Design, synthesis and biological evaluation of 2,4-disubstituted oxazole derivatives as potential PDE4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.01.047

In this study, a series of pyrazole derivatives containing 4-phenyl-2-oxazole moiety were designed and synthesized in a concise way, some of which exhibited considerable inhibitory activity against PDE4B and blockade of LPS-induced TNF-α release. Compound

Full text of DOI:10.1016/j.bmc.2017.01.047

Accepted Manuscript
Design, Synthesis and Biological Evaluation of 2,4-Disubstituted Oxazole De-
rivatives as Potential PDE4 Inhibitors
Ya-Sheng Li, De-Kun Hu, Dong-Sheng Zhao, Xing-Yu Liu, Hong-Wei Jin,
Gao-Peng Song, Zi-Ning Cui, Lian-Hui Zhang
PII:
S0968-0896(16)31240-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.047
BMC 13526
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Accepted Date:
19 November 2016
31 January 2017
Please cite this article as: Li, Y-S., Hu, D-K., Zhao, D-S., Liu, X-Y., Jin, H-W., Song, G-P., Cui, Z-N., Zhang, L-
H., Design, Synthesis and Biological Evaluation of 2,4-Disubstituted Oxazole Derivatives as Potential PDE4
Inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.01.047
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Design, Synthesis and Biological Evaluation of 2,4-Disubstituted Oxazole
Derivatives as Potential PDE4 Inhibitors
Ya-Sheng Li a,#, De-Kun Hu a,#, Dong-Sheng Zhao d,#, Xing-Yu Liu a,
Hong-Wei Jin e, Gao-Peng Song c,*, Zi-Ning Cui a,b,*, Lian-Hui Zhang a
a
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Integrative
Microbiology Research Centre, Guangdong Province Key Laboratory of Microbial Signals and Disease
Control, South China Agricultural University, Guangzhou 510642, China
b
Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou
University, Guiyang 550025, China
c College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China;
d Department of Pharmacy, Quanzhou Medical College, Quanzhou 362100, China
e
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China
#Both authors contributed equally to this paper.
*Corresponding author. Tel.: +86-20-85288229; Fax: +86-20-85288229
E-mail address: ziningcui@scau.edu.cn (Z. C.), vinsin1021@126.com (G. S.)
1

Abstract: In this study, a series of pyrazole derivatives containing 4-phenyl-2-oxazole moiety were
designed and synthesized in a concise way, some of which exhibited considerable inhibitory activity
against PDE4B and blockade of LPS-induced TNF-α release. Compound 4c displayed the strongest
inhibition activity (IC50 = 1.6±0.4 μM) and good selectivity against PDE4B. Meanwhile, compound 4c
showed good in vivo activity in animal models of asthma/COPD and sepsis induced by LPS. The primary
structure–activity relationship study showed the 3,5-dimethylpyrazole residue was essential for the
bioactivity, and the substituted group R1 at the benzene ring also affected the activity. Docking results
showed that compound 4c played a key role to form integral hydrogen bonds and a π-π stacking
interaction, using hydrazide scaffold (-CO-N-N-) and pyrazole ring respectively, with PDE4B protein.
While the rest part of the molecule extended into the catalytic domain to block the access of cAMP and
formed the foundation for inhibition of PDE4B. Compound 4c would be great promise as a lead
compound for further study based on the preliminary structure-activity relationship and molecular
modeling studies.
Keywords: synthesis; 4-phenyl-2-oxazole; pyrazole derivatives; PDE4 inhibitor; molecular simulation
2

1. Introduction
Cyclicadenosine monophosphate (cAMP) and cyclicguanosine monophosphate (cGMP), the secondary
signal messengers, are involved in the regulation of almost all physiological processes. In airway smooth
muscle, inflammatory cells, and immune cells, these second messengers play an important role in the
regulation of the function. It is well known that cyclic nucleotide phosphodiesterases (PDEs) include 11
diverse families according to their structures and properties.1,2 PDE4 plays a key role in the hydrolysis of
cAMP and cGMP, which can selectively catalyze the hydrolysis of a 3’-phosphodiester bond, forming an
inactive 5’-monophosphate.3,4 Thus, inhibition of PDE4 enzymes can result in increasing cellular levels of
cAMP, which contributes to both the relaxation of airway smooth muscle and the prevention of
proinflammatory cell activation. Therefore PDE4 inhibitors are considered to be potent anti-inflammatory
agents for the treatment of inflammatory related diseases, such as asthma and chronic obstructive
pulmonary disease (COPD).5-7
For past few decades, PDE4 inhibitors have been extensively studied as anti-inflammatory drugs since
the discovery of rolipram and piclamilast (Fig. 1) in the early 1990s.8,9 Structurally, rolipram and
piclamilast belong to diether derivatives of catechol class, some of which have been evaluated in clinic for
the treatment of patients with depression, asthma, COPD, dermatitis, neuroinflammation10,11 and
rheumatoid arthritis.12 Further structural optimization demonstrated that the 8-methoxyquinoline-5-
carboxamides moiety (such as SCH365351, Fig. 1)13 had excellent PDE4 inhibitory activity. Molecular
simulation studies suggested that the quinoline moiety of SCH365351 could bind to the adenosine
recognition site. In our previous work,14 we reported the compounds possessed 7-(cyclopentyloxy)-6-
methoxy 1,2,3,4-tetrahydroisoquinoline (Fig. 1) showed potential inhibitory activity against PDE4.
It became important to establish selectivity towards one subtype, e.g., PDE4A and PDE4B for PDE4
inhibitory activity to obtain potent and selective inhibitors with an improved therapeutic index and
reduced emetic side effects. Recent studies of selective PDE4 inhibitors declared introduction of some
groups, such as a short amide linker (−CONHCH2−) or a 4-methoxy benzyl group, was helpful to
enhance the selectivity over other PDE family members.15 In addition, a five-membered heterocyclic
3

oxazole moiety was explored as a possible linker to replace the amide portion, which was found to be a
highly versatile linker and the derivatives exhibited significantly potent PDE4 inhibitory activity,
suggesting it became a key core of PDE4 inhibitors (Fig. 1).16-18 In our recent study,19 we reported
pyrazole and triazole derivatives containing 5-phenyl-2-furan functionality (Fig. 1) exhibited considerable
inhibitory activity against PDE4B, the subtype of PDE4. The primary SAR results showed that a five-
membered heterocycles including oxazole and furan rings could be a key pharmacophore of PDE4
inhibitors. In this study, pyrazole derivatives containing oxazole moiety as the bioisosteric surrogates of
the pyrazole and triazole derivatives were further designed and synthesized (Fig. 1) in order to obtain the
detailed structure-activity relationship, of which inhibitory activity against PDE4B and blockade of LPS-
induced TNF-α release was evaluated. Meanwhile the preliminary structure-activity relationship and
docking studies were carried out.
4

