyrosine kinase inhibition (18), muscarinic agonism (19), histamine H3 antagonism (20), antiinflammation (21), anticancer (22–24), and monoamine oxidase inhibition (25). The 1,2,4-oxadiazoles are also widely used as heterocyclic amide or ester bioisosters (26) and in the design of dipeptidomimetics as peptide building blocks (27,28). Incorporation of 1,2,4-oxadiazole ring in the second generation PDE4 inhibitors may improve their metabolic stability and inhibition activities (Figure 1).

image

Figure 1.  Second generation phosphodiesterase4 inhibitors.

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To design selective PDE4 inhibitors devoid of side-effects, we have synthesized a new series of 3,5-disubstituted-1,2,4-oxadiazoles while maintaining the crucial catechol ring with cyclopentyl and methyl groups (Figure 2).

image

Figure 2.  Schematic view for the rational in designing 1,2,4-oxadiazoles as phosphodiesterase4 inhibitors.

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

General

All the laboratory grade reagents were obtained commercially. Melting points were determined on EZ-Melt (Stanford Research Systems, Sunnyvale, CA, USA) automated melting point apparatus and are uncorrected. The arylnitriles 6 were prepared according to the literature procedures (29,30). All the reactions were monitored by thin layer chromatography, which was performed on Merck precoated plates (silica gel 60 F254, 0.25 mm) and visualized by fluorescence quenching under UV light (254 nm). Column chromatography was performed using 200–400 mesh silica gel and a mixture of hexane and ethyl acetate for elution. 1H NMR spectra were recorded on Bruker Avance II (400 MHz) and Bruker (200 MHz) NMR spectrometers. Mass spectra were obtained on a ‘Hewlett-Packard’ HP GS/MS 5890/5972 mass spectrometer.

Experimental

Synthesis of amidoximes (7)

To a mixture of appropriate benzonitrile 6 (10 mmol) and hydroxylamine hydrochloride (20 mmol) in ethanol (50 mL) was added drop wise an aqueous solution of sodium hydroxide (20 mmol, 10 mL) while maintaining the temperature at 0 °C. The resulting mixture was allowed to reflux with stirring for 18 h. Ethanol was distilled off under reduced pressure. and the remaining crude product was taken into water (50 mL). The pH (∼2) of the solution was adjusted with 1N HCl. and the aqueous phase was washed with ethyl acetate (2 × 25 mL). Upon cooling (0 °C) and neutralization with sodium carbonate gave off white precipitate that was filtered, washed, and air dried at 60 °C to afford pure amidoxime 7.

General procedure for the synthesis of 3,5-disubstituted-1,2,4-oxadiazoles (8a-s)

A solution of appropriate carboxylic acid (0.8 mmol) in dry DMF (1 mL) was cooled to 0 °C and added dicyclohexylcarbodiimide (1.2 mmol) under nitrogen atmosphere, and the reaction mixture was stirred at the same temperature for 1 h. To this reaction mixture, appropriate amidoxime 7 (0.8 mmol) was added and stirred at 0 °C for 0.5 h. The reaction mixture was slowly brought up to 30 °C and continued stirring for another 3 h; gradually, reaction temperature was raised to 110 °C, and stirred for another 10 h. The reaction mixture was cooled to 25 °C and poured into ice-cold water (25 mL). Upon addition of ethyl acetate (25 mL) and stirring for 10 min, crystals of dicyclohexylurea were separated out and removed by filtration. The separated aqueous phase was extracted with ethyl acetate (2 × 20 mL), and the combined organic phase was washed with brine and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off, and the residue thus obtained was purified by flash column chromatography using ethyl acetate–hexane (0–25%) as eluent to afford pure 8.

Synthesis of ethyl 4-(3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (10a)

A solution of 3-(3-cyclopentyloxy-4-methoxy-phenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole 9a (0.2 mmol) in dry dichloromethane (5 mL) was cooled to 0 °C and added triethylamine (0.25 mmol) followed by ethyl chloroformate (0.2 mmol) under nitrogen atmosphere, and the reaction mixture was stirred for 5 h at 30 °C. The reaction mixture was poured into ice-cold water (5 mL), and separated aqueous phase was extracted with dichloromethane (3 × 10 mL). The combined organic phase was washed with brine, dried over anhydrous sodium sulfate, and distilled off. The residue so obtained was purified by flash column chromatography using ethyl acetate–hexane (0–50%) as eluent to afford pure compound 10a.

