Synthesis and bioactivity of phenyl substituted furan and oxazole carboxylic acid derivatives as potential PDE4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112795

In this present study, a series of 5-phenyl-2-furan and 4-phenyl-2-oxazole derivatives were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. In vitro results showed that the synthesized compounds exhibited considerable inhibitory ac

Full text of DOI:10.1016/j.ejmech.2020.112795

European Journal of Medicinal Chemistry 207 (2020) 112795
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and bioactivity of phenyl substituted furan and oxazole
carboxylic acid derivatives as potential PDE4 inhibitors
,
,
,
, c, **
Yinuo Lin a 1, Wasim Ahmed a 1, Min He a 1, Xuwen Xiang a, Riyuan Tang b
,
Zi-Ning Cui a
, b, *
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 Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
c Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this present study, a series of 5-phenyl-2-furan and 4-phenyl-2-oxazole derivatives were designed and
synthesized as phosphodiesterase type 4 (PDE4) inhibitors. In vitro results showed that the synthesized
Received 29 July 2020
Received in revised form
30 August 2020
Accepted 30 August 2020
Available online 4 September 2020
compounds exhibited considerable inhibitory activity against PDE4B and blockade of LPS-induced TNF-
release. Among the designed compounds, Compound 5j exhibited lower IC50 value (1.4 M) against PDE4
than parent rolipram (2.0 M) in in vitro enzyme assay, which also displayed good in vivo activity in
a
m
m
animal models of asthma/COPD and sepsis induced by LPS. Docking results suggested that introduction
of methoxy group at para-position of phenyl ring, demonstrated good interaction with metal binding
pocket domain of PDE4B, which was helpful to enhance inhibitory activity.
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
5-phenyl-2-furan
4-phenyl-2-oxazole
2-cyanoimino-1,3-thiazolidine
Synthesis
PDE4 inhibitors
Molecular simulation
1. Introduction
messengers [3,4]. Out of these 11 different members, PDE4 is
considered as one of the most important enzyme for targeting the
Physiological processes of animals, plants and microbes are
regulated by secondary messengers. Cyclic adenosine mono-
phosphate (cAMP) and cyclic guanosine monophosphate (cGMP)
plays a cardinal regulatory role in airway epithelium, inflammatory
cells, airway smooth muscle cells and immune cells in animal
secondary messengers. Balance of these two important messengers
(cAMP and cGMP) are disrupted by the group of enzymes called
cyclic nucleotide phosphodiesterases (PDEs) [1,2]. These groups of
enzymes consist of 11 contrasting families, segregated on the basis
of their structures and properties during catalysis of secondary
cAMP, and the presence of PDE4 is also reported in immune cells.
PDE4 members target the catalysis of cAMP by hydrolysis of 30-
phosphodiester bond resulting in an inactive 50-monophosphate
[5] and thus inhibition of the PDE4 is contemplated an important
enzymatic aspect to control it. PDE4 inhibition accelerates the
increasing amount of cAMP which is helpful for physiological
processes like airway muscle relaxation as well to stop the activa-
tion of proinflammatory cell activation. Hence, there is an acute
need to develop potential inhibitor of PDE4 that can act as anti-
inflammatory agent.
These types of chemical compounds can be further used for
control of disease like asthma and pulmonary diseases [6,7]. In
former times, researchers reported the development of novel in-
hibitors (rolipram and piclamilast) as anti-inflammatory drugs in
1990s (Fig. 1). Inhibition of PDE4 elevates the cellular cAMP levels,
due to which the activation of specific phosphorylation cascade,
that inhibit the release of tumor necrosis factors-a (TNF-a), acti-
vation of inflammatory cells and cytokines (interleukin-2,
interleukin-12 and leukotriene B4).
* Corresponding author. State Key Laboratory for Conservation and Utilization of
Subtropical Agro-bioresources, Integrative Microbiology Research Centre, Guang-
dong Province Key Laboratory of Microbial Signals and Disease Control, South China
Agricultural University, Guangzhou, 510642, China..
