Design, synthesis, anti-inflammatory activity, and molecular docking studies of perimidine derivatives containing triazole

Article abstract of DOI:10.1016/j.bmcl.2017.08.014

We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a–s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, ¹H NMR, ¹³C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-α and interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-κB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity.

Full text of DOI:10.1016/j.bmcl.2017.08.014

Accepted Manuscript
Design, synthesis, anti-inflammatory activity, and molecular docking studies of
perimidine derivatives containing triazole
Hong-Jian Zhang, Xiu-Zhi Wang, Qi Cao, Guo-Hua Gong, Zhe-Shan Quan
PII:
S0960-894X(17)30815-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.014
BMCL 25208
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 February 2017
6 June 2017
8 August 2017
Please cite this article as: Zhang, H-J., Wang, X-Z., Cao, Q., Gong, G-H., Quan, Z-S., Design, synthesis, anti-
inflammatory activity, and molecular docking studies of perimidine derivatives containing triazole, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, anti-inflammatory activity, and molecular docking
studies of perimidine derivatives containing triazole
Hong-Jian Zhang 'a', Xiu-Zhi Wang 'b', Qi Cao 'c', Guo-Hua Gong 'b',*, Zhe-Shan Quan 'a',*
'a'Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain,Affiliated Ministry of
Education, College of Pharmacy, Yanbian University, No. 977, Park road, Yanji, Jilin, 133002, China
'b' Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, China
'c'Department of Pathology, 306 Hospital of PLA, Beijing, 100101, China
Abstract
We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine
derivatives containing triazole (5a-s). The chemical structures of the synthesized compounds have
been assigned on the basis of IR, 1H-NMR, 13C-NMR, and HRMS spectral analyses. The
anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a
lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound
7-(3-methylbenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5h) and compound
7-(2-fluorobenzyl)-7H-[1,2,4]triazolo[4,3-a]perimidine (hereafter referred to as 5n) caused a
reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-α and
interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n
was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the
most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This
activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that
of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects,
LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK)
phosphorylation were studied. The results of western blotting showed that the extract obtained from
compound 5n inhibited NF-κB (p65) activation and MAPK (extracellular signal-regulated kinase
(ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking
study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar
*Corresponding author: Zhe-Shan Quan Email adress: zssquan@ybu.edu.cn. Tel: +86-433-2436020, Fax: +
86-433-2436020; Guo-Hua Gong: E-mail address: gongguohua0211@163.com, Tel: +86-475-8314245.

to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed
it could significantly inhibit COX-2 activity.
Keywords: Synthesis, Perimidine, Anti-inflammatory activity, Molecular docking study
Inflammation is a common condition in which body tissues are affected by heat, redness,
swelling, and pain.1 Inflammation is generally caused by hay fever, periodontitis, atherosclerosis,
rheumatoid arthritis, and even cancer.2 Commonly, non-steroidal anti-inflammatory drugs (NSAIDs)
are used as analgesics and antipyretics, as well as anti-inflammatory agents at higher doses.
Nevertheless, the long-term use of NSAIDs is known to cause gastro-intestinal complications, such as
bleeding, ulcer, perforation, obstruction, and so on.3 Therefore, it is necessary to study and develop
safe anti-inflammatory drugs.
As previously reported, various structures of compounds containing triazole have shown
anti-inflammatory properties, such as 2-ethylthio-6-formyl-7-(4-methoxyphenylamino)-5-oxo-
1-phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (I), 6-(m-tolyloxy)-[1,2,4]triazolo
[3,4-a]phthalazine-3-carboxamide (II), (1H-perimidin-2-ylthio)(phenyl)methanol (III), and
(Z)-2-(1-(6-methoxynaphthalene-2-yl)ethyl)-5-((5-methylthiophene-2-yl)methylene)thiazolo[3,2-b]-
1,2,4-triazole-6(5H)-one (IV).4-7 Bassyouni et al. reported that1H-perimidine derivatives had
anti-inflammatory activities (V). In search for new potentially active substances, we committed to
obtaining target compounds containing 1H-perimidine and triazole molecules. Accordingly, the
target compounds 5a-s were designed (Figure 1).8
Figure 1. Structures of some compounds in containing triazole and design of target compounds
- 2 -

