Synthesis, activity and mechanism for double-ring conjugated enones

Article abstract of DOI:10.1016/j.bmcl.2021.128315

The relationship between TLR4 and inflammation-related diseases has been paid more and more attention. The studies have shown that TLR4/NF-κB signaling pathway plays an important role in the transmission of inflammatory signals. A large number of pro-inflammatory factors, chemokines, adhesion factors, TLR4 and its ligands interact with each other, and jointly promote the development of diseases. In this work, 8 target compounds were synthesized to screen the inhibitory activity of TLR4 in vitro. The results of TLR4 inhibition test in vitro showed that the double-ring conjugated enones had a good inhibitory activity, and the IC₅₀ value of compound 4f was 0.56 ± 0.10 μM, and it was superior to the positive control methotrexate. To further study the anti-inflammatory effect and mechanism of double-ring conjugated enones by using LPS induced rat synovial cell inflammation model. The results of the mechanism test showed that compound 4f could effectively promote the apoptosis of rat synovial cells, and the mechanism might be related to the up-regulation of the expression of apoptosis-related protein Caspase-3. In addition, compound 4f could significantly inhibit the increase of inflammatory factors TNF-α, IL-1β and IL-6 in rat synovial cells induced by LPS, showing a good anti-inflammatory activity. In the TLR4/NF-κB signaling pathway test of rat synovial cells, compound 4f can effectively regulate the expression levels of TLR4, MyD88, NF-κB and IκB related proteins in TLR4/NF-κB signaling pathway, which may be due to its inhibition of LPS-induced inflammation in rat synovial cells. At the same time, it inhibits the abnormal proliferation of cells and its important mechanism promoted of apoptosis.