** Corresponding author. Guangdong Laboratory for Lingnan Modern Agriculture,
Guangzhou, 510642, China.
E-mail addresses: rytang@scau.edu.cn (R. Tang), ziningcui@scau.edu.cn
(Z.-N. Cui).
1
These authors contributed equally to this paper.
https://doi.org/10.1016/j.ejmech.2020.112795
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
Fig. 1. The designed strategy for the title compounds [18e21].
Rolipram and piclamilast are well known for their high inhibi-
tory activity against PDE4 and good curative effects, however,
treatment with rolipram can cause nausea, sweating and other
unbearable side effects for patients, as a structural analogue,
piclamilast also contains aforementioned side effects, which limits
their application. Hence, we have been focusing on the construction
of new PDE4 inhibitor skeletons. As shown in our previous reports
[8], 4-(3,4-dialkoxyphenyl) moiety was essential for PDE4 inhibi-
tion where the catechol ether oxygens played an important role in
binding to the enzyme. Further study shows that the modification
of the 4-(3,4-dialkoxyphenyl) moiety to the 8-methoxyquinoline-
5-carboxamides (such as SCH 365351) could enhance the PDE4
inhibitory activity. Replacement of the amide moiety of SCH 365351
with five-membered heterocyclic rings (oxazole or furan ring) and
the cyclization of imide moiety into pyrazole ring are both helpful
to enhance PDE4 inhibitory activity. Further investigation found
that simplification from 8-methoxyquinoline moiety to benzene
ring was also beneficial to the enhancement of PDE4 inhibitory
activity. Based on our previous findings described above, com-
pounds 1 (pyrazole derivative containing furan ring) and 2 (pyr-
azole derivative containing oxazole ring) was designed to show
significant PDE4 inhibitor activity [8,9] (Fig. 1). With these two
skeletons in hand, we wish to disclose here design and synthesis of
three novel series of compounds 3 (2-alkyl-5-phenyl furan deriv-
ative), 4 (2-carbonyl-5-phenyl furan derivative) and 5 (2-phenyl-4-
carbonyl oxazole derivative) and speculated the effect of new
introduced heterocyclic N-(thiazolidin-2-ylidene) cyanamide in the
design. As demonstrating in our previous research, five membered
heterocyclic moiety, forming hydrophobic interaction with protein,
was essential for the bioactivity. A series of designed molecules
derived from furan and oxazole scaffolds that are further inflated
with superior PDE4 activity and selectively conducted in wet lab
experiments and supported with the significant in vivo efficacy
than rolipram.
2. Material and methods
2.1. General
Mass spectra were checked with a Bruker APEX IV spectrometer
(Bruker, Fallanden, Switzerland). 1H NMR and 13C NMR spectra
were measured on Bruker DPX600 (Bruker, Fallanden,
2

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
Switzerland), while tetramethylsilane was used as an internal
standard. Melting points were recorded with a Cole-Parmer
melting point apparatus (ColeParmer, Vernon Hills, Illinois, USA).
Elemental analyses were performed on a Vario EL elemental
analyzer. Analytical thin-layer chromatography was carried out on
silica gel 60 F254 plates. The promoter activity of hpa1 was checked
by a FACS-Caliber flow cytometer (CytoFLEX USA). RNA concen-
tration and purity were monitored using the Nanovue UV-Vs
spectrophotometer (GE Healthcare Bio-Science, Sweden). The
cDNA levels were quantified by Applied Biosystems 7500 Real-Time
PCR System (Thermo,USA). The growth rates were recorded using a
Bioscreen (Bioscreen, Finland).
10 mL of acetonitrile solution containing 2 mmol of 2-cyanoimino-
1,3-thiazolidine and 2 mmol of K2CO3. The mixture was stirred for
3e6 h at 75 ꢁC, then acetonitrile was removed under reduced
pressure. An excess of water was added and the reaction mixture
was extracted three times with dichloromethane. After that, it was
washed successively with 10% HCl, 10% NaHCO3 and water, dried
over anhydrous MgSO4. The product was purified by column
chromatography (40 ꢂ 250 mm) on silica gel using dichloro-
methane and methanol (v/v 95:5) as the eluent to yield the title
compounds.