Synthesis of 3-(3-cyclopentyloxy-4-methoxy-phenyl)-5-(1-methylsulfonylpiperidin-4-yl)-1,2,4-oxadiazole (10b)

The 1,2,4-oxadiazole 10b was prepared according to the procedure described for 10a except that methanesulfonyl chloride was used instead of ethyl chloroformate.

PDE4B2 inhibitory activity

The PDE4B2 Inhibition was measured using PDE4B2 catalytic domain and HitHunter cAMP assay kit according to the manufacturer’s instructions (DiscoveRx Corp., Fremont, CA, USA). All assay reactions were performed at 30 °C in 96-well plates. Compounds were dissolved in 100% dimethylsulfoxide (DMSO) as a 1 mm stock and diluted in 5% DMSO buffer (20 mm Tris–Cl, 10 mm MgCl2 at pH 7.4) to obtain a range of concentrations in the assay. %Inhibition was determined at 10 μm inhibitor concentration; dose-dependent inhibition was determined in the presence of 1 μm concentrations of cAMP over a range of inhibitor compound concentrations using vehicle control as DMSO and reference control as Piclamilast.

Screening of analgesic activity: formalin-induced pain in mice model

Swiss Albino mice of either sex weighing between 20 and 25 g were selected for the experiment. All mice were divided in six groups (control, test, and standard in duplicates). At zero time, all mice in control group were injected with saline solution (vehicle), while those in standard group were injected with Aspirin (100 mg/kg) intraperitoneally, 30 min prior to formalin injection (31). Test group animals were injected with compound 9a at dose of and 20 mg/kg/10 mL. After 30 min, 25 μL of 2% formalin was injected in each mouse by subcutaneous route in hind paw. After 5 min, every mouse was observed for response to formalin-induced pain in terms of number of flinching, licking, biting, and lifting of its limbs. All readings were noted down. The results are expressed as mean ± SEM. Differences in mean values between groups were analyzed by a one-way analysis of variance (anova) followed by a post hoc dunnet test.

Screening of antiinflammatory activity: carrageenan-induced paw edema in rat model

The antiinflammatory activity of compound 9a was determined using carrageenan-induced rat paw edema test according to the method of winter et al. (32). Forty-two male Wistar rats (180–220 g) were randomly divided into six groups and fasted overnight before the experiment with free access to water. Compound 9a in 10% PEG at doses of 10 and 20 mg/kg was administered intraperitoneally to rats, 30 min before subcutaneous injection of carrageenan (1%) into the plantar surface of the left hind paw. The control group received an equivalent volume of vehicle (10% PEG), and the positive-control group received Diclofenac 10 mg/kg i.p. After the carrageenan injection, the paw volumes were measured at 60 min using a plethysmometer (Model 7150; UGO Basile, Comerio, Italy). Edema was expressed as the mean increase in paw volume relative to control animals. The results are expressed as mean ± SEM. Differences in mean values between groups were analyzed by a one-way analysis of variance (anova) followed by a post hoc dunnet test.

Docking study

The different ligand-binding modes of the compound 9a and Piclamilast with the PDE4B enzyme (PDB ID: 1XM4) were investigated by using an advanced molecular docking program glide (Schrödinger Inc., New York, NY, USA). Inhibitor geometry was optimized by gamess interface in CHEMBIO-3D using semi-empirical method PM3. Protein preparation was performed in Maestro 8.5 protein preparation wizard (Schrödinger, LLC, New York, NY, USA); the center and the size for the docking grid box were selected with respect to the co-crystallized ligand, and the default parameters were used for flexible ligand docking using the SP level of accuracy.

Analytical data

3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-phenyl-1,2,4-oxadiazole (8a)

White solid. Yield 39%, m.p. 102–103 °C. 1H NMR (CDCl3, 400 MHz): 8.22 (2H, dd, J = 5.20, 0.9 Hz), 7.76 (1H, dd, J = 6.24, 1.44 Hz), 7.67 (1H, d, J = 1.44 Hz), 7.61–7.54 (3H, m), 6.97 (1H, d, J = 6.3 Hz), 4.94–4.91 (1H, m), 3.92 (3H, s), 2.17–1.84 (6H, m), 1.67–1.62 (2H, m). MS (ESI, m/z): 337.2 (M+H)+.