Figure 1. The designed strategy for the title compounds.
2. Results and discussion
2.1. Chemistry
The synthetic route of title compounds 3a-3k and 4a-4k was described in Scheme 1. The oxazole
ethyl esters 1a-1k were obtained by cyclization of benzoyl amides in the presence of ethyl bromopyruvate
under reflux in ethanol for about 5 hours. Then the oxazole ethyl esters were hydrolyzed with 2 M NaOH
in water to get the corresponding carboxylic acids 2, which was reacted with thionyl chloride and
pyrazole or 3,5-dimethylpyrazole in anhydrous dichloromethane to obtain the title compounds 3 and 4.
1
13
The title compounds have been characterized by melting point, H NMR, C NMR, mass spectrometry
5

and elementary analysis. All spectral data were consistent with the assigned structures (the spectra of 1H
NMR and 13C NMR were shown in supplementary materials).
Scheme 1. The synthetic route of the title compounds 3a-3k and 4a-4k. Reagents and conditions: (a)
ethyl bromopyruvate, ethanol, reflux, 5 h; (b) 2 M NaOH, r.t., 10 h; (c) SOCl2, anhydrous toluene,
reflux, 4 h; (d) pyrazole or 3,5-dimethylpyrazole, anhydrous dichloromethane, reflux, 4 h, 80-92% in two
steps. R1 = 3a: 4-Cl; 3b: 2-OH; 3c: 4-OCH3; 3d: 4-CH3; 3e: 3-OCH3; 3f: H; 3g: 4-Br; 3h: 4-F; 3i: 3-OH;
3j: 4-OH; 3k: 3-F; 4a: 4-Cl; 4b: 2-OH; 4c: 4-OCH3; 4d: 4-CH3; 4e: 3-OCH3; 4f: H; 4g: 4-Br; 4h: 4-F;
4i: 3-OH; 4j: 4-OH; 4k: 3-F.
2.2. Biological evaluation and SAR studies
In vitro activity for the inhibition of PDE4B and blockade of LPS-induced TNF-α release were listed
in Table 1. Rolipram was chosen as the positive control. From the results in Table 1, it revealed that the
activity of title compounds containing 3,5-dimethylpyrazole (series 4) was better than that of the
compounds containing pyrazole (series 3). For example, the IC50 values of compounds 4c (R1 = 4-OCH3,
R2 = Me) and 4d (R1 = 4-CH3, R2 = Me) against PDE4B were 1.6 μM and 6.7 μM respectively, which
were better than that of the compounds 3c (R1 = 4-OCH3, R2 = H, IC50 = 3.2 μM) and 3d (R1 = 4-CH3,
R2 = H, IC50 = 17.5 μM).
The different activities of compounds 3a-3k and 4a-4k with various substitution effects on a benzene
ring demonstrated that the modification of the substituted group R1 at the benzene was significant.
Generally, the compounds without any substituted group (3f and 4f, R1 = H) gave the poorest activity.
Among all the title compounds, 4c was the most potent inhibitor, and the IC50 value of 4c was 1.6 μM
and 10.3 μM respectively against PDE4B and TNF-α, which showed comparable or better activity than
the positive control rolipram (1.8 μM and 18.6 μM) (Table 1). Compound 3c, with the same substituted
6

group at the same position (R1 = 4-OCH3), exhibited considerable IC50 values (3.2 μM against PDE4B
and 20.8 μM against TNF-α) to that of rolipram. Compounds 3e and 4e, with the same methoxyl group
as 3c and 4c, but at the different positon, demonstrated lower bioactivities (3e: IC50 = 5.9 μM against
PDE4B and IC50 = 45.8 μM against TNF-α; and 4e: IC50 = 4.5 μM against PDE4B and IC50 = 18.7 μM
against TNF-α). It was suggested that a methoxyl group on a benzene ring displayed good activities
against PDE4B, and a methoxyl group at para-position of a benzene ring had been proven to be the best
bioactivity against PDE4B in our recent study19 and confirmed to the conclusion of earlier research14.
When R1 was a hydroxyl group, bioactivity of compounds 3b (R1 = 2-OH, IC50 = 85.6 μM against
PDE4B and IC50 = 205.4 μM against TNF-α), 3i (R1 = 3-OH, IC50 = 39.7 μM against PDE4B and IC50 =
88.4 μM against TNF-α), 3j (R1 = 4-OH, IC50 = 18.4 μM against PDE4B and IC50 = 75.5 μM against
TNF-α) and 4b (R1 = 2-OH, IC50 = 54.8 μM against PDE4B and IC50 = 138.7 μM against TNF-α), 4i (R1
= 2-OH, IC50 = 28.9 μM against PDE4B and IC50 = 78.4 μM against TNF-α), 4j (R1 = 2-OH, IC50 = 15.8
μM against PDE4B and IC50 = 66.4 μM against TNF-α) with respect to substitution at a benzene ring
follows the trend: 4->3->2-. When R1 was a halogen atom at the para-position, activity with respect to
substitution follows the trend: 4-Cl (3a and 4a: IC50 = 5.3 μM and 4.1 μM) >4-F (3h and 4h: IC50 = 9.8
μM and 8.4 μM) >4-Br (3g and 4g: IC50 = 58.7 μM and 54.8 μM). It was suggested that the electron-
donating group (EDG) and the halogen atom at the para-position would be favored to the activities.
Preliminary structure-activity relationship study showed that substituent at para-position of a benzene
ring was a crucial point to the bioactivity and a methoxyl group at para-position of a benzene ring
revealed the promising lead compound 4c in this study.
Since inhibition of the subtype PDE4D was associated with the emetic response, therefore we
evaluated the selected in vitro active compounds 3c and 4c for their PDE4B selectivity over PDE4D. The
results demonstrated that 3c and 4c displayed inhibitory effects on PDE4D with IC50 values of 8.4 μM
and 10.6 μM (Table 1), indicating their obvious selectivity towards PDE4B (IC50 = 3.2μM and 1.6 μM).
Table 1. Impact on enzymatic potency (PDE4B) and inhibition of TNF-α release from human blood
mononuclear cells stimulated with lipopolysaccharide a
7