The synthesis of the target compounds is presented in Scheme 1. Compound 1 was prepared by
reacting 1,8-diaminonaphthalene with carbamide via a melting reaction. Compound 1 reacted under
POCl3 conditions and yielded compound 2. Compound 2 was then reacted with hydrazine hydrate in
ethanol to produce compound 3. Compound 3 reacted with formic acid under reflux conditions to
yield compound 4. Compound 4 was then reacted with alkyl halide or substituted with benzyl
chloride in the presence of sodium hydride in N,N-dimethylformamide (DMF) to produce compounds
5a-s. The structures of the synthesized compounds were confirmed via IR, 1H-NMR, 13C-NMR, and
high-resolution mass spectroscopy (HRMS) analyses.
Scheme 1. Synthesis of the target compounds.
The in vitro anti-inflammatory activities of the synthesized compounds were evaluated using a
LPS-stimulated inflammation model. The results of the perimidine derivatives containing triazole
were presented in Figure 2. Compound 5a, 5d, 5e, 5f, 5g, 5i, 5l, 5m, and 5s showed cell toxicity, and
the other seven compounds reduced cell viability significantly, except for 5a and 5l (p < 0.001) at a
dose of 50 μg/mL. Obviously, the length of the carbon chain was closely related with the compounds’
- 3 -

toxicity, especially when the length of the carbon chain was greater than five. Furthermore, when
RAW264.7 cells were treated with LPS and synthesized compounds at the same time, the viability of
cells treated with compounds 5b, 5e, and 5l decreased to a greater extent than when treated with other
compounds.
Figure 2. The effect of compound 5a-s and LPS for cell viability.
Black column: only compound, Gray column: compound + LPS; Data are means ± SD; *:0.01 <p< 0.05,***: p < 0.001 compared
with Control group; #: 0.01 <p< 0.05, ##:0.001 <p< 0.01, ###: p < 0.001compared with LPS group.
TNF-α, IL-6, and other pro-inflammatory cytokines play an important role in biological injury
and inflammation.9 TNF-α has a wide range of biological activities, and participates in inflammation,
immune response, anti-tumor activities, and engages in the pathological process of endotoxin shock.
It can cause fever, shock, among other abnormalities, as well as increase the levels of IL-6 and other
cytokines, thus aggravating the inflammatory response if an excessive amount is released or
produced.10 Generally, IL-6 is produced after TNF-α stimulation, and is closely related to the
pathological and physiological processes of various diseases. It is also an important, sensitive
indicator which reflects the severity of the inflammation and tissue damage.11,12 TNF-α and IL-6 are
involved in the body's inflammatory response as important inflammatory mediators.13 Under the
physiological conditions, TNF-α and IL-6 levels remain low; however, in a pathological state, a large
- 4 -

number of TNF-α and IL-6 molecules are secreted. The preliminary screening showed compounds 5c,
5h, 5j, 5k, 5n, 5o, 5p, 5q, and 5r displayed lower toxicity by MTT assay. Then, we measured their
concentration of TNF-α and IL-6 at dose of 50 μg/mL, respectively. We found only two compounds
5h and 5n could decrease the concentration of two inflammatory factors. Thus, compounds 5h and
5n were chose for the following test. The effects of compounds 5h and 5n on pro-inflammatory
cytokine production are shown in Figure 3. With regard to TNF-α and IL-6, levels in the control
group were regard as 100 %. The fold of test group was the ratio of the content of test group with
that of the control group. There were statistically significant differences between the control group
and the LPS group. This suggests that LPS induced an increase in TNF-α and IL-6 secretion.
Fortunately, compounds 5h and 5n could inhibit this increase in TNF-α and IL-6 in a dose-dependent
manner. Compounds 5h or 5n at doses of 25 μg/mL, and 50 μg/mL significantly reduced the levels of
TNF-α. Among them, TNF-α levels reduced most noticeably at a treatment dose of 50 μg/mL, with
the mean fold of TNF-α at 146.24 % and 124.87 %. The positive drug group had no significant
difference compared with that of LPS group. Compounds 5h and 5n reduced IL-6 levels in the same
conditions, as was expected. The effects of compounds 5n were obviously stronger than those of
compound 5h. IL-6 fold were reduced by compound 5n to 71.80 %, 68.23 %, and 47.21 % at doses of
12.5 μg/mL, 25 μg/mL, and 50 μg/mL, respectively.
Figure 3. The effect of compound 5h and 5n on pro-inflammatorycytokine production in RAW
264.7 cells.
The TNF-α and IL-6 production in the supernatants was determined by ELISA.Values were the mean ± S.D. (fold) of triplicate
experiments.###: p < 0.001 compared with Control group; *:0.01 <p< 0.05, **: 0.001<p< 0.01, ***: p < 0.001 compared with LPS
group.
- 5 -

The xylene-induced ear-edema test was used to evaluate acute anti-inflammatory activity.14,15
The anti-inflammatory activities of compounds 5h and 5n were also evaluated in vivo and the results
are shown in Table 1. In this test, compounds were screened in a xylene-induced ear-edema test in
mice, with ibuprofen and indomethacin used as the reference drugs. Anti-inflammatory activity was
assessed based on the ability of the test compounds to prevent edema. The data revealed that
compounds 5h and 5n showed significant anti-inflammatory effects at