3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-(pyridin-3-yl)-1,2,4-oxadiazole (8b)

Light cream solid. Yield 35%, m.p. 117–118 °C. 1H NMR (CDCl3, 400 MHz): 9.45 (1H, dd, J = 2.08, 0.56 Hz), 8.84 (1H, dd, J = 4.88, 1.68 Hz), 8.48 (1H, dt, J = 8.04, 2.0 Hz), 7.76 (1H, dd, J = 8.4, 2.0 Hz), 7.67 (1H, d, J = 1.92 Hz), 7.52 (1H, ddd, J = 8, 4.8, 0.6 Hz), 6.98 (1H, d, J = 8.44 Hz), 4.93–4.92 (1H, m), 3.93 (3H, s), 2.04–1.85 (6H, m), 1.71–1.62 (2H, m). MS (ESI, m/z): 338.2 (M+H)+.

3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-(pyridin-4-yl)-1,2,4-oxadiazole (8c)

Light cream solid. Yield 32%, m.p. 97–98 °C. 1H NMR (CDCl3, 400 MHz): 8.88 (2H, dd, J = 4.48, 1.68 Hz), 8.06 (2H, dd, J = 4.08, 1.68 Hz), 7.76 (1H, dd, J = 8.40, 2.04 Hz), 7.66 (1H, d, J = 1.96 Hz), 6.98 (1H, d, J = 8.40 Hz), 4.94–4.90 (1H, m), 3.93 (3H, s), 2.06–1.85 (6H, m), 1.68–1.61 (2H, m). MS (ESI, m/z): 338.2 (M+H)+.

3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-(4-fluorophenyl)-1,2,4-oxadiazole (8d)

White solid. Yield 41%, m.p. 83–84 °C. 1H NMR (CDCl3, 400 MHz): 8.25–8.21 (2H, m), 7.74 (1H, dd, J = 8.36, 2.04 Hz), 7.66 (1H, d, J = 1.92 Hz), 7.27–7.22 (2H, m), 6.97 (d, 1H, J = 8.4 Hz), 4.93–4.90 (1H, m), 3.92 (3H, s), 2.04–1.84 (6H, m), 1.67–1.62 (2H, m). MS (ESI, m/z): 355.2 (M+H)+.

5-(4-Chlorophenyl)-3-(3-(cyclopentyloxy)-4-methoxyphenyl)-1,2,4-oxadiazole (8e)

White solid. Yield 42%, m.p. 119–120 °C. 1H NMR (CDCl3, 400 MHz): 8.16 (2H, dt, J = 8.64, 2.16 Hz), 7.74 (1H, dd, J = 8.4, 1.92 Hz), 7.65 (1H, d, J = 1.92 Hz), 7.54 (2H, dt, J = 8.64, 2.16 Hz), 6.97 (1H, d, J = 8.40 Hz), 4.93–4.89 (1H, m), 3.92 (3H, s), 2.06–1.84 (6H, m), 1.67–1.64 (2H, m). MS (ESI, m/z): 371.1 (M+H)+.

4-(3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)phenol (8f)

White solid. Yield 23%, m.p. 156–157 °C. 1H NMR (CDCl3, 400 MHz): 8.01 (2H, dt, J = 8.8, 2.34 Hz), 7.74 (1H, dd, J = 8.28, 1.92 Hz), 7.66 (1H, d, J = 1.92 Hz), 7.01–6.95 (3H, m), 6.50 (1H, s(br)), 4.94–4.89 (1H, m), 3.91 (3H, s), 2.05–1.81 (6H, m), 1.71–1.59 (2H, m).

5-((1H-Indol-3-yl)methyl)-3-(3-(cyclopentyloxy)-4-methoxyphenyl)-1,2,4-oxadiazole (8g)

Light cream solid. Yield 37%, m.p. 126–127 °C. 1H NMR (CDCl3, 400 MHz): 8.22 (1H, s), 7.70 (1H, d, J = 8.84 Hz), 7.64 (1H, dd, J = 8.32, 1.92 Hz), 7.57 (1H, d, J = 1.96 Hz), 7.37 (1H, d, J = 8.08 Hz), 7.24–7.13 (3H, m), 6.91 (1H, d, J = 8.44 Hz), 4.87–4.84 (1H, m), 4.43 (2H, s), 3.88 (3H, s), 2.04–1.80 (6H, m), 1.67–1.59 (2H, m). MS (ESI, m/z): 390.3 (M+H)+.