Full text of DOI:10.1016/j.bmcl.2021.128315

Bioorg. Med. Chem. Lett. 49 (2021) 128315
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, activity and mechanism for double-ring conjugated enones
,
b
,
,
,
Shiyang Zhoua c, Gangliang Huangc *, Guangying Chena *, Jian Liud
a Key Laboratory of Tropical Medicinal Resource Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, China
b Chongqing Chemical Industry Vocational College, Chongqing 401228, China
c Active Carbohydrate Research Institute, Chongqing Key Laboratory of Inorganic Functional Materials, College of Chemistry, Chongqing Normal University, Chongqing
401331, China
d The First Hospital, Hunan University of Chinese Medicine, Changsha 410007, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The relationship between TLR4 and inflammation-related diseases has been paid more and more attention. The
studies have shown that TLR4/NF-κB signaling pathway plays an important role in the transmission of inflam-
matory signals. A large number of pro-inflammatory factors, chemokines, adhesion factors, TLR4 and its ligands
interact with each other, and jointly promote the development of diseases. In this work, 8 target compounds were
synthesized to screen the inhibitory activity of TLR4 in vitro. The results of TLR4 inhibition test in vitro showed
that the double-ring conjugated enones had a good inhibitory activity, and the IC50 value of compound 4f was
TLR4/NF-κB
Rheumatoid arthritis
Mechanism
Signaling pathway
0.56 ± 0.10 μM, and it was superior to the positive control methotrexate. To further study the anti-inflammatory
effect and mechanism of double-ring conjugated enones by using LPS induced rat synovial cell inflammation
model. The results of the mechanism test showed that compound 4f could effectively promote the apoptosis of rat
synovial cells, and the mechanism might be related to the up-regulation of the expression of apoptosis-related
protein Caspase-3. In addition, compound 4f could significantly inhibit the increase of inflammatory factors
TNF-α, IL-1β and IL-6 in rat synovial cells induced by LPS, showing a good anti-inflammatory activity. In the
TLR4/NF-κB signaling pathway test of rat synovial cells, compound 4f can effectively regulate the expression
levels of TLR4, MyD88, NF-κB and IκB related proteins in TLR4/NF-κB signaling pathway, which may be due to
its inhibition of LPS-induced inflammation in rat synovial cells. At the same time, it inhibits the abnormal
proliferation of cells and its important mechanism promoted of apoptosis.
Rheumatoid arthritis (RA) treatment is mainly to relieve joint
inflammation and joint pain.1 Inhibitory the further development of RA
and the irreversible destruction of bone, and strive to protect the normal
function of joints and muscles.2–4 So, as to achieve the goal of
completely alleviating arthritis or completely being treated, improve the
overall function of patients.5–8 At present, the commonly used for
treatment of RA drugs can be divided into five categories: non-steroidal
anti-inflammatory drugs, slow-acting anti-rheumatic drugs, glucocorti-
coids, biological agents targeted therapy, botanical preparation.9–11
Current, plant drugs was used already appeared much kind in cure RA,
wait for on the market medicine such as total glucoside of paeony,
sinomenine, tripterygium wilfordii. Plant drugs have certain clinical
efficacy in the treatment of RA, but the mechanism of action of the drugs
still needs further research.12–14 Dysdensiol K is a kind of monomer
compound with anti-rheumatoid arthritis activity isolated from Fissis-
11.8 ± 0.23
μ
M.15,16 In the previous work, Dysdensiol K was used as the
lead compound to design and synthesize four series of double-ring
conjugated enones, and the anti-rheumatoid arthritis activity was
evaluated. The results of anti-rheumatoid arthritis activity evaluation
showed that double-ring conjugated enones were identified as potential
candidates drug for treatment of RA. In the screening of rat synovial cell
formation in vitro, the double-ring conjugated enones showed ideal
inhibitory activity, among which the IC50 values of low
μ
M level.17
Meanwhile, in the CIA rat model, the double-ring conjugated enones
showed in vivo therapeutic and ameliorative effects on RA compared
with methotrexate, achieving the same therapeutic level. The double-
ring conjugated enones inhibited the production of IL-6 and appropri-
ately reduced the formation of TNF-
α
during in vivo treatment.18–20
Molecular docking simulation showed that the target compound had a
good match with TLR4 receptor. TLR4/NF-κB plays an important role in
tigma oldhamii. Its inhibitory effect on synovial cells in vitro was IC50
=
the pathogenesis of RA by regulating the expression of cytokines (TNF-α,
* Corresponding authors.
E-mail addresses: huangdoctor226@163.com (G. Huang), chgying123@163.com (G. Chen).
https://doi.org/10.1016/j.bmcl.2021.128315
Received 9 June 2021; Received in revised form 22 July 2021; Accepted 8 August 2021
Available online 11 August 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
Scheme 1. The synthetic route of the double-ring conjugated enones 4a-4h. Reagents and conditions: (a) ethanediol, benzene, p-TsOH, reflux for 48 h; (b)
substituted cyclopentanone or cyclohexanone, C2H5OH, C2H5ONa, reflux for 12 h; (c) HCl, H2O, 60 ◦C, 2 h, (d) KOH, C2H5OH, reflux for 12 h.
IL-1β), matrix metalloproteinases (MMP-3, MMP-9), angiogenic factors
(VEGF), inducible enzymes (COX-2, iNOS), and other genes.21,22
Involved in the pathological process of arthroinflammatory response
and injury response.23 Based on the existing activity evaluation, this
work we synthesized eight double-ring conjugated enones, and further
studied the effect target and the mechanism of action against RA.24–26
The TLR4 receptor inhibitory test was used in vitro to determine the
receptor of action.27 The effects on the apoptosis of primary rat synovial
cells, the expression of apoptotic-related proteins in primary rat synovial
cells, the effects on the inflammatory factors in primary rat synovial cells
induced by LPS and the effects on the TLR4/NF-κB signaling pathway in
primary rat synovial cells induced by LPS were determined to study the
mechanism of action.28–30 The mechanism of anti-rheumatoid arthritis
was studied to provide theoretical guidance for the drug pharmacody-
namics research, and provides a certain experimental basis for further
research and development of anti-rheumatoid arthritis drugs in the
future.
adiposus to change the logP and pKa of the target compounds, so as to
achieve the goal of changing the anti-rheumatoid arthritis activity of the
target compounds 4a-4h. In the synthesis of the target compounds 4a-
4h we chose an efficient and simple synthesis method. According to the
pathway described in Scheme 1, a series of double-ring conjugated
enones were synthesized. We used ethyl acetoacetate or ethyl levulinate
as the starting material, and synthesize the target compounds through
four steps: acetylcarbonyl protection, condensation reaction, hydrolysis
reaction and ring formation reaction. During the protection of ace-
tylcarbonyl group, ethyl acetoacetate (compound 1a) and ethylene
glycol were used for condensation reaction in benzene. In this step, p-
methylbenzene sulfonic acid (p-TsOH) was used as reaction catalyst, and
the reduced ketals (compound 2a) could be obtained within 48 h after
reflux (yield 99%). The general method were used to synthesis com-
pounds 2b. After the acquisition of compound 2a and 2b, the key in-
termediates (compounds 3a-3h) were obtained by condensation
reaction and hydrolysis reaction with substituted cyclopentanone and
cyclohexanone. Anhydrous ethanol was used as the reaction solvent, and
solid sodium hydroxide (NaOH) was used as the basic substance. After
reflux reaction for 12 h, the key intermediates compounds 3a-3h were
obtained by hydrolysis and removal of the carbonyl protection. Finally,
the compounds 3a-3h were dissolved in anhydrous ethanol to generate a
ring reaction. The solid potassium hydroxide (KOH) was used as the
basic material, and the target compounds (compounds 4a-4h) could be
obtained after reflux reaction for 12 h. The structures of all target
The Dysodensiol K which has an anti-rheumatoid arthritis activity
(IC50 = 11.8 ± 0.23 μM) and it was isolated from the Fissistigma oldhamii.
The chemical structures of the lead compound was analyzed and it was
found that all of them had the double-ring structures. Through the
analysis of SAR, it was found that the double-ring structure was the
parent nuclear structure of the compound, and the activity of the com-
pound against rheumatoid arthritis was determined by the substituents
on the ring. On the basis of this, the Dysodensiol K was used as the lead
compound to design the double-ring conjugated enones with similar
parent nucleus structure. From the chemical structure analysis, it found
that the target compounds and the Dysodensiol K also have the structure
of double-ring conjugated enone. In such a structural design, the target
compounds should have a similar spatial structure of the lead com-
pound, which may also show a good anti-rheumatoid arthritis activity in
the biological activity. Even after the chemical structure modification of
the lead compound, some of the target compounds have a better bio-
logical activity than the lead compound. During the structural modifi-
cation of the lead compound, we introduced two different substituents of
electron-absorbing group and electron-donating group at position arcus
1
compounds were characterized by H NMR, 13C NMR and HR-ESI-MS
spectra. The synthesis route was characterized by simple operation,
mild reaction conditions, cheap reagents and raw materials, and the
total yield was moderate to good.
Toll-like receptor 4 (TLR4) was belong to the pattern receptor family.
They were highly conserved receptor families that recognize conserved
pathogen associated submodes and therefore represent the first line of
defense. TLR4 was thought to be a recognition receptor for lipopoly-
saccharide in gram-negative bacteria. In addition, TLR4 has been widely
shown to be involved in the recognition of endogenous molecules that
were released by damaged tissues and necrotic cells, and these damage-
2