2.2.3. General procedure for the synthesis of title compound 5a-5k
Intermediates ethyl 2-phenyloxazole-4-carboxylate were pre-
pared following the reported literature [11], followed by the hy-
drolysis of ester group to get 2-substituted phenyl-4-oxazole
carboxylic acids as described below.
Ethyl 2-phenyloxazole-4-carboxylate was dissolved in THF-H2O
(1:1, 20 mL) and 2 M aqueous NaOH (2 equiv.) solution was added
dropwise at 0 ꢁC. Reaction mixture was stirred at room temperature
for 2e4 h and monitored by TLC. Upon completion of reaction, THF
was removed in vacuo and aqueous layer was washed with ethyl
acetate and pH of aqueous layer was adjusted to 2 by the slow
addition of 2 M aqueous HCl at 0 ꢁC, which gives the precipitate at
this stage. Solid was filtered, washed with water and dried in vacuo
and used for the next step without further purification.
Mixture of 2-phenyloxazole-4-carboxylic acid (1 equiv.) and
thionyl chloride (5 mL) was refluxed for 1 h under nitrogen at-
mosphere. Reaction was cooled to room temperature and excess of
thionyl chloride was removed under vacuum. Crude mass was
dissolved in acetonitrile, followed by the addition of K2CO3 (3
equiv.) and 2-cyanoimino-1,3-thiazolidine (1 equiv.) at room tem-
perature. Reaction mixture was stirred for 3e6 h at room temper-
ature and monitored by TLC. Upon completion of reaction, it was
diluted with water and organic compounds were extracted with
dichloromethane. Organic layer was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. Product was purified
by silica gel (200e300 mesh) column chromatography using
methanol/DCM solvents as an eluent to give the title compound 5 in
moderate to good yield.
2.2. Synthesis of title compounds 3, 4 and 5
2.2.1. General procedure for the synthesis of title compounds 3a-3k
According to the reported literature [10aec], Intermediates 5-
substituted phenyl-2-furoic acid 7a-7k were prepared from
substituted aniline by Meerwein arylation reaction, and the cor-
responding 5-phenylfuran-2-carbonyl chloride were prepare using
SOCl2 as solvent and reactant. A solution of 5-phenylfuran-2-
carbonyl chloride (5.0 mmol, 1 equiv) was prepared in dry THF
and cooled to ꢀ10 ꢁC under nitrogen atmosphere. Sodium boro-
hydride (6.0 mmol, 1.2 equiv) was added to the THF solution and
the reaction mixture was stirred for 2 h at 0 ꢁC. The reaction was
quenched by adding 10% aqueous ammonium chloride solution.
THF was distilled off and the residue was diluted by adding chlo-
roform and water. The product was extracted with three portions of
chloroform and the organic layers were combined, dried over
anhydrous MgSO4, filtered and concentrated to get the crude
alcohol. It was purified by column chromatography (silica gel,
hexane and chloroform as eluents) to isolate a colorless liquid (80%
yield).
A mixture of (5-phenylfuran-2-yl) methanol (4.0 mmol) in dry
dichloromethane (20 mL) and pyridine (4.0 mmol) was cooled in an
ice bath. Solution of thionyl chloride (15.0 mmol) in dry dichloro-
methane (10 mL) was added under N2 atmosphere, at such a rate to
keep the temperature between ꢀ10 and 0 ꢁC. After complete
addition, the reaction mixture was stirred at room temperature for
2 h. Ice was added and the reaction mixture was stirred for further
5 min. A small amount of NaHCO3 was added to adjust pH 6.0. The
organic layer was separated and dried over MgSO4. Filtration and
evaporation of the solvent gave 2-(chloromethyl)-5-phenylfuran
(40% yield).