PDE4B
TNF-α
PDE4B
TNF-α
Compd.
R1
R2
Compd.
R1
R2
IC50
IC50
IC50 (μM)
IC50
(μM)
(μM)
(μM)
4-Cl
2-OH
H
H
H
5.3±0.2
40.1±1.2
4-Cl
2-OH
4-OCH3 Me
Me
4.1±0.6
27.6±1.3
138.7±4.8
10.3±0.9
3a
3b
3c
4a
4b
4c
85.6±2.4 205.4±5.2
Me 54.8±2.6
4-OCH3
3.2±0.2
(8.4±0.5)b
17.5±1.1
5.9±0.3
20.8±1.1
1.6±0.4
(10.6±1.1)b
6.7±0.3
4-CH3
3-OCH3
H
H
H
H
H
H
H
H
H
72.4±3.4
45.8±1.6
4-CH3
Me
36.7±1.5
18.7±1.2
198.8±5.8
167.8±5.2
29.7±1.4
78.4±2.0
66.4±1.9
83.4±2.1
3d
3e
3f
4d
4e
4f
3-OCH3 Me
4.5±0.2
88.7±2.9 198.6±5.2
58.7±2.4 187.4±4.8
H
Me 75.8±3.1
Me 54.8±2.2
4-Br
4-Br
4-F
3g
3h
3i
4g
4h
4i
4-F
9.8±0.4
39.7±0.9
18.4±0.7
45.1±1.4
1.8±0.2
54.8±2.3
88.4±3.5
75.5±2.7
99.6±3.1
18.6±0.8
Me
8.4±0.4
3-OH
4-OH
3-F
3-OH
4-OH
3-F
Me 28.9±1.1
Me 15.8±0.6
Me 40.8±1.5
3j
4j
3k
4k
rolipram
(3.5±0.3)b
a Results are the average of at least three assays. b IC50 values against PDE4D
LPS induced sepsis model for the measurement of TNF-α inhibition (in female Swiss Albino mice) and
neutrophilia inhibition for asthma and COPD (in male Sprague Dawley rats) with selected in vitro active
compounds 3c and 4c were performed. The details such as oral dosage and number of animals grouped
for the experiments were listed in Table 2. The results showed that compound 4c demonstrated better
inhibitory activity against TNF-α release (42.6%) and LPS induced neutrophilia inhibition (39.1%) than
the positive control rolipram (40.8% and 30.9%) and compound 3c (26.1% and 22.4%).
Table 2. LPS induced TNF-α in SA mice and neutrophil influx in BALF of SD rats
Swiss Albino mice (n = 6)
Sprague Dawley rats (n = 6)
Does
TNF-α
Does
LPS induced
neutrophilia (%
inhibition)
Compd. R2
R1
(mg/kg, po)
(mg/kg, po)
Inhibition (%)
8

H
4-OCH3
4-OCH3
10
10
10
26.1
42.6
40.8
10
10
10
22.4
39.1
30.9
3c
4c
H
rolipram
2.3. Docking simulation
Considering the inhibitory activity of title compounds, it was of interest to explore the binding to the
PDE4 structure. The bioassay results demonstrated that compounds containing a para-methoxy group
(3c and 4c) showed the great promise as novel lead compounds for further discovery. Therefore docking
simulation of compounds 3c and 4c at PDE4B (PDB ID: 1XMY) was conducted using Surflex-Dock in
Sybyl 8.0 and the docking contour maps were shown in Figure 2.
The docking orientation demonstrated that the five-membered heterocyclic moiety as the pivotal
pharmacophore formed integral hydrogen bonds with the conserved glutamine residue (Gln443) (Fig. 2)
and the heterocyclic ring was evidently positioned between the phenylalanine (Phe446) and isoleucine
(Ile410) (Fig. 2C and 2E), which formed the cavity accommodating the hydrophobic moiety of
compounds 3c and 4c. Particularly, oxygen atom and nitrogen atom of hydrazide moiety (-CO-N-N-) in
compounds 3c and 4c formed two steady hydrogen bonds with the same hydrogen atom of amide (-CO-
NHH) in Gln443, which composed a five element ring (Fig. 2C and 2E). In compound 4c, the oxygen
atom of carbonyl group and the nitrogen atom of pyrazole ring interacted with Gln443, which played
the same role as the oxygen atoms of methoxy group and cyclopentyloxy group in rolipram respectively
(Fig. 2G and 2H). The pyrazole ring in 4c oriented the similar position with the cyclopentane ring in
rolipram. Meanwhile, the pyrazole ring formed a π-πstacking interaction with benzene ring of Phe446
(Fig. 2C and 2E), which enhanced the interaction with PDE4B. Compared with the pyrazole derivative
3c, the 3,5-dimethylpyrazole moiety of 4c formed stronger π-π stacking interaction with benzene ring (3c:
3.59 Å and 4c: 3.43 Å, Fig. 2C and 2E) in the phenylalanine (Phe446), and the two methyl groups of 4c
could play a supporting role in the cavity of enzyme, which could also enhance the binding affinity with
the enzyme. The remainder of the molecule was displayed to extend into the catalytic domain in close to
9

both the Zn2+ and Mg2+ cations (Fig. 2D and 2F), which played a vital role in the catalytic mechanism of
cAMP hydrolysis. The para-methoxy group (-OMe) formed coordinate bonds with the Zn2+ (3c: 2.78 Å
and 4c: 2.19 Å, Fig. 2D and 2F) and Mg2+ (3c: 3.14 Å and 4c: 3.18 Å, Fig. 2D and 2F) cations. Such
orientations and interactions would block the access of cAMP to the catalytic domain and form the
foundation for inhibition of PDE4.
In general, compound 4c exhibited the stronger π-π stacking interaction with phenylalanine (Phe446)
and metal coordination with catalytic Zn2+ cation than that of compound 3c. That could give the reason
why most title compounds containing 3,5-dimethylpyrazole (series 4) showed better bioactivity against
PDE4B than that of the compounds containing pyrazole (series 3).
A
B
C
D
E
F
10