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
Table 1
TLR4 in vitro inhibition data.
Compounds
Structure
TLR4
IC50
(
μ
M)a
±
SD
4a
2.53 ±
O
H
0.31
O
4b
4c
2.71 ±
O
H
0.30
O
Cl
2.82 ±
O
0.28
H
Fig. 1. The changes in the morphology of the synovial cells in rats (Tunel
staining, 100×).
O
immunosorbent assay (ELISA). TLR4 was placed in the coated micro-
pores of the captured antibody, and the detection antibodies to be tested
and HRP-labeled was added successively. The antibodies were incubated
and thoroughly washed. The substrate TMB, catalyzed by peroxidase,
was transformed into blue and the final yellow under the action of acid.
The shade of the color was positively correlated with the TLR4 in the
sample. The absorbance was measured at 450 nm with a microplate
analyzer and the concentration of the sample was calculated.31 To
further investigate the in vitro anti-rheumatoid arthritis activity of these
target compounds, we tested compounds 4a-4h for TLR4 inhibition in
vitro. As could be seen from the test data in Table 1, compounds 4a-4h
showed good inhibitory activity against TLR4. The IC50 values of these
4d
4e
1.54 ±
O
H
0.21
O
COOCH2CH3
0.73 ±
O
0.15
H
compounds inhibited TLR4 in vitro were at the level of
compounds 4e and 4f were the most prominent, and the IC50 values of
TLR4 inhibition activities were 0.73 ± 0.15 and 0.56 ± 0.10 M,
respectively, which were better than the positive control methotrexate
μM, among which
O
4f
0.56 ±
μ
O
0.10
H
H
O
N
(1.04 ± 0.22 μM).These results suggest that the target compound can
bind well to TLR4, inhibit its activity and reduce the release of inflam-
matory factors, and ultimately improve or treatment RA.
O
O
Cytomorphology, also known as cytological examination. It is a
traditional and classic clinical diagnosis method with a long history.
Cytomorphology is a science that studies the microstructure and sub-
microstructure of cells and their components, including the structure of
biological macromolecules that represent the phenomena of cell life.32
Because of its simplicity, practicality and the fact that seeing directly is
better than hearing, cytomorphology is often use in testing the activity
of compounds. Based on the results of LTR4 inhibition test in vitro, we
selected the target compound 4ffor the morphological observation of rat
synovial cells (Fig. 1). The results showed that, compared with the
normal group, the synovial cells of rats induced by LPS were signifi-
cantly proliferated, and the cell bodies were significantly enlarged, and
were round, oval or irregular spindle shaped. However, after treatment
4g
1.85 ±
O
H
0.26
O
4h
2.66 ±
O
H
0.26
O
methotrexateb
1.04 ±
0.22
with 1 μM methotrexate and 1 μM compound 6, the number of cells was
a
b
significantly reduced, the refractive ability was reduced, and the cell
body was irregular spindle or round, suggesting that compound 4f could
significantly inhibit the abnormal proliferation of rat synovial cells
induced by LPS.32
Values are the average of 3 independent experiments run in triplicate.
Methotrexate is use as positive control.
related molecular pattern molecules induce and promote inflammatory
response activation by interacting with TLR4.31 Thus, TLR4 was a key
receptor that was induced by exogenous and endogenous ligand proin-
flammatory responses in infectious stimulation-mediated interactions.
Apoptosis is a basic phenomenon that exists widely in the biological
world. It is plays a very important role as well as cell growth, develop-
ment and proliferation. Currently, it is believe that the extracellular
stimulation to induce apoptosis must be transmitted through a series of
TLR4 test kits was
a double-antibody sandwich enzyme-linked
3