2.3. Bioassay
2.3.1. Assay of human PDE4 activity
Enzyme PDE4 was isolated from the sample as previously
described method [10aec,12]. The enzyme was prepared from
U937 cells which was derived from human monocytes, and was
stored at ꢀ20 ꢁC after preparation. Dilution of enzyme was done
with triple distilled water containing bovine serum albumin and
measurement of PDE4 activity was performed using this stored
enzyme. The substrate solution was prepared by adding [3H] cAMP
2-cyanoimino-1,3-thiazolidine (2.0 mmol) and potassium car-
bonate (3.0 mmol) were dissolved in 5 mL tetrahydrofuran and
stirred at room temperature. To the solution, 2-(chloromethyl)-5-
phenylfuran in acetonitrile was added dropwise. The reaction
mixture was refluxed for 3 h. After the completion of the reaction,
the solvent was evaporated under reduced pressure, water was
added, and the mixture was extracted with ethyl acetate. The
combined organic layer was washed with brine, dried over Na2SO4,
and concentrated. The residue was purified by silica gel chroma-
tography to give the corresponding product in 64% yield.
(300,000 dpm (5000 Bq)/assay) and 100
100 mmol/L TriseHCl (pH 8.0) containing 5 mmol/L ethylene
glycol-bis (
-aminoethyl ether) and O,O0-bis(2-aminoethyl) ethyl-
mmol/L cAMP solution to
b
eneglycol- N,N,N0,N0-tetraacetic acid. The substrate solution was
mixed with the enzyme solution containing a test compound dis-
solved in DMSO, and incubation was done for 30 min at 30 ꢁC.
Assays were performed in duplicate at different concentrations of
each test compound.
2.2.2. General procedure for the synthesis of title compounds 4a-4l
Intermediates 5-substituted phenyl-2-furoic acids 7a-7l were
prepared from substituted aniline by Meerwein arylation reaction
according to the reported literature [10aec].
Thionyl chloride (15 mL) was added to 0.01 mol of 5-substituted
phenyl-2-furoic acid. The mixture was refluxed at 80 ꢁC for 3 h. The
reaction was monitored by periodic thin layer chromatography
(TLC). When the reaction was completed, excess of thionyl chloride
was removed under reduced pressure. The crude product was
dissolved in 20 mL of anhydrous acetonitrile and added to the
2.3.2. Assay of TNF-a release
The blood is mixed with saline at a ratio of 1:1, and the pe-
ripheral blood mononuclear cells (PBMCs) were isolated from buffy
coats using Lymphoprep tubes [13]. The PBMCs were suspended in
RPMI 1640 with 0.5% human serum albumin, pen/strep, and 2 mM
L
-glutamine at 5 ꢂ 105 cells/mL. The cells were pre-incubated with
3

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
the test compounds in 96-well plates for 30 min and stimulated for
3. Result and discussion
18 h with 1 mg/mL lipopolysaccharide. TNF-a concentration in the
supernatants was measured by homogeneous time-resolved fluo-
rescence resonance (TR-FRET). The assay is quantified by measuring
3.1. Chemistry
fluorescence at 665 nm (proportional to TNF-
620 nm (control). Results are expressed as IC50 values (
a
concentration) and
M).
Based on our previous research work, we have designed three
new and novel modified structures, 2,5-disubstituted furan (3 and
4) and 2,4-disubstituted oxazole (5) as shown in Fig. 1. We wish to
analyze the effect of different heterocyclic rings, therefore thio-
zolidine ring was selected to attach with furan or oxazole moieties,
the effect of the reduction of amide to amine was also planned to
analyze. Introduction of a heterocyclic ring 2-cyanoimino-1,3-
thiazolidine was considered to be a new parameter of analysis.
In order to prepare the title compounds 3 and 4, intermediate 5-
substituted phenyl-2-furoic acids 7a-7l were synthesized (Meer-
wein arylation) following our previous reports [10aec] (Scheme 1).