1-(3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)propan-2-one (8h)

Colorless gum. Yield 45%. 1H NMR (CDCl3, 400 MHz): 7.65 (1H, dd, J = 8.40, 2.04 Hz), 7.57 (1H, d, J = 1.96 Hz), 6.94 (1H, d, J = 8.44 Hz), 4.89–4.86 (1H, m), 4.1 (2H, s), 3.91 (3H, s), 2.35 (3H, s), 2.17–1.80 (6H, m), 1.64–1.58(2H, m). MS (ESI, m/z): 339.3 (M+H)+.

3-(3-Cyclopentyloxy-4-methoxyphenyl)-5-(3,4-dimethoxystyryl)-1,2,4-oxadiazole (8i)

White solid. Yield 34%, m.p. 102–103 °C. 1H NMR (CDCl3, 400 MHz): 7.80 (1H, d, J = 16.28 Hz), 7.70 (1H, dd, J = 8.4, 1.92 Hz), 7.62 (1H, d, J = 1.92 Hz), 7.20 (1H, dd, J = 8.28, 1.88 Hz), 7.14 (1H, d, J = 1.88 Hz), 6.97–6.91 (3H, m), 4.92–4.90 (1H, m), 3.96 (3H, s), 3.94 (3H, s), 3.92 (3H, s), 2.03–1.84 (6H, m), 1.66–1.62 (2H, m). MS (ESI, m/z): 423.4 (M+H)+.

5-(5-(4-Fluorophenyl)-1,2,4-oxadiazol-3-yl)-2-methoxyphenol (8j)

Off white solid. Yield 36%, m.p. 152–153 °C. 1H NMR (CDCl3, 400 MHz): 8.24–8.20 (2H, m), 7.73–7.69 (2H, m), 7.25–7.22 (2H, m), 6.96 (1H, d, J = 8.20 Hz), 5.72 (1H, s), 3.97 (3H, s). MS (ESI, m/z): 287.0 (M+H)+.

3-(4-(Benzyloxy)-3-methoxyphenyl)-5-(pyridin-4-yl)-1,2,4-oxadiazole (8k)

Cream solid. Yield 40%, m.p. 150–151 °C. 1H NMR (CDCl3, 400 MHz): 8.88 (2H, d, J = 5.16 Hz), 8.06 (2H, dd, J = 4.56, 1.56 Hz), 7.72 (1H, dd, J = 8.36, 1.92 Hz), 7.67 (1H, d, J = 1.96 Hz), 7.46 (2H, d, J = 8.68 Hz), 7.41–7.37 (2H, m), 7.34–7.32 (1H, m), 7.00 (1H, d, J = 8.4 Hz), 5.25 (2H, s), 4.00 (3H, s).

5-Cyclopentyl-3-(3-cyclopentyloxy-4-methoxy-phenyl)-1,2,4-oxadiazole (8m)

Colorless gum. Yield: 47%. 1H NMR (CDCl3 400 MHz): 7.67 (1H, d, J = 8.36, 1.91 Hz), 7.57 (1H, d, J = 1.96 Hz), 6.92 (1H, d, J = 8.4 Hz), 4.91–4.87 (1H, m), 3.92 (3H, s), 2.00–1.82 (11H, m), 1.77–1.30 (6H, m). MS (ESI, m/z): 329.09 (M  +  H)+.

5-Cyclohexyl-3-(3-cyclopentyloxy-4-methoxy-phenyl)-1,2,4-oxadiazole (8n)

Colorless gum. Yield: 45%. 1H NMR (CDCl3 400 MHz): 7.65 (1H, d, J = 8.36, 1.91 Hz), 7.58 (1H, d, J = 1.96 Hz), 6.93 (1H, d, J = 8.4 Hz), 4.90–4.87 (1H, m), 3.90 (3H, s), 2.00–1.82 (11H, m), 1.75–1.58 (4H, m), 1.44–1.34 (4H, m). MS (ESI, m/z): 343.11 (M+H)+.

t-Butyl 4-(3-(3-cyclopentyloxy-4-methoxy-phenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-carboxylate (8o)

Light cream gum. Yield: 37%. 1H NMR (CDCl3, 200 MHz): 7.63 (1H, dd, J = 8.00, 2.00 Hz), 7.55 (1H, d, J = 2.00 Hz), 6.91 (1H, d, J = 8.00 Hz), 4.89–4.78 (1H, m), 4.16–4.09 (2H, m), 3.89 (3H, s), 3.08–2.87 (3H, m), 2.14–1.85 (10H, m), 1.65–1.62 (3H, m), 1.46 (9H, s). 13C NMR (100 MHz, CDCl3) δ 180.93, 168.10, 154.64, 152.50, 147.83, 120.68, 119.24, 113.19, 111.54, 80.52, 79.82, 56.02, 43.51, 34.51, 32.79, 29.16, 28.42, 24.10.