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
Fig. 2. Apoptosis of rat synovial cells (Tunel staining, 200×).
Fig. 4. Comparison of the expression levels of apoptosis-related protein
Caspase-3 in rat synovial cells (n = 3). Compared with the LPS group, the
significance symbols is ## = p < 0.01.
nuclear pyknosis and chromatin concentration, suggesting that com-
pound 4f can effectively promote the apoptosis of rat synovial cells.33
Caspase-3 is a protease, a member of the Caspase family which plays
an irreplaceable role in cell apoptosis. Caspase-3 can be activate by a
variety of factors. In CTL cell killing, Caspase-3 can be activate by both
the Fas/FasL pathway and the granulosase B pathway. Granulosase B is a
serine esterase in CTL cell granules, and it is the only mammalian pro-
tease that cleaves after Asp except the Caspase protease. Caspase-3
normally exists in the cytoplasm in the form of proenzyme (32 kDa).
In the early stage of apoptosis, it is activate.34 The activate Caspase-3
consists of two large subunits (17 kDa) and two small subunits (12
kDa), cleavages the corresponding cytoplasmic and nuclear substrates,
and eventually leads to apoptosis. However, the activity of caspase-3
decrease significantly in the late stage of apoptosis and in the dead
cells. In order to verify the mechanism of compound 4f promoting the
apoptosis of rat synovial cells, we further studied the effect of compound
4fon the expression level of apoptosis-related protein Caspase-3 (Fig. 3
and Fig. 4). The results of Caspase-3 expression test showed that the
expression of apoptosis-related protein Caspase-3 was increased in the
LPS group compared with the normal group, but there was no significant
Fig. 3. Expression of apoptosis-related protein Caspase-3 in rat synovial cells
(Immunofluorescence, 200×).
intracellular signals.33 Apoptosis was induced by endogenous and
exogenous factors in the induction stage. Endogenous factors include the
activation of apoptosis inducing mechanisms (such as Fas ligands, tumor
necrosis factor, etc.) and the inactivation of inhibition mechanisms
(such as growth factors, hormones, receptor factors and other prolifer-
ative factors). Exogenous factors include physical factors such as radi-
ation and heat shock, chemical factors such as drugs and poisons,
biological factors such as viruses and bacteria. The reason of apoptosis
has become a hot spot of research is largely determined by the close
relationship between apoptosis and clinical virus. This relationship is
not only manifest in the study of apoptosis and its mechanism, which
elucidates the pathogenesis of a large class of immune diseases, but also
leads to the emergence of new therapies for diseases. In particular, the
close relationship between apoptosis and tumor, AIDS and RA has
attracted much attention.33 The clinical manifestation of RA was syno-
vitis, so inhibiting the growth of synovial cells and promoting their
apoptosis could be used as an evaluation index. In the process of rat
synovial cell apoptosis induced by compound 4f, the positive apoptotic
cells were stained green after Tunel staining (Fig. 2). The results of cell
apoptosis showed that there were almost no positive apoptotic cells in
difference34. Compared with LPS group, 1
μM methotrexate and 1 μM
compound group 4f significantly increased the expression level of
apoptosis-related protein Caspase-3 in synovial cells (p < 0.01), which
suggested that compound 4fgroup could effectively promote the
apoptosis of rat synovial cells, and the mechanism of promoting
apoptosis might be related to the up-regulation of apoptosis-related
protein Caspase-3.
RA is an autosystemic immune disease with symmetrically and multi-
articular synovitis as the main clinical manifestation. With progression
of synovitis, the synovial cells proliferate abnormally and form pannus
of synovial vessels.35 There is a large number of neovascularization and
infiltration of various inflammatory cells inside, which erode cartilage
and bone and lead to joint destruction. In RA patients, synovial cells are
significantly increase, including a large number of fibroblast synovial
cells and inflammatory cells are infiltrate around proliferative FLS. The
produce a large number of inflammatory factors, such as TNF-α, IL-1β,
IL-6, etc., leading to synovial inflammation and cartilage and bone
destruction.35 In this experiment, LPS induced synovial cell inflamma-
the normal group. After LPS (1
apoptotic cells increased, but there was no significant difference
compared with the normal group. However, after treatment with 1
methotrexate and 1 M compound 4f, the number of apoptotic cells
increased significantly, showing typical apoptotic characteristics such as
μg/mL) induction, the number of
tion model, and the content of factors (TNF-α, IL-1β, IL-6) in the su-
pernatant of cell culture was detected by ELISA (Fig. 5a-5c). Compared
μ
M
with the normal group, the contents of TNF-α, IL-1β and IL-6 in the sy-
μ
novial cell supernatant of rats in LPS group were significantly increased
(p < 0.01). Compared with LPS group, the contents of inflammatory
4