G
H
Figure 2. Model of PDE4 and docking of compounds 3c and 4c. (A, B) The entire PDE4B structure
(N-terminal domain, a catalytic domain and a C-terminal domain) bound to 3c. (C, D) The catalytic
domain bound to 3c. (E, F) The catalytic domain bound to 4c. (G, H) The catalytic domain bound to 4c
overlaid with rolipram (orange).
3. Conclusion
In this study, we reported the design, synthesis, and biological evaluation of new phosphodiesterase
type 4 inhibitors, containing 2-phenyl-4-oxazole moiety. Their in vitro bioactivity against
phosphodiesterase type 4 and TNF-α, and in vivo activity in animal models of asthma/COPD and sepsis
induced by LPS were evaluated. Compound 4c showed the highest inhibitory activity against PDE4B and
blockade of LPS-induced TNF-α release among all the title compounds. A primary structure-activity
relationship study showed that the 3,5-dimethylpyrazole residue was favored for the inhibitory activity
and substituent at para-position of benzene ring in the molecule had an important effect on the inhibitory
activity. The docking results suggested that title compounds interacted well with PDE4B protein where a
five element ring composing of two intermolecular hydrogen bonds, the π-π stacking interaction, the
hydrophobic interaction and the metal coordination in the ligand-receptor complex were vital for the
binding affinity. Such efforts were helpful for the further development of novel and effective PDE4
inhibitors.
4. Experimental procedure
4.1. Chemistry
1
All solvents and reagents were obtained from commercial sources without further purification. H
13
NMR and C NMR spectra were recorded on Bruker Avance DRX spectrometer at 600 and 150 MHz.
11

Chemical shifts are reported as δ values in parts per million (ppm), while tetramethylsilane (TMS) was
used as an internal standard. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet;
q, quartet; dd, doublet of doublets; td, triplet of doublets; ddd, doublet of doublet of doublets; brs, broad
singlet; m, multiplet. Coupling constants (J values) are given in hertz (Hz). Compounds were dissolved in
DMSO-d6 or in CDCl3. Mass spectra were recorded on a Q-TOF Global mass spectrometer. Elemental
analysis was carried out with a Flash EA 1112 elemantal analyzer. All the melting points were determined
with a Cole-Parmer melting point apparatus while the thermometer was uncorrected. Thin-layer
chromatography (TLC) was performed using Merck 60 F254 silica gel plates. Column chromatography
was performed using silica gel (200-300 mesh, Qingdao, China) with a linear solvent gradient.
4.1.1. General synthesis of ethyl aryl-1,3-oxazole-4-carboxylates (1a-1k).
Amide (1.0 equiv) and ethyl bromopyruvate (1.2 equiv) were dissolved in ethanol and heated to 70 °C
for 5 h. The reaction was monitored with TLC until completion. The solvent was evaporated, dried over
anhydrous MgSO4. The ethyl ester (1a-1k) was purified by flash column chromatography (ethyl acetate/
cyclohexane 3:7 (v/v) ).
4.1.2. General synthesis of 2-substituted phenyl-4-oxazolecarboxylic acid (2a-2k).
The ethyl ester (1a-1k) was hydrolyzed by dissolving in EtOH/H2O, adding 2 M NaOH (5 equiv) and
stirring at room temperature for 10 h. Upon completion of the reaction, the solvent was evaporated in
vacuum and the reaction mixture was extracted with ethyl acetate. The aqueous layer was acidified with
2 M HCl to pH 2 and the product precipitated. The precipitate was filtered, washed with water and dried
to afford the carboxylic acids 2a-2k.
4.1.3. General synthesis of the title compounds 3 and 4.
A mixture of 2-substituted phenyl-4-oxazolecarboxylic acid 2 (100 mmol) and thionyl chloride (500
mmol) was refluxed in anhydrous benzene for 4 h. The excess of thionyl chloride and the solvent were
distilled off, and the residue was dissolved in anhydrous dichloromethane. The solution of 2-phenyl-4-
oxazolecarbonyl chloride in anhydrous dichloromethane was added into pyrazole (110 mmol) or 3,5-
dimethylpyrazole (110 mmol). The mixture was stirred and refluxed for 4 h. After cooling, the solvent
12

was evaporated off under reduced pressure, and the solid was recrystallized from ethanol to obtain the
title compounds 3a-3k and 4a-4k.
4.1.3.1. [2-(4-chlorophenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3a):
1
Compound 3a was obtained (82%) as a white solid. mp: 156-157 ℃; H NMR (600 MHz, CDCl3) δ
6.55 (dd, J = 2.9, 1.5 Hz, 1H), 7.48-7.51 (m, 2H), 7.82 (dd, J = 1.4, 0.6 Hz, 1H), 8.12-8.15 (m, 2H),
13
8.52 (dd, J = 2.9, 0.7 Hz, 1H), 9.10 (s, 1H); C NMR (150 MHz, CDCl3) δ 109.80, 124.67, 128.34,
129.24, 129.88, 133.59, 137.61, 144.56, 147.99, 157.78, 161.07; ESIMS(m/z): 274.3 [M + H]+. Anal.
Calcd. (%) for C13H8ClN3O2: C, 57.21; H, 3.10; N, 15.11. Found: C, 57.05; H, 2.95; N, 15.35.
4.1.3.2. [2-(2-hydroxyphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3b):
Compound 3b was obtained (80%) as a yellow solid. mp: 175-176 ℃; 1H NMR (600 MHz, DMSO-d6)
δ 6.56 (dd, J = 2.8, 1.5 Hz, 1H), 7.00 (ddd, J = 8.0, 7.3, 1.1 Hz, 1H), 7.14 (dd, J = 8.4, 0.8 Hz, 1H),
10.79 (s, 1H), 7.42-7.46 (m, 1H), 7.83 (dd, J = 1.4, 0.6 Hz, 1H), 7.90 (dd, J = 7.9, 1.6 Hz, 1H), 8.47
13
(dd, J = 2.9, 0.6 Hz, 1H), 9.08 (s, 1H); C NMR (150 MHz, DMSO-d6) δ 109.96, 109.98, 117.61,
119.61, 126.36, 129.73, 131.91, 133.40, 144.66, 146.55, 156.93, 157.70, 161.51; ESIMS(m/z): 256.1
[M + H]+. Anal. Calcd. (%) for C13H9N3O3: C, 60.99; H, 3.47; N, 16.58. Found: C, 61.18; H, 3.55; N,
16.46.
4.1.3.3. [2-(4-methoxyphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3c):
1
Compound 3c was obtained (91%) as a white solid. mp: 171-172 ℃; H NMR (600 MHz, CDCl3) δ
3.87 (s, 3H), 6.52 (dd, J = 2.8, 1.4 Hz, 1H), 6.99 (d, J = 8.9 Hz, 2H), 7.80 (s, 1H), 8.11 (d, J = 8.9 Hz,
13
2H), 8.50 (d, J = 2.8 Hz, 1H), 9.02 (s, 1H); C NMR (150 MHz, CDCl3) δ 55.44, 109.63, 114.28,
118.89, 128.83, 129.88, 133.32, 144.42, 147.52, 158.04, 162.07, 162.09; ESIMS(m/z): 270.1 [M + H]+.
Anal. Calcd. (%) for C13H9N3O3: C, 62.68; H, 4.37; N, 15.41. Found: C, 62.45; H, 4.12; N, 15.61.
4.1.3.4. [2-(4-methylphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3d):
Compound 3d was obtained (90%) as a white solid. mp: 91-92 ℃; 1H NMR (600 MHz, CDCl3) δ 2.42
(s, 3H), 6.53 (dd, J = 2.8, 1.5 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 7.80 (d, J = 0.8 Hz, 1H), 8.07 (d, J =
8.2 Hz, 2H), 8.51 (dd, J = 2.8, 0.5 Hz, 1H), 9.06 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 21.60, 109.64,
13