t-Butyl 4-(3-(4-cyclopentyloxy-3-methoxy-phenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-carboxylate (8p)

Off white solid. Yield: 39%, m.p. 78–81 °C. 1H NMR (CDCl3, 200 MHz): 7.62 (1H, dd, J = 8.00, 2.00 Hz), 7.54 (1H, d, J = 2.00 Hz), 6.92 (1H, d, J = 8.00 Hz), 4.85–4.78 (1H, m), 4.16–4.09 (2H, m), 3.91 (3H, s), 3.19–2.90 (3H, m), 2.12–1.73 (12H, m), 1.46 (9H, s). 13C NMR (100 MHz, CDCl3) 180.98, 168.11, 154.68, 150.34, 149.98, 120.75, 118.94, 114.06, 110.39, 80.43, 79.81, 79.59, 56.13, 42.01, 34.51, 32.85, 28.42, 24.14.

t-Butyl 4-(3-(3,4-dimethoxyphenyl)-1,2,4-oxa-diazol-5-yl)piperidin-1-carboxylate (8q)

Light cream solid. Yield: 37%, m.p. 93–94 °C. 1H NMR (CDCl3, 400 MHz): 7.68 (1H, dd, J = 8.40, 1.96 Hz), 7.56 (1H, d, J = 1.92 Hz), 6.95 (1H, d, J = 8.40 Hz), 4.14–4.12 (2H, m), 3.97 (3H, s), 3.95 (3H, s), 3.19–3.13 (1H, m), 3.02–2.96 (2H, m), 2.13–2.09 (2H, m), 1.94–1.85 (2H, m), 1.48 (9H, s). MS (ESI, m/z): 332.12 (M-C(CH3)3)+.

t-Butyl 4-(3-(3-ethoxy-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-carboxylate (8r)

Colorless gum. Yield: 36%. 1H NMR (CDCl3, 400 MHz): 7.66 (1H, dd, J = 8.32, 1.92 Hz), 7.56 (1H, d, J = 1.92 Hz), 6.95 (1H, d, J = 8.44 Hz), 4.21–4.11 (4H, m), 3.93 (3H, s), 3.18–3.13 (1H, m), 3.02–2.96 (2H, m), 2.12–2.10 (2H, m), 1.94–1.87 (2H, m), 1.52–1.47 (12H, m). MS (ESI, m/z): 404.20 (M+H)+.

t-Butyl 4-(3-(3-butoxy-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-carboxylate (8s)

Light cream solid. Yield: 36%, m.p. 78–81 °C. 1H NMR (CDCl3, 400 MHz): 7.66 (1H, dd, J = 8.36, 1.92 Hz), 7.56 (1H, d, J = 1.92 Hz), 6.94 (1H, d, J = 8.44 Hz), 4.13–4.08 (4H, m), 3.92 (3H, s), 3.17–3.13 (1H, m), 2.99–2.97 (2H, m), 2.13–2.09 (2H, m), 1.91–1.82 (4H, m), 1.54–1.49 (2H, m), 1.47 (9H, s), 1.00 (3H, t, J = 7.36 Hz). MS (ESI, m/z): 432.14 (M+H)+.

3-(3-Cyclopentyloxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9a)

Light cream solid. Yield: 87%, m.p. 92–93 °C. 1H NMR (CDCl3, 200 MHz): 7.63 (dd, 1H, J = 8.00, 2.00 Hz), 7.54 (d, 1H, J = 2.00 Hz), 6.92 (d, 1H, J = 8.00 Hz), 4.86–4.78 (m, 1H), 3.88 (s, 3H), 3.50–2.87 (m, 10H), 2.16–1.61 (m, 8H). MS (ESI, m/z): 344.20 (M+H)+.