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
Fig. 5. The content of inflammatory factors TNF-
α
, IL-1βand IL-6 in rat synovial cells were determined (n = 3). Compared with the normal group, the significance
symbols is ** = p < 0.01; compared with the LPS group, the significance symbols is ## = p < 0.01.
Fig. 6. The TLR4 protein expression and content in rat synovial cells (Immunofluorescence, 200×, n = 3). Compared with the normal group, the significance symbols
is ** = p < 0.01; compared with the LPS group, the significance symbols is ## = p < 0.01.
5

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
Fig. 7. The MyD88 protein expression and content in rat synovial cells (Immunofluorescence, 200×, n = 3). Compared with the normal group, the significance
symbols is ** = p < 0.01; compared with the LPS group, the significance symbols is ## = p < 0.01.
Fig. 8. The NF-κB protein expression and content in rat synovial cells (Immunofluorescence, 200×, n = 3). Compared with the normal group, the significance
symbols is ** = p < 0.01; compared with the LPS group, the significance symbols is ## = p < 0.01.
Fig. 9. The IκB protein expression and content in rat synovial cells (Immunofluorescence, 200×, n = 3). Compared with the normal group, the significance symbols is
** = p < 0.01; compared with the LPS group, the significance symbols is ## = p < 0.01.
6

S. Zhou et al.
Bioorganic & Medicinal Chemistry Letters 49 (2021) 128315
factors TNF-
α
, IL-1β and IL-6 in cell supernatant of 1
μ
M methotrexate
Laboratory of Tropical Medicinal Resource Chemistry of Ministry of
Education (RDZH2021002).
and 1μM compound 4f group were significantly decreased (p < 0.01),
which indicated that compound 4f group could significantly inhibit the
LPS-induced increase of inflammatory factors TNF-
α, IL-1β and IL-6 in
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could significantly inhibit the inflammatory factors TNF-α, IL-1β and IL-
6 induced by LPS in rat synovial cells, and effectively regulate the
expression levels of TLR4, MyD88, NF-κB and IκB related proteins in
TLR4/NF-κB signaling pathway. These tests data suggest that this may
be an important molecular mechanism to improve the abnormal pro-
liferation of synovial cells in rats with rheumatoid arthritis, as well as
anti-inflammatory and pro-apoptotic effects. In vitro TLR4 inhibition test
and mechanism study, for further study of double-ring conjugated
enones anti-rheumatoid arthritis efficacy to provide certain theoretical
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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The project was sponsored by the National Natural Science Foun-
dation of China (21662012, 41866005). The project was also sponsored
by the Postgraduate Research and Innovation Project of Hainan Normal
University (Hsyx2018-8) and Open Foundation Project of Key
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