123.51, 127.04, 129.57, 133.38, 129.89, 141.79, 144.43, 147.68, 157.98 162.22; ESIMS(m/z): 254.1 [M
+ H]+. Anal. Calcd. (%) for C14H11N3O2: C, 66.24; H, 4.61; N, 16.32. Found: C, 66.40; H, 4.38; N,
16.59.
4.1.3.5. [2-(3-methoxyphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3e):
1
Compound 3e was obtained (87%) as a white solid. mp: 134-135 ℃; H NMR (600 MHz, CDCl3) δ
3.91 (s, 3H), 6.54 (dd, J = 2.8, 1.5 Hz, 1H), 7.07 (ddd, J = 8.3, 2.6, 0.9 Hz, 1H), 7.41 (t, J = 8.0 Hz,
1H), 7.72 (dd, J = 2.5, 1.6 Hz, 1H), 7.74-7.77 (m, 1H), 7.81 (d, J = 0.8 Hz, 1H), 8.51 (dd, J = 2.8, 0.5
13
Hz, 1H), 9.09 (s, 1H); C NMR (150 MHz, CDCl3) δ 55.62, 109.73, 111.50, 118.17, 119.50, 127.38,
129.90, 129.97, 133.47, 144.51, 147.95, 157.95, 159.95, 161.92; ESIMS(m/z): 270.1 [M + H]+. Anal.
Calcd. (%) for C13H9N3O3: C, 62.68; H, 4.37; N, 15.41. Found: C, 62.45; H, 4.12; N, 15.61.
4.1.3.6. (2-phenyl-1,3-oxazol-4-yl)(1H-pyrazol-1-yl)methanone (3f):
1
Compound 3f was obtained (89%) as a white solid. mp: 139-140 ℃; H NMR (600 MHz, CDCl3) δ
6.53 (dd, J = 2.8, 1.5 Hz, 1H), 7.80 (d, J = 0.8 Hz, 1H), 7.48-7.52 (m, 3H), 8.16-8.19 (m, 2H), 8.50 (d,
13
J = 2.8 Hz, 1H), 9.08 (s, 1H); C NMR (150 MHz, CDCl3) δ 109.70, 126.18, 127.04, 128.84, 129.86,
131.30, 133.46, 144.48, 147.91, 157.88, 161.95; ESIMS(m/z): 240.1 [M + H]+. Anal. Calcd. (%) for
C13H9N3O2: C, 65.42; H, 3.54; N, 17.81. Found: C, 65.27; H, 3.79; N, 17.56.
4.1.3.7. [2-(4-bromophenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3g):
1
Compound 3g was obtained (87%) as a yellow solid. mp: 167-168 ℃; H NMR (600 MHz, CDCl3) δ
6.54 (dd, J = 2.7, 1.4 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.81 (s, 1H), 8.05 (d, J = 8.5 Hz, 2H), 8.50 (d,
13
J = 2.8 Hz, 1H), 9.08 (s, 1H); C NMR (150 MHz, CDCl3) δ 109.79, 125.10, 126.03, 128.48, 129.86,
132.19, 133.61, 144.55, 148.00, 157.74, 161.13; ESIMS(m/z): 318.1 [M + H]+. Anal. Calcd. (%) for
C13H8BrN3O2: C, 49.25; H, 2.37; N,13.07. Found: C, 49.08; H, 2.53; N, 13.21.
4.1.3.8. [2-(4-fluorophenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3h):
1
Compound 3h was obtained (90%) as a white solid. mp: 157-158 ℃; H NMR (600 MHz, CDCl3) δ
6.54 (dd, J = 2.7, 1.4 Hz, 1H), 7.21-7.17 (m, 2H), 7.81 (s, 1H), 8.16-8.21 (m, 2H), 8.50 (d, J = 2.8 Hz,
2
1H), 9.07 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 109.76, 116.15 (d, JC-F = 22.2 Hz), 122.57 (d, 4JC-F
=
14