Obtained carboxylic acids was refluxed in SOCl2 for 1 h to form
corresponding acid chlorides, which was coupled with N-(thiazo-
lidin-2-ylidene) cyanamide in the presence of base K2CO3 in
acetonitrile to furnish the title compounds 4a-4l (Scheme 1). In
order to probe the influence of amine group instead of amide,
structure 3 was designed which is the reduced form of amide 4. To
synthesize alcohol precursor 8a-8k, reduction of carboxylic acid
7a-7k was first performed with lithium aluminium hydride, but
due to low yield of the products, different methods were explored.
Hence, carboxylic acid was converted into its corresponding acid
chloride first by treatment with thionyl chloride and then it was
reduced with sodium borohydride in THF/DMF solvent at ꢀ10 to
m
2.3.3. LPS induced sepsis for measurement of TNF-a inhibition in
mice
The LPS induced sepsis model in mice was performed following
the literature [14]. Female Swiss albino mice were selected ac-
cording 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 sus-
pended in vehicle containing 0.5% Tween 80 in 0.25% sodium salt of
carboxymethyl cellulose. The control mice were performed the
vehicle alone. After 30 min of oral dosing, the mice were injected
into intraperitoneal cavity with 500
mg 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
blood samples at 4000 rpm for 20 min, which were stored over-
night at 4 ꢁC. Immediately, the serum samples were checked for
TNF-a levels using commercial mouse TNF-a ELISA kit (Amersham
Biosciences) and assay was carried out following the manufacturer
instruction.
0
ꢁC. Alcohol was obtained in moderate to good yield by applying
this reduction method. In order to couple this key intermediate
with N-(thiazolidin-2-ylidene) cyanamide, alcohol was trans-
formed into the corresponding chloride by reacting with thionyl
chloride, and then nucleophilic substitution with N-(thiazolidin-2-
ylidene) cyanamide in presence of K2CO3 in acetonitrile, title
compounds 3a-3k were obtained. (Scheme 1).
2.3.4. LPS induced neutrophilia model for asthma and COPD
LPS induced neutrophilia in Sprague Dawley rats was assessed
according to the described protocol [15]. 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
Following the reported literature, carboxylic acid precursors of
title compounds 5a-5k were obtained by treating corresponding
substituted formamides 9a-9k with ethyl 3-bromopyruvate [11],
followed by the hydrolysis of esters with sodium hydroxide. With
the carboxylic acid in hand, it was planned to couple with N-
(thiazolidin-2-ylidene) cyanamide, hence, different coupling
methods were tried such as i. EDCI, HOBt; ii. DCC, DMAP, but the
yield was not satisfactory. The best result was obtained by treat-
ment of carboxylic acids with thionyl chloride and then coupled
with N-(thiazolidin-2-ylidene) cyanamide (Scheme 2).
the rats were exposed to 100 mg/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.
All the new compounds were characterized by 1H, 13C NMR and
mass spectroscopy. The structures of compounds 3i, 4l and 5e were
confirmed by X-ray single crystal diffraction and shown in Fig. 2.
(See the supporting information data for details).
2.4. Molecular docking
Molecular docking was performed on Surflex-Dock module of
Sybyl 8.0 to know the exact active site of amino acids [10aec,16].
Crystal structure of PDE4B (PDB ID:1XMY) obtained from Protein
Date Bank was used as the receptor for molecular docking study.
The 3D structure of compounds 3j, 4j and 5j was drawn and opti-
mized 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 mo-
lecular docking, the interactions of the docked domain with ligand
were analyzed.