3-(4-Cyclopentyloxy-3-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9b)

White solid. Yield: 83%, m.p. 110–112 °C. 1H NMR (CDCl3, 200 MHz): 7.62 (1H, dd, J = 8.00, 2.00 Hz), 7.54 (1H, d, J = 2.00 Hz), 6.92 (1H, d, J = 8.00 Hz), 4.86–4.78 (1H, m), 3.91 (3H, s), 3.37–3.32 (6H, m), 2.94–2.81 (2H, m), 2.17–2.00 (2H, m), 1.97–1.62 (8H, m). 13C NMR (100 MHz, CDCl3) 180.85, 168.13, 150.36, 149.98, 120.77, 118.92, 114.07, 110.42, 80.44, 56.14, 44.72, 33.99, 32.85, 29.05, 24.14. MS (ESI, m/z): 344.1 (M+H)+.

3-(3,4-Dimethoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9c)

Off white solid. Yield: 84%, m.p. 190–192 °C. 1H NMR (DMSO-d6, 400 MHz): 9.70 (1H, s(br)), 7.66 (1H, dd, J = 8.32, 1.40 Hz), 7.54 (1H, d, J = 1.48 Hz), 6.98 (1H, d, J = 8.4 Hz), 3.95 (3H, s), 3.94 (3H, s), 3.50–3.36 (3H, m), 3.16–3.01 (2H, m), 2.42–2.39 (2H, m), 2.32–2.24 (2H, m). MS (ESI, m/z): 290.08 (M+H)+.

3-(3-Ethoxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9d)

Off white solid. Yield: 78%, m.p. 137–138 °C. 1H NMR (CDCl3, 400 MHz): 9.70 (1H, s(br)), 7.66 (1H, dd, J = 8.36, 1.88 Hz), 7.56 (1H, d, J = 1.88 Hz), 6.96 (1H, d, J = 8.44 Hz), 4.19 (2H, q, J = 6.96 Hz), 3.94 (3H, s), 3.52–3.47 (2H, m), 3.37–3.34 (1H, m), 3.21–3.14 (2H, m), 2.46–2.41 (2H, m), 2.36–2.30 (2H, m), 1.51 (3H, t, J = 6.96 Hz). 13C NMR (100 MHz, CDCl3) 178.90, 168.26, 151.94, 148.55, 120.86, 118.82, 111.26, 111.07, 64.49, 56.00, 42.26, 31.51, 25.66, 14.69. MS (ESI, m/z): 304.09 (M+H)+.

3-(3-n-Butoxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9e)

White solid. Yield: 86%, m.p. 130–131 °C. 1H NMR (CDCl3, 400 MHz): 9.70 (1H, s(br)), 7.66 (1H, dd, J = 8.40, 1.92 Hz), 7.55 (1H, d, J = 1.88 Hz), 6.95 (1H, d, J = 8.44 Hz), 4.10 (2H, t, J = 6.76 Hz), 3.92 (3H, s), 3.52–3.46 (2H, m), 3.36–3.34 (1H, m), 3.20–3.14 (2H, m), 2.46–2.41 (2H, m), 2.36–2.30 (2H, m), 1.90–1.83 (2H, m), 1.55–1.49 (2H, m), 1.00 (3H, t, J = 7.4 Hz). 13C NMR (100 MHz, CDCl3): 178.87, 168.28, 152.09, 148.83, 120.84, 118.80, 111.37, 111.21, 68.81, 56.02, 42.26, 31.52, 31.16, 25.63, 19.19, 13.86. MS (ESI, m/z): 332.12 (M+H)+.

Ethyl 4-(3-(3-cyclopentyloxy-4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (10a)

White solid. Yield 78%, m.p. 55–56 °C. 1H NMR (CDCl3, 200 MHz): 7.62 (1H, dd, J = 8.00, 2.00 Hz), 7.55 (1H, d, J = 2.00 Hz), 6.92 (1H, d, J = 8.00 Hz), 4.89–4.82 (1H, m), 4.20–4.09 (4H, m), 3.89 (3H, s), 3.22–2.94 (3H, m), 2.16–1.85 (10H, m), 1.62–1.58 (2H, m), 1.27 (3H, t, J = 6 Hz). MS (ESI, m/z): 416.13 (M+H)+.