3
1
3.1 Hz), 129.32 (d, JC-F = 8.8 Hz), 129.89, 133.51, 144.53, 147.89, 157.86, 161.16, 164.65 (d, JC-F
=
252.6 Hz); ESIMS(m/z): 258.2 [M + H]+. Anal. Calcd. (%) for C13H8FN3O2: C, 60.92; H, 2.98; N, 16.11.
Found: C, 60.70; H, 3.13; N, 16.34.
4.1.3.9. [2-(3-hydroxyphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3i):
Compound 3i was obtained (80%) as a white solid. mp: 199-200 ℃; 1H NMR (600 MHz, DMSO-d6) δ
6.74 (dd, J = 2.8, 1.5 Hz, 1H), 7.01 (ddd, J = 8.1, 2.5, 0.9 Hz, 1H), 7.40 (t, J = 7.9 Hz, 1H), 7.48-7.50
(m, 1H), 7.50-7.52 (m, 1H), 8.04 (d, J = 0.9 Hz, 1H), 8.65 (dd, J = 2.8, 0.5 Hz, 1H), 9.27 (s, 1H), 9.96
13
(s, 1H); C NMR (150 MHz, DMSO-d6) δ 110.83, 113.35, 117.64, 119.16, 127.27, 130.68, 130.99,
133.05, 145.56, 149.20, 157.69, 158.37, 161.36; ESIMS(m/z): 256.1 [M + H]+. Anal. Calcd. (%) for
C13H9N3O3: C, 59.92; H, 3.78; N, 16.24. Found: C, 61.18; H, 3.55; N, 16.46.
4.1.3.10. [2-(4-hydroxyphenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3j):
Compound 3j was obtained (89%) as a white solid. mp: 209-210 ℃; 1H NMR (600 MHz, DMSO-d6)
δ 6.73 (dd, J = 2.8, 1.5 Hz, 1H), 6.97-6.94 (m, 2H), 10.26 (s, 1H), 7.90-7.93 (m, 2H), 8.02 (d, J = 0.9
13
Hz, 1H), 8.64 (d, J = 2.8 Hz, 1H), 9.20 (s, 1H); C NMR (150 MHz, DMSO-d6) δ 110.77, 116.52,
117.14, 128.92, 130.70, 132.89, 145.51, 148.58, 157.81, 160.95, 161.81; ESIMS(m/z): 256.1 [M + H]+.
Anal. Calcd. (%) for C13H9N3O3: C, 61.31; H, 3.67; N, 16.71. Found: C, 61.18; H, 3.55; N, 16.46.
4.1.3.11. [2-(3-fluorophenyl)-1,3-oxazol-4-yl](1H-pyrazol-1-yl)methanone (3k):
1
Compound 3k was obtained (92%) as a white solid. mp: 146-147 ℃; H NMR (600 MHz, CDCl3) δ
6.54 (dd, J = 2.8, 1.5 Hz, 1H), 7.22 (tdd, J = 8.3, 2.5, 0.6 Hz, 1H), 7.48 (td, J = 8.0, 5.6 Hz, 1H), 7.81
(d, J = 0.6 Hz, 1H), 7.88 (ddd, J = 9.4, 2.3, 1.7 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 8.50 (d, J = 2.8 Hz,
2
2
1H), 9.10 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 109.79, 114.16 (d, JC-F = 24.2 Hz), 118.38 (d, JC-F
=
4
3
3
21.3 Hz), 122.87 (d, JC-F = 3.0 Hz), 128.27 (d, JC-F = 9.1 Hz), 129.93, 130.69 (d, JC-F = 8.2 Hz),
4
1
133.77, 144.56, 148.11, 157.74, 160.89 (d, JC-F = 3.3 Hz), 162.99 (d, JC-F = 247.2 Hz); ESIMS(m/z):
258.1 [M + H]+. Anal. Calcd. (%) for C13H8FN3O2: C, 60.88; H, 3.01; N, 16.10. Found: C, 60.70; H,
3.13; N, 16.34.
4.1.3.12. [2-(4-chlorophenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4a):
15

1
Compound 4a was obtained (82%) as a white solid. mp: 158-159 ℃; H NMR (600 MHz, CDCl3) δ
13
2.30 (s, 3H), 2.65 (s, 3H), 6.05 (s, 1H), 7.44-7.50 (m, 2H), 8.08-8.13 (m, 2H), 8.97 (s, 1H); C NMR
(150 MHz, CDCl3) δ 13.92, 14.52, 111.30, 124.95, 128.22, 129.17, 134.61, 137.32, 145.48, 147.20,
152.83, 159.31, 160.59; ESIMS(m/z): 302.3 [M + H]+. Anal. Calcd. (%) for C15H12ClN3O2: C, 59.92; H,
3.86; N, 14.14. Found: C, 59.71; H, 4.01; N, 13.93.
4.1.3.12. [2-(2-hydroxyphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4b):
Compound 4b was obtained (82%) as a white solid. mp: 177-178 ℃; 1H NMR (600 MHz, DMSO-d6)
δ 2.28 (s, 3H), 2.57 (s, 3H), 6.32 (s, 1H), 7.04-7.07 (m, 1H), 7.11 (d, J = 8.3 Hz, 1H), 7.50-7.46 (m,
13
1H), 7.88 (dd, J = 7.8, 1.5 Hz, 1H), 9.25 (s, 1H), 10.82 (s, 1H); C NMR (150 MHz, DMSO-d6) δ
14.02, 14.48, 111.04, 112.15, 117.56, 120.34, 127.64, 132.70, 133.74, 145.01, 147.79, 153.12, 157.02,
158.58, 160.68; ESIMS(m/z): 284.2 [M + H]+. Anal. Calcd. (%) for C15H13N3O3: C, 63.41; H, 4.82; N,
15.03. Found: C, 63.60; H, 4.63; N, 14.83.
4.1.3.14. [2-(4-methoxyphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4c):
1
Compound 4c was obtained (92%) as a white solid. mp: 125-126 ℃; H NMR (600 MHz, CDCl3) δ
2.30 (s, 3H), 2.65 (s, 3H), 3.87 (s, 3H), 6.04 (s, 1H), 6.97-7.00 (m, 2H), 8.09-8.12 (m, 2H), 8.92 (s, 1H);
13C NMR (150 MHz, CDCl3) δ 13.91, 14.53, 55.41, 111.15, 114.21, 119.21, 128.68, 134.33, 145.41,
146.70, 152.63, 159.61, 161.59, 161.89; ESIMS(m/z): 298.1 [M + H]+. Anal. Calcd. (%) for C16H15N3O3:
C, 64.85; H, 4.89; N, 14.35. Found: C, 64.64; H, 5.09; N, 14.13.
4.1.3.15. [2-(4-methylphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4d):
1
Compound 4d was obtained (85%) as a white solid . mp: 125-126 ℃; H NMR (600 MHz, CDCl3) δ
2.30 (s, 3H), 2.41 (s, 3H), 2.65 (s, 3H), 6.04 (s, 1H), 7.28 (d, J = 8.0 Hz, 2H), 8.05 (d, J = 8.2 Hz, 2H),
8.94 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 13.91, 14.53, 21.57, 111.18, 123.79, 126.92, 129.51,
134.38, 141.48, 145.42, 146.88, 152.67, 159.54, 161.73; ESIMS(m/z): 282.1 [M + H]+. Anal. Calcd. (%)
for C16H15N3O2: C, 68.18; H, 5.64; N, 14.69. Found: C, 68.31; H, 5.37; N, 14.94.
4.1.3.16. [2-(3-methoxyphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4e):
16