3.2. Biological evaluation and SAR studies
The in vitro activity of the inhibition of PDE4 and blockade of LPS
induced TNF-a in human blood monolayer cells were listed in
Table 1. Rolipram was used as positive marker during experiment. It
was observed that inhibition activity was highly influenced by the
substitution at different position of phenyl ring. Compounds with
chloro substitution were found to be more active than unsub-
stituted for PDE4B inhibition. Among the chloro substitution at
different position, para substituted compound 3d (IC50 ¼ 15.7
was found to be more effective than 3b (IC50 ¼ 101.6 M) and 3c
(IC50 ¼ 62.8 M). On comparison with same substituted at series 4
and 5, compound 5d (IC50 ¼ 3.6 M) showed better activity. It was
observed that chloro substituents compounds showed better ac-
tivity (TNF- inhibition) than unsubstituted. Among these chloro
substituted compounds, para substituted compound 5d showed
mM)
m
m
m
a
4

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
Scheme 1. Synthetic route to title compounds 3 and 4. Reagents and conditions: (a) i. NaNO2, HCl; ii. furan-2-carboxylic acid, cat. CuCl2, acetone, H2O; (b) i. SOCl2, reflux, 1h; ii.
K2CO3, N-(thiazolidin-2-ylidene) cyanamide, CH3CN, rt, 3e6 h; (c) i. SOCl2, reflux, 1h; ii. NaBH4, THF, DMF, ꢀ10 to 0 ꢁC; (d) i. SOCl2, Py, DCM, ꢀ10 to ꢀ5 ꢁC; ii. K2CO3, N-(thiazolidin-2-
ylidene) cyanamide, CH3CN, rt, 3e6 h.
Scheme 2. Synthetic route to title compounds 5a-5k. Reagents and conditions: (a) i. ethyl 3-bromopyruvate, NaHCO3, THF, 60 ꢁC, 23 h; ii. (CF3CO)2O, THF, rt, 12 h; (b) 2 M aq. NaOH,
THF, H2O, 0 ꢁC to rt, 2e4 h; (c) i. SOCl2, reflux, 1 h; ii. K2CO3, N-(thiazolidin-2-ylidene) cyanamide, CH3CN, rt, 3e6 h.
Fig. 2. Single crystal structures of compounds 3i, 4l and 5e.
better results. Same pattern was observed for the fluoro substituted
M) and little variation was found in
comparison with para chloro substituted compound (3d). When
donating group) compound was found to be more promising. IC50
value of compound 5j was found 1.4 M which was better than 3j
(IC50 ¼ 9.6 M) and 4j (IC50 ¼ 2.8 M). Also the inhibition activity
against TNF- M) was better than any
for compound 5j (IC50 ¼ 11.8
compounds 3g (IC50 ¼ 18.9
m
m
m
a
m
the substituent was changed to bromo at para position, inhibition
m
activity was further decreased (3i: IC50 ¼ 52.7
with series 3, 4 and 5, compound 5i (IC50 ¼ 46.7
activity than 3i (IC50 ¼ 52.7 M) and 4i (IC50 ¼ 49.6
of TNF-
also followed the similar pattern and 5i (IC50 ¼ 131.2
showed better activity than 3i (IC50 156.2 M) and 4i
(IC50 ¼ 138.9 M). 2, 6-Difluoro substituent was also analyzed and
found that 5k (IC50 ¼ 16.3 M) revealed modest activity against
PDE4B in comparison with 3k (IC50 45.7 M) and 4k
(IC50 ¼ 22.1 M), also the same pattern was observed against TNF-
M). When
m
M). In comparison
M) exhibit better
M). Inhibition
M)
other compound of series 3 and 4. We also speculated the selected
in vitro active compounds for their PDE4B selectivity over PDE4D
and it was concluded that compounds with all three series had
obvious selectivity towards PDE4B. Compound 5j showed better
selectivity (PDE4D/B ¼ 11.21) than reference rolipram and also with
other tested compounds. Selected in vitro active compounds 3j, 4j
and 5j were tested for LPS induced sepsis model for the measure-
m
m
m
a
m
¼
m
m
m
¼
m
ment of TNF-a inhibition (in female Swiss Albino mice) and neu-
m
a
trophilia inhibition for asthma and COPD (in male Sprague Dawley
rats). Table 2 demonstrated the results of experiment including
details such as oral dosages and number of animals grouped. The
results showed that compound 5j revealed better inhibitory activity
(5k: IC50 ¼ 49.3
m
M, 4k: IC50 ¼ 60.1
m
M, 3k: IC50 ¼ 82.3
m
the substituent at para position was changed from electron with-
drawing to electron donating group, significance change was
observed against PDE4B. IC50 values of 5h was found 9.2
comparison to 3h and 4h (IC50 ¼ 20.1 M and 12.5 M respectively).
