Design and Synthesis of 3,5-Disubstituted-1,2,4-Oxadiazoles as Potent Inhibitors of Phosphodiesterase4B2

Article abstract of DOI:10.1111/j.1747-0285.2011.01304.x

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=5.28μm). 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.

Full text of DOI:10.1111/j.1747-0285.2011.01304.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01304.x
Chem Biol Drug Des 2012; 79: 810–818
Research Letter
Design and Synthesis of 3,5-Disubstituted-1,2,4-
Oxadiazoles as Potent Inhibitors of
Phosphodiesterase4B2
Dalip Kumar1*, Gautam Patel1, Lalitha
neutrophils, monocytes, and macrophages (2–9). Inhibitors of PDE4
enzyme exhibit broad range of antiinflammatory and antibroncho-
constrictor effects in animal models (10). The diverse biological
effects have implicated PDE4 as a potential target for a range of
Vijayakrishnan2, Sunanda G. Dastidar2 and
Abhijit Ray2
1Department of Chemistry, Birla Institute of Technology and
inflammatory diseases including asthma and chronic obstructive pul-
monary disease (2). However, the therapeutic window of orally
administered PDE4 inhibitors in clinical trials is limited by gastroin-
testinal side-effects such as nausea, vomiting, abdominal pain, diar-
rhea, 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 gener-
ation 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).
Science, Pilani, Rajasthan 333 031, India
2Department of Pharmacology, New Drug Discovery Research,
Ranbaxy Research Laboratories, R & D-III, Plot No 20 Sector 18,
Gurgaon 122015, Haryana, India
*Corresponding author: Dalip Kumar, dalipk@bits-pilani.ac.in
A series of 3,5-disubstituted-1,2,4-oxadiazoles has
been prepared and evaluated for phosphodiester-
ase inhibition (PDE4B2). Among the prepared 3,5-
disubstituted-1,2,4-oxadiazoles, compound 9a is
the most potent inhibitor (PDE4B2 IC50 = 5.28 lM).
Structure–activity relationship studies of 3,5-
disubstituted-1,2,4-oxadiazoles revealed that sub-
stituents 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-ox-
adiazoles with PDE4B has shown similar interac-
tions of 3-cyclopentyloxy-4-methoxyphenyl group;
however, heteroatom ring is slightly deviating
when compared to Piclamilast. 3-(3-Cyclopentyl-
oxy-4-methoxyphenyl)-5-(piperidin-4-yl)-1,2,4-ox-
adiazole (9a) exhibited good analgesic and
antiinflammatory activities in formalin-induced
pain in mice and carrageenan-induced paw edema
model in rat.
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 tyro-
sine 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).
Key words: antiinflammatory, phosphodiesterase, phosphodiester-
ase4B2 inhibitor, 1,2,4-oxadiazole.
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).
Received 6 May 2010, revised and accepted for publication 5 December
2011
Materials and Methods
Secondary messengers, cyclic adenosine monophosphate (cAMP)
and cyclic guanosine monophosphate (cGMP), are of immense inter-
est among chemists and biologists since their discovery (1). Phos-
phodiesterase4 (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 benefi-
cial in controlling the proinflammatory cytokines like tumor necrosis
factor-a, interleukins, leukotrienes, and interferons, and it also
reduces the recruitment and activation of inflammatory cells like
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 pre-
810

Design and Synthesis of 3,5-Disubstituted-1,2,4-Oxadiazoles
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–hex-
ane (0–25%) as eluent to afford pure 8.
Figure 1: Second generation phosphodiesterase4 inhibitors.
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 com-
bined organic phase was washed with brine, dried over anhydrous
sodium sulfate, and distilled off. The residue so obtained was puri-
fied by flash column chromatography using ethyl acetate–hexane
(0–50%) as eluent to afford pure compound 10a.
Figure 2: Schematic view for the rational in designing 1,2,4-ox-
adiazoles as phosphodiesterase4 inhibitors.
coated plates (silica gel 60 F254, 0.25 mm) and visualized by fluo-
rescence quenching under UV light (254 nm). Column chromatogra-
phy 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.
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.
Experimental
Synthesis of amidoximes (7)
To a mixture of appropriate benzonitrile 6 (10 mmol) and hydroxyl-
amine 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 ace-
tate (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.
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 dis-
solved 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 lM inhibitor concentration; dose-dependent inhibi-
tion was determined in the presence of 1 lM concentrations of
cAMP over a range of inhibitor compound concentrations using vehi-
cle control as DMSO and reference control as Piclamilast.
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 mix-
ture, 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
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 lL of 2% formalin was
Chem Biol Drug Des 2012; 79: 810–818
811