1
Compound 4e was obtained (92%) as a white solid. mp: 133-134 ℃; H NMR (600 MHz, CDCl3) δ
2.30 (s, 3H), 2.65 (s, 3H), 3.89 (s, 3H), 6.04 (s, 1H), 7.04 (dd, J = 8.0, 2.2 Hz, 1H), 7.38 (t, J = 7.9 Hz,
1H), 7.75-7.69 (m, 2H), 8.97 (s, 1H); 13C NMR (150 MHz, CDCl3) δ 13.89, 14.51, 55.55, 111.24,
111.31, 117.94, 119.33, 127.63, 129.87, 134.45, 145.43, 147.17, 152.74, 159.46, 159.58, 161.39;
ESIMS(m/z): 298.1 [M + H]+. Anal. Calcd. (%) for C16H15N3O3: C, 64.47; H, 5.31; N, 14.29. Found: C,
64.64; H, 5.09; N, 14.13.
4.1.3.17. (2-phenyl-1,3-oxazol-4-yl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone (4f):
Compound 4f was obtained (82%) as a white solid. mp: 125-126 ℃; MW: 267.10; 1H NMR (600 MHz,
CDCl3) δ 2.29 (s, 3H), 2.65 (s, 3H), 6.04 (s, 1H), 7.46-7.50 (m, 3H), 8.15-8.18 (m, 2H), 8.97 (s, 1H);
13C NMR (150 MHz, CDCl3) δ 13.90, 14.52, 111.21, 126.45, 126.92, 128.77, 131.04, 134.47, 145.40,
147.11, 152.70, 159.43, 161.47; ESIMS(m/z): 268.1 [M + H]+. Anal. Calcd. (%) for C15H13N3O2: C,
67.66; H, 5.10; N, 15.48. Found:C, 67.40; H, 4.90; N, 15.72.
4.1.3.18. [2-(4-bromophenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4g):
1
Compound 4g was obtained (85%) as a white solid. mp: 167-168 ℃; H NMR (600 MHz, CDCl3) δ
13
2.30 (s, 3H), 2.65 (s, 3H), 6.05 (s, 1H), 7.61-7.65 (m, 2H), 8.02-8.05 (m, 2H), 8.97 (s, 1H); C NMR
(150 MHz, CDCl3) δ 13.92, 14.52, 111.31, 125.38, 125.74, 128.38, 132.14, 134.64, 145.48, 147.23,
152.83, 159.29, 160.65; ESIMS(m/z): 347.1 [M + H]+. Anal. Calcd. (%) for C15H12BrN3O2: C, 52.31; H,
3.24; N, 12.31. Found: C, 52.04; H, 3.49; N, 12.14.
4.1.3.19. [2-(4-fluorophenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4h):
1
Compound 4h was obtained (85%) as a white solid. mp: 131-132 ℃; H NMR (600 MHz, CDCl3) δ
13
2.30 (s, 3H), 2.65 (s, 3H), 6.05 (s, 1H), 7.14-7.19 (m, 2H), 8.14-8.18 (m, 2H), 8.96 (s, 1H); C NMR
2
4
(150 MHz, CDCl3) δ 13.91, 14.52, 111.28, 116.06 (d, JC-F = 22.2 Hz), 122.84 (d, JC-F = 3.2 Hz),
1
129.17 (d, 3JC-F = 8.8 Hz), 134.52, 145.47, 147.10, 152.80, 159.40, 160.68, 164.50 (d, JC-F = 251.9 Hz);
ESIMS(m/z): 286.1 [M + H]+. Anal. Calcd. (%) for C15H12FN3O2: C, 62.97; H, 4.08; N, 14.92. Found: C,
63.15; H, 4.24; N, 14.73.
4.1.3.20. [2-(3-hydroxyphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4i):
17

Compound 4i was obtained (92%) as a yellow solid. mp: 193-194 ℃; 1H NMR (600 MHz, DMSO-d6)
δ 2.27 (s, 3H), 2.56 (s, 3H), 6.29 (s, 1H), 6.98 (ddd, J = 8.1, 2.3, 0.7 Hz, 1H), 7.39 (t, J = 7.9 Hz, 1H),
13
7.45-7.50 (m, 2H), 9.16 (s, 1H), 9.93 (s, 1H); C NMR (150 MHz, DMSO-d6) δ 14.00, 14.53, 111.97,
113.25, 117.54, 119.00, 127.49, 130.98, 134.03, 144.89, 148.63, 152.80, 158.37, 159.11, 160.91;
ESIMS(m/z): 284.2 [M + H]+. Anal. Calcd. (%) for C15H13N3O3: C, 63.81; H, 4.79; N, 14.58. Found: C,
63.60; H, 4.63; N, 14.83.
4.1.3.21. [2-(4-hydroxyphenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4j):
Compound 4j was obtained (91%) as a yellow solid. mp: 207-208 ℃; 1H NMR (600 MHz, DMSO-d6)
δ 2.26 (s, 3H), 2.56 (s, 3H), 6.29 (s, 1H), 6.93-6.96 (m, 2H), 7.87-7.90 (m, 2H), 9.09 (s, 1H), 10.22 (s,
1H); 13C NMR (150 MHz, DMSO-d6) δ 14.02, 14.55, 111.92, 116.50, 117.40, 128.79, 133.86, 144.85,
148.00, 152.71, 159.29, 160.81, 161.36; ESIMS(m/z): 284.1 [M + H]+. Anal. Calcd. (%) for C15H13N3O3:
C, 63.79; H, 4.48; N, 14.61. Found: C, 63.60; H, 4.63; N, 14.83.
4.1.3.22. [2-(3-fluorophenyl)-1,3-oxazol-4-yl](3,5-dimethyl-1H-pyrazol-1-yl)methanone (4k):
1
Compound 4k was obtained (88%) as a white solid. mp: 141-142 ℃; H NMR (600 MHz, CDCl3) δ
2.30 (s, 3H), 2.65 (d, J = 0.7 Hz, 3H), 6.05 (s, 1H), 7.22 (ddd, J = 10.7, 8.4, 0.7 Hz, 1H), 7.25-7.29 (m,
13
1H), 7.50-7.46 (m, 1H), 8.21 (td, J = 7.6, 1.7 Hz, 1H), 9.03 (s, 1H); C NMR (150 MHz, CDCl3) δ
4
13.88, 14.48, 111.26, 114.81 (d, 3JC-F = 11.0 Hz), 116.82 (d, 2JC-F = 21.2 Hz), 124.39 (d, JC-F = 3.7 Hz),
3
4
130.33, 132.77 (d, JC-F = 8.4 Hz), 134.29, 145.42, 147.42, 152.77, 158.31 (d, JC-F = 3.8 Hz), 159.25,
160.26 (d, 1JC-F = 257.9 Hz); ESIMS(m/z): 286.1 [M + H]+. Anal. Calcd. (%) for C15H12FN3O2: C, 63.37;
H, 4.01; N, 14.88. Found: C, 63.15; H, 4.24; N, 14.73.
4.2. Bioassay
4.2.1. Assay of human PDE4 activity
Phosphodiesterase type 4 isozyme (PDE4) was isolated with the modified method.19,20 The enzyme
was prepared from U937 cells which was derived from human monocytes, and was stored at -20 ºC after
preparation. Measurement of PDE4 activity was performed using this stored enzyme after it was diluted
with distilled water containing bovine serum albumin. The substrate solution was prepared by adding
18