3-(3-cyclopentyloxy-4-methoxy-phenyl)-5-(1-methylsulfonylpiperidin-4-yl)-1,2,4-oxadiazole (10b)

White solid. Yield 72%, m.p. 78–79 °C. 1H NMR (CDCl3, 200 MHz): 7.63 (1H, dd, J = 8.00, 2.00 Hz), 7.55 (1H, d, J = 2.00 Hz), 6.92 (1H, d, J = 8.00 Hz), 4.90–4.82 (1H, m), 3.89 (3H, s), 3.79–3.71 (2H, m), 3.20–2.94 (3H, m), 2.81 (3H, s), 2.26–1.79 (10H, m), 1.62–1.58 (2H, m). 13C NMR (100 MHz, CDCl3) 180.13, 168.20, 152.62, 147.86, 120.73, 119.04, 113.20, 111.56, 80.60, 56.04, 44.87, 35.26, 33.48, 32.80, 28.77, 24.11. MS (ESI, m/z): 422.08 (M+H)+.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

Chemistry

Compounds 8a-s were synthesized according to Scheme 1. Reaction of aldehyde 4 with hydroxylamine hydrochloride followed by formic acid catalyzed dehydration produced arylnitriles 5 (33,34) in very good yields (85%). Alkylation of arylnitriles 5 with appropriate alkyl halide in presence of potassium carbonate in DMF resulted in the formation of arylnitriles 6. Reaction of 6 with hydroxylamine hydrochloride in presence of sodium carbonate in aqueous ethanol afforded amidoximes 7. Finally, coupling of amidoximes 7 with appropriate carboxylic acid using DCC in DMF and heating the resulting O-acylamidoxime at 110 °C afforded 1,2,4-oxadiazoles 8a–s in moderate yields. Compounds 9a–e were synthesized according to Scheme 2. The t-Boc protective group in 8o–s was removed by trifluoroacetic acid to afford 1,2,4-oxadiazoles 9a-e in excellent yields. Reaction of 9a with ethyl chloroformate in presence of triethylamine led to the formation of 10a. Under similar conditions, reaction of 9a with methanesulfonylchloride produced 10b in good yield. All the synthesized compounds were characterized by their NMR and MS spectral data.

image

Figure Scheme 1:.  Synthesis of 1,2,4-oxadiazoles 8: Reagents and conditions: (i) NH2OH.HCl, HCOONa, HCOOH 125 °C, 3 h; (ii) K2CO3, R2X alkyl halid, DMF; (iii) NH2OH.HCl, Na2CO3, aqueous ethanol, reflux, 5 h; (iv) R3COOH, DCC, DMF, 0–25 °C 3 h; (v) heating 110 °C.

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image

Figure Scheme 2:.  Synthesis of 1,2,4-oxadiazoles 9 and 10. Reagents and condition: (i) trifluoroacetic acid, DCM, 25 °C, 3 h; (ii) ethyl chloroformate of methanesulfonyl chloride, triethylamine, DCM, 0–25 °C, 8 h.

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PDE4B2 inhibition activity

Compounds (8a–s, 9a–e, 10a–b) were evaluated for their PDE4B2 inhibitory activity (Table 1). Some of the compounds exhibited moderate to good inhibition (4–66% at 10 μm). The 1,2,4-oxadiazole 8a with a phenyl group at 5-position showed 48% inhibition. Further substitution at the 4-position of 5-phenyl with chloro and hydroxy groups resulted in loss of activity (compounds 8e and 8f), whereas 4-fluoro substituent exhibited 18% inhibition (8d). Introduction of a heteroatom in the phenyl derivative 8a led to the 3-pyridyl and 4-pyridyl analogs (8b and 8c) with reduced activity (19% inhibition). Compounds (8g and 8i) having indol-3-ylmethyl and 3,4-dimethoxystyrenyl at 5-position were equipotent (38–41% inhibition). Compound 8h with 2-oxopropyl moiety at 5-position showed very good inhibition (57%, IC50 = 7.21 μm). Interestingly, piperidin-4-yl moiety at 5-position is beneficial for the activity (compound 9a, 66% inhibition), although N-ethoxycarbonyl (10a) and N-methanesulfonyl (10b) derivatives were devoid of inhibitory activity. This indicated that the presence of free N-H in the six-membered ring is vital for the activity, which may have favorable interaction with the PDE4 receptor residue or surrounding water molecules. Cycloalkyl substituents at 5-position are detrimental for the inhibitory activity (compounds 8m and 8n, no inhibition). The five-membered heterocyclic ring, pyrrolidin-2-yl is also not tolerable (compound 8l, 9% inhibition). The 3-cyclopentyloxy-4-methoxyphenyl group is optimal for the activity as any modification resulted in either complete loss or poor activity (compounds 8j, 8k, 8o–s, and 9b–e). As inhibition of PDE4 also leads to the suppression of cell proliferation (35,36), an independent anticancer activity study of a few 1,2,4-oxadiazole derivatives was conducted, and surprisingly compound 9a exhibited appreciable cytotoxicity (24).