Among the para substitutions, methoxy substituted (electron
m
M in
against TNF-a release (52.6%) and LPS induced neutrophilia inhi-
bition (42.1%) than the positive control rolipram (45.3% and 36.8%),
compound 3j (31.2% and 28.7%) and 4j (40.5% and 33.8%).
m
m
5

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
a
Table 1
Inhibition (IC50
,
m
M) of enzymatic potency (PDE4B and PDE4D) and TNF-
a
release from human blood mononuclear cells stimulated with lipopolysaccharide
.
Compd.
R
PDE4B IC50
(
m
M)
PDE4D
Selectivity (D/B)
TNF-a IC50 (mM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
162.5 5.8
101.6 3.9
62.8 2.9
15.7 0.9
85.6 2.8
59.7 2.1
18.9 1.1
20.1 1.1
52.7 1.8
9.6 0.8
45.7 1.4
111.3 4.5
62.8 2.4
29.4 1.5
6.6 1.0
62.4 1.2
37.8 0.9
11.3 0.7
12.5 1.1
49.6 1.2
2.8 0.6
22.1 1.1
71.2 2.9
40.3 1.8
18.1 0.9
3.6 0.6
NTb
e
198.6 6.4
149.1 4.2
89.7 2.8
44.2 1.5
121.4 3.7
89.5 3.1
45.8 1.8
53.9 1.8
156.2 6.6
36.8 1.5
82.3 2.5
169.5 3.9
122.5 3.7
74.3 1.9
35.6 1.0
109.8 3.1
72.6 2.5
29.8 0.9
41.3 1.8
138.9 4.4
15.3 0.9
60.1 1.9
141.5 5.7
101.4 4.2
62.5 2.1
27.2 1.2
92.8 3.7
64.3 3.1
22.1 1.0
35.2 1.4
131.2 5.2
11.8 0.6
49.3 1.8
18.9 0.9
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
4-CH3
4-Br
4-OCH3
2,6-di-F
H
2-Cl
3-Cl
4-Cl
2-F
NTb
e
NTb
e
58.3 1.2
3.71
e
NTb
NTb
e
110.5 3.1
121.5 4.2
NTb
5.85
6.04
e
3j
32.8 1.2
159.6 4.8
NTb
3.42
3.49
e
3k
4a
4b
4c
4d
4e
4f
4g
4h
4i
NTb
e
99.8 3.3
26.4 1.7
NTb
3.39
4.00
e
3-F
4-F
NTb
e
55.7 1.8
54.3 2.1
NTb
4.93
4.34
e
4-CH3
4-Br
4-OCH3
2,6-di-F
H
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
4-CH3
4-Br
4-OCH3
2,6-di-F
4j
18.7 1.0
88.6 1.9
NTb
6.68
4.01
e
4k
5a
5b
5c
5d
5e
5f
5g
5h
5i
NTb
e
102.5 3.1
26.8 1.1
NTb
5.66
7.44
e
52.8 1.4
27.6 1.0
6.8 0.8
9.2 0.7
46.7 1.4
1.4 0.3
NTb
e
45.9 1.3
66.9 1.8
NTb
15.7 0.8
98.7 2.5
2.8 0.7
6.75
7.27
e
5j
11.21
6.06
1.40
5k
rolipram
16.3 1.0
2.0 0.3
a
Results are the average of at least three assays.
NT, not tested.
b
Table 2
LPS induced TNF-a in SA mice and neutrophil influx in BALF of SD rats.
Compd.