[3H]-cAMP (300,000 dpm (5000 Bq)/assay) and 100 µmol/L cAMP solution to 100 mmol/ L Tris–HCl
(pH 8.0) containing 5 mmol/ L ethylene glycol-bis (β-aminoethyl ether) and O,O'-bis(2-
aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid. The substrate solution was mixed with the enzyme
solution containing a test compound dissolved in DMSO, and incubation was done for 30 min at 30 ºC.
Assays were performed in duplicate at different concentrations of each test compound.
4.2.2. Assay of TNF-α release.
The blood is mixed with saline at a ratio of 1:1, and the peripheral blood mononuclear cells (PBMCs)
were isolated from buffy coats using Lymphoprep tubes. The PBMCs were suspended in RPMI 1640
with 0.5% human serum albumin, pen/ strep, and 2 mM L-glutamine at 5 × 105 cells/mL. The cells were
pre-incubated with the test compounds in 96-well plates for 30 min and stimulated for 18 h with 1
mg/mL lipopolysaccharide. TNF-α concentration in the supernatants was measured by homogeneous
time-resolved fluorescence resonance (TR-FRET). The assay is quantified by measuring fluorescence at
665 nm (proportional to TNF-α concentration) and 620 nm (control). Results are expressed as IC50
values (μM).
4.2.3. LPS induced sepsis for measurement of TNF-α inhibition in mice
The LPS induced sepsis model in mice was evaluated following the literature.21 Female Swiss albino
mice were selected according to the body weights, which were equivalent within each group. The mice
were fasted for 20 h with free access to water and dosed for oral administration (po) with the test
compounds suspended in vehicle containing 0.5% Tween 80 in 0.25% sodium salt of
carboxymethylcellulose. The control mice were performed the vehicle alone. After 30 min of oral
dosing, the mice were injected into intraperitoneal cavity with 500 μg of lipopolysaccharide
(Escherichia coli, LPS: B4 from Sigma) in phosphate buffer. Then the mice were bled via retro-orbital
sinus puncture after 90 min of LPS administration. Serum samples were collected by centrifuging the
o
blood samples at 4000 rpm for 20 min, which were stored overnight at 4 C. Immediately, the serum
samples were checked for TNF-α levels using commercial mouse TNF-α ELISA kit (Amersham
Biosciences) and assay was carried out following the manufacturer instruction.
19

4.2.4. LPS induced neutrophilia model for asthma and COPD
LPS induced neutrophilia in Sprague Dawley rats was assessed according to the protocol described.22
Male Sprague Dawley rats were acclimatized to laboratory conditions for one week prior to the
experiment. According to the body weight, the rats were distributed to various groups randomly. Except
normal group, all the rats were exposed to 100 μg/mL lipopolysaccharide (Escherichia coli, LPS: B4
from Sigma) for 40 min. The rats were dosed for oral administration (po) with the test compounds
suspended in the vehicle containing 0.25% sodium salt of carboxymethylcellulose before half an hour
of LPS exposure. Bronchoalveolar lavage (BAL) was performed 6 h after LPS exposure, total cell count
and DLC (differential leukocyte count) were checked and compared with control.
4.4. Molecular docking
Molecular docking was performed on Surflex-Dock module of Sybyl 8.0.14,23,24 Crystal structure of
PDE4B (PDB ID: 1XMY) obtained from Protein Data Bank was used as the receptor for molecular
docking study. The 3D structure of compounds 3c and 4c was drawn and optimized with SYBYL
package. The docking procedure was started with the protomol generation, which was created using a
ligand-based approach (native ligand for PDE4B structure). Proto threshold was set to 0.5 and proto
bloat was kept at 0 as a default parameter. For docking, max conformation and max rotation values were
20 and 100, respectively. Pre-dock and post-dock energy minimization methods were also applied.
Docking results were compared by the total score values. The pose with the higher total-score value was
considered as the best one. After the end of molecular docking, the interactions of the docked domain
with ligand were analyzed.
Acknowledgments
We acknowledge the financial supports from the Special Fund for the National Natural Science
Foundation of China (31570122), the National Key Project for Basic Research (973 Program,
2015CB150600), the Pearl River S&T Nova Program of Guangzhou (201506010029), the Research and
Construction of Public Service Ability (2014A020210019 and 2016A020217014), the Opening
20

Foundation of the Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University (2015GDGP0101).
References and notes
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Figure captions
Figure 1. The designed strategy for the title compounds.
Figure 2. Model of PDE4 and docking of compounds 3c and 4c. (A, B) The entire PDE4B structure
(N-terminal domain, a catalytic domain and a C-terminal domain) bound to 3c. (C, D) The catalytic
domain bound to 3c. (E, F) The catalytic domain bound to 4c. (G, H) The catalytic domain bound to 4c
overlaid with rolipram (orange).
Scheme 1. The synthetic route of the title compounds 3a-3k and 4a-4k.
Table 1 Impact on enzymatic potency (PDE4B) and inhibition of TNF-α release from human blood
mononuclear cells stimulated with lipopolysaccharide
Table 2 LPS induced TNF-α in SA mice and neutrophil influx in BALF of SD rats
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Graphical Abstract
Design, Synthesis and Biological Evaluation of 2,4-Disubstituted Oxazole
Derivatives as Potential PDE4 Inhibitors
Ya-Sheng Li a,#, De-Kun Hu a,#, Dong-Sheng Zhao c,#, Xing-Yu Liu a,
Hong-Wei Jin d, Gao-Peng Songb,* , Zi-Ning Cui a,*, Lian-Hui Zhang a
24