Table 1.   PDE4B2 inhibitory activity of 1,2,4-oxadiazoles 8-10 (Inhibition @10 μM) Thumbnail image of
Analgesic and antiinflammatory activity

To investigate the effect of potent PDE4 inhibitor 9a in pain and inflammation symptoms, we have performed formalin-induced pain study in mice with Aspirin as positive control (37,38). The compound 9a was administered at 10 and 20 mg/kg i.p. dosage. A significant reduction in pain threshold was observed in the test group. With respect to control group, at 10 and 20 mg/kg dose, 9a exhibited 38.34% and 52.86% reduction in pain symptoms (Table 2, Figure 3A).

Table 2.   Analgesic activity exhibited by 9a, Aspirin, and control group animals
Treatment groupDose mg/kg (i.p.)No. of flinching% Reduction in pain symptoms
  1. All the data were analyzed by one-way anova post hoc test Dunnet, p < 0.05, n = 6 per group and result are expressed as Mean ± SEM.

Formalin control98.66 ± 12.34
Aspirin100 mg/kg34.50 ± 5.6460.97
9a10 mg/kg60.83 ± 12.6038.34
9a20 mg/kg46.50 ± 6.8352.86
image

Figure 3.  (A) Histograms representing the effects of 9a (doses in mg/kg, n = 5 each dose) on formalin test flinching behavior of mice. (B) Antiinflammatory activity exhibited by 9a, Diclofenac, and control group animals in carrageenan-induced rat paw edema.

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The encouraging analgesia exhibited by compound 9a propelled us to study its antiinflammatory activity. The antiinflammatory activity was carried out using carrageenan-induced rat paw edema model. With respect to control group, at 10 and 20 mg/kg dose, 9a exhibited 42.1% and 50.29% reduction in paw swelling, respectively (Table 3, Figure 3B).

Table 3.   Effect of 9a on carrageenan-induced rat paw edema
Treatment groupDose mg/kg (i.p.)Change in paw volume (mL)% reduction in paw swelling
  1. All the data were analyzed by one-way anova post hoc test Dunnet, p < 0.05, n = 5 per group and result were expressed as Mean ± SEM.

Carrageenan control0.342 ± 0.045
Diclofenac100.096 ± 0.01371.92
9a100.198 ± 0.02542.10
9a200.170 ± 0.04650.29

Docking study

To rationalize the activity and binding mode of most active compound 9a, docking studies were performed with PDE4B (PDB ID: 1XM4). As shown in Figure 4A,B, the probable binding mode of 9a is nearly similar to Piclamilast; key interactions of 3-cyclopentyloxy-4-methoxyphenyl group with Gln-443 and proximity of piperidinyl nitrogen with water-1005 and Mg-1002 support its PDE4B inhibitory activity. Relative deviation of piperidinyl ring in compound 9a may be responsible for its reduced activity when compared with Piclamilast.

image

Figure 4.  Docked structural conformation of Piclamilast (2) and 3-(3-cyclopentyloxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-oxadiazole (9a) in the active site of phosphodiesterase4 (PDB ID: 1XM4) using Glide (Schrodinger), the compound 2 is shown as orange colored and compound 9a shown as gray green stick model. The metal atoms Mg and Zn are shown as magenta, and gray spheres are displayed in ribbon presentation (A) and important active site amino acid residues are displayed in (B).

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Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

A new chemical class of 3,5-disubstituted-1,2,4-oxadiazoles has been identified as potent inhibitors of PDE4B2. Docking study of most active 3,5-disubs-tituted-1,2,4-oxadiazole 9a supported its binding similarity with the Piclamilast. Encouraging in-vivo antiinflammatory activity results in two different models of 9a further supports PDE4 inhibition. The preliminary investigations of the synthesized compounds indicated that 1,2,4-oxadiazole heterocycle can act as pseudo amide, and there is a vast scope for the structural modification of 3,5-disubstituted-1,2,4-oxadiazoles to optimize the PDE4B2 inhibition. Further, exploration of structure–activity relationship and correlation of PDE4B2 inhibition with observed cytotoxicity of 9a are underway in our laboratory.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

The authors thank CSIR, New Delhi for the financial support and SAIF, Panjab University for providing analytical support.

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  3. Materials and Methods
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  5. Conclusion
  6. Acknowledgments
  7. References
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