Kumar et al.
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.
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)
Screening of antiinflammatory activity:
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)+.
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 intraperi-
toneally to rats, 30 min before subcutaneous injection of carra-
geenan (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.
3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-(4-
fluorophenyl)-1,2,4-oxadiazole (8d)
1
White solid. Yield 41%, m.p. 83–84 ꢀC. H 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)+.
Docking study
The different ligand-binding modes of the compound 9a and Pic-
lamilast with the PDE4B enzyme (PDB ID: 1XM4) were investigated
by using an advanced molecular docking program GLIDE (Schrçdinger
Inc., New York, NY, USA). Inhibitor geometry was optimized by ga-
mess interface in CHEMBIO-3D using semi-empirical method PM3.
Protein preparation was performed in MAESTRO 8.5 protein prepara-
tion wizard (Schrçdinger, 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.
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).
Analytical data
5-((1H-Indol-3-yl)methyl)-3-(3-(cyclopentyloxy)-4-
methoxyphenyl)-1,2,4-oxadiazole (8g)
3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-5-
phenyl-1,2,4-oxadiazole (8a)
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)+.
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)+.
1-(3-(3-(Cyclopentyloxy)-4-methoxyphenyl)-
1,2,4-oxadiazol-5-yl)propan-2-one (8h)
Colorless gum. Yield 45%. H 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,
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,
1
812
Chem Biol Drug Des 2012; 79: 810–818

Design and Synthesis of 3,5-Disubstituted-1,2,4-Oxadiazoles
s), 2.17–1.80 (6H, m), 1.64–1.58(2H, m). MS (ESI, m ⁄ z): 339.3
t-Butyl 4-(3-(4-cyclopentyloxy-3-methoxy-
phenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-
carboxylate (8p)
(M+H)+.
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.
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)+.
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)+.
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)
t-Butyl 4-(3-(3-ethoxy-4-methoxyphenyl)-1,2,4-
oxadiazol-5-yl)piperidin-1-carboxylate (8r)
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).
Colorless gum. Yield: 36%. H NMR (CDCl3, 400 MHz): 7.66 (1H, dd,
1
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)+.
5-Cyclopentyl-3-(3-cyclopentyloxy-4-methoxy-
phenyl)-1,2,4-oxadiazole (8m)
Colorless gum. Yield: 47%. H 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)+.
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)+.
1
5-Cyclohexyl-3-(3-cyclopentyloxy-4-methoxy-
phenyl)-1,2,4-oxadiazole (8n)
1
Colorless gum. Yield: 45%. H NMR (CDCl3 400 MHz): 7.65 (1H, d,
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)+.
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)
3-(4-Cyclopentyloxy-3-methoxyphenyl)-5-
(piperidin-4-yl)-1,2,4-oxadiazole (9b)
1
Light cream gum. Yield: 37%. H NMR (CDCl3, 200 MHz): 7.63 (1H,
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)+.
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) d 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.
Chem Biol Drug Des 2012; 79: 810–818
813

Kumar et al.
3-(3,4-Dimethoxyphenyl)-5-(piperidin-4-yl)-1,2,4-
oxadiazole (9c)
1
Off white solid. Yield: 84%, m.p. 190–192 ꢀC. H 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)
Scheme 1: Synthesis of 1,2,4-oxadiazoles 8: Reagents and con-
ditions: (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.
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)+.
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)+.
3-(3-n-Butoxy-4-methoxyphenyl)-5-(piperidin-4-
yl)-1,2,4-oxadiazole (9e)
Results and Discussion
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)+.
Chemistry
Compounds 8a-s were synthesized according to Scheme 1. Reac-
tion 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 pres-
ence 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 character-
ized by their NMR and MS spectral data.
Ethyl 4-(3-(3-cyclopentyloxy-4-methoxyphenyl)-
1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate
(10a)
1
White solid. Yield 78%, m.p. 55–56 ꢀC. H 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. H 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,
PDE4B2 inhibition activity
Compounds (8a–s, 9a–e, 10a–b) were evaluated for their
PDE4B2 inhibitory activity (Table 1). Some of the compounds exhib-
1
Scheme 2: Synthesis of 1,2,4-oxadiazoles 9 and 10. Reagents and condition: (i) trifluoroacetic acid, DCM, 25 ꢀC, 3 h; (ii) ethyl chlorofor-
mate of methanesulfonyl chloride, triethylamine, DCM, 0–25 ꢀC, 8 h.
814
Chem Biol Drug Des 2012; 79: 810–818

Design and Synthesis of 3,5-Disubstituted-1,2,4-Oxadiazoles
O
O
S
N
CH3
Chem Biol Drug Des 2012; 79: 810–818
815

Kumar et al.
ited moderate to good inhibition (4–66% at 10 lM). The 1,2,4-
oxadiazole 8a with a phenyl group at 5-position showed 48% inhi-
bition. 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 lM). Interestingly,
piperidin-4-yl moiety at 5-position is beneficial for the activity (com-
pound 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 inhibi-
tory activity (compounds 8m and 8n, no inhibition). The five-mem-
bered 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).
120
100
80
60
40
20
0
A
Control
Aspirin 100 mg/kg 9a 10 mg/kg
9a 20 mg/kg
Dosage (i.p.)
B
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
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 com-
pound 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).
Control Diclofenac 10 mg/kg 9a 10 mg/kg
9a 20 mg/kg
Dosage (ip)
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.
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 exhib-
ited 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
group
Dose
mg ⁄ kg (i.p.)
Change in
paw volume (mL)
% reduction in
paw swelling
Carrageenan
control
Diclofenac
9a
–
0.342 € 0.045
–
10
10
20
0.096 € 0.013
0.198 € 0.025
0.170 € 0.046
71.92
42.10
50.29
Table 2: Analgesic activity exhibited by 9a, Aspirin, and control
group animals
9a
Treatment
group
Dose
mg ⁄ kg (i.p.)
% Reduction in
pain symptoms
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.
No. of flinching
98.66 € 12.34
Formalin
control
Aspirin
9a
–
–
Docking study
100 mg ⁄ kg
10 mg ⁄ kg
20 mg ⁄ kg
34.50 € 5.64
60.83 € 12.60
46.50 € 6.83
60.97
38.34
52.86
To rationalize the activity and binding mode of most active com-
pound 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
9a
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.
816
Chem Biol Drug Des 2012; 79: 810–818

Design and Synthesis of 3,5-Disubstituted-1,2,4-Oxadiazoles
A
B
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 com-
pound 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).
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 Piclami-
last.
4. Zhang K.Y.J., Ibrahim P.N., Gillette S., Bollag G. (2005) Phospho-
diesterase-4 as a potential drug target. Expert Opin Ther Tar-
gets;9:1283–1305.
5. Burnouf C., Pruniaux M.P. (2002) Recent advances in PDE4 inhibi-
tors as immunoregulators and anti-inflammatory drugs. Curr
Pharm Des;8:1255–1296.
Conclusion
6. Kodimuthali A., Jabaris S.S., Pal M. (2008) Recent advances on
phosphodiesterase 4 inhibitors for the treatment of asthma and
chronic obstructive pulmonary disease. J Med Chem;51:5471–
5489.
7. Gopalan B., Gharat X., Lakdawala A.D., Karunakaran U. (2004)
WO patent application 2004 ⁄ 089940.
8. Chakraborti A.K., Sarin S., Rudrawar S.V., Kumar R., Chankeshw-
ara S.V., Ray A., Dastidar S.G. (2008) US patent 2008 ⁄ 0207659.
9. Houslay M.D., Schafer P., Zhang K.Y. (2005) Keynote review:
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 antiin-
flammatory activity results in two different models of 9a further
supports PDE4 inhibition. The preliminary investigations of the syn-
thesized 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 relation-
ship and correlation of PDE4B2 inhibition with observed cytotoxicity
of 9a are underway in our laboratory.
phosphodiesterase-4 as
Today;10:1503–1519.
a therapeutic target. Drug Discov
10. Bundschuh D.S., Eltze M., Barsig J., Wollin L., Hatzelmann A.,
Beume R. (2001) In vivo efficacy in airway disease models of
roflumilast, a novel orally active PDE4 inhibitor. J Pharmacol Exp
Ther;297:280–290.
11. Burnouf C., Pruniaux M.-P., Szilagyi C.M., James A.B. (1998)
Phosphodiesterases 4 inhibitors. Annu Rep Med Chem;33:91–
109.
Acknowledgments
12. Lipworth B.J. (2005) Phosphodiesterase-4 inhibitors for asthma
and chronic obstructive pulmonary disease. Lancet;365:167–175.
13. Rabe K.F., Bateman E.D., O'Donnell D., Witte S., Bredenbroker
D., Bethke T.D. (2005) Roflumilast - an oral anti-inflammatory
treatment for chronic obstructive pulmonary disease: a rando-
mised controlled trial. Lancet;366:563–571.
The authors thank CSIR, New Delhi for the financial support and
SAIF, Panjab University for providing analytical support.
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