R
Swiss Albino mice (n ¼ 6)
Sprague Dawley rats (n ¼ 6)
ꢀ
Does(mg/kg, po)
TNF-a Inhibition (%)
Does(mg/kg, po)
LPS induced neutrophilia (% inhibition)
3j
4j
5j
4-OCH3
4-OCH3
4-OCH3
10
10
10
10
31.2
40.5
52.6
45.3
10
10
10
10
28.7
33.8
42.1
36.8
rolipram
3.3. Docking results
Further, compound 5j also depicted more stability within the
binding pocket due to the hydrophobic interaction formed between
Phe 446 and thiazolidine ring of the compound. Figure 4 G and 4 H
revealed that compound 4j form more stable interaction with
PDE4B compare with rolipram by extra coordinate bonds. Oxygen
of p-methoxy group constituted coordinate bonds with the Zn2þ
(3j: 4.56 Å, 4j: 4.55 Å and 5j: 3.40 Å, Fig. 4) and Mg2þ (3j: 2.45 Å 4j:
3.46 Å and 5j: 3.31 Å, Fig. 3) cations.
The binding mode of the synthesized active compounds at the
PDE4B cavity was simulated with docking analysis by using Surflex-
Dock in Sybyl 8.0 software Fig. 3. For the docking tasks, crystal
structures of the PDE4B with (R)-rolipram was employed (PDB ID:
1XMY, 2.4 Å resolution) [17]. In docking studies, it was observed
that 4-methoxy phenyl ring of the compounds extended into the
metal binding pocket domain, thiazolidine ring into Q switch and P
clamp pocket [17]. Amide moiety of compound 4j exhibited H-
bonding interaction with the conserved glutamine (Gln443) res-
idue which was involved in bidentate H-bonding with eCO- at 1.7 Å
and 2.63 Å respectively Fig. 3C and 3D. Similarly, compound 5j
exhibited same interaction with the conserved Gln443 residue at
1.87 Å and 3.13 Å respectively Fig. 3E and 3F. Fig. 3 (A and B)
demonstrated that reduction of the amide bond (compound 3j)
decreased its ability in establishing crucial interactions with
Gln443, and only unveiled hydrophobic interaction between thia-
zolidine ring and phenylalanine (Phe 446) at a distance of 3.07 Å.
4. Summary
In the continuation of our effort towards the finding of new
PDE4 inhibitors, herein, we designed and synthesized three novel
series of compounds containing 2,5-disubstituted furan (3 and 4)
and 2,4-disubstituted oxazole (5) moieties. Synthesized com-
pounds were evaluated for in vitro activity against phosphodies-
terase type 4 and TNF-
show moderate to good inhibitory activity against PDE4 and TNF-
Further these compounds were tested for in vivo activity in animal
a. Compounds 3j, 4j and 5j were found to
a.
6

Y. Lin, W. Ahmed, M. He et al.
European Journal of Medicinal Chemistry 207 (2020) 112795
Fig. 3. Interaction of PDE4B in complex with compound 3j (A, B), compound 4j (C, D) and compound 5j (E, F). The catalytic domain bound to 4j overlaid with rolipram (violet color)
(G, H). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
models of asthma/COPD and sepsis induced by LPS in which com-
pound 5j displayed good in vivo activity in animal models. A pri-
mary SAR study revealed that incorporation of 2-cyanoimino-1,3-
thiazolidine ring was effective and favored the inhibitory activity
and substituent at para position of phenyl ring in the molecule had
an important effect of inhibitory and it can be concluded that the
effect of substitutions at para position in the structures were found
to be crucial. The docking results demonstrated that title com-
pounds interacted well with PDE4B protein by intermolecular
hydrogen bonding (eCOeN, Gln443), hydrophobic interaction
(thiazolidine Phe 446) and the metal coordination in the ligand-
receptor complex (p-OMe, Zn2þ and Mg2þ). We believe that such
efforts will be beneficent in future towards the development of
advanced and effective PDE4 inhibitors.
Cooperation Program in Guangdong (2020A0505100048), the Na-
tional Key Project for Basic Research (973 Program, 2015CB150600).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112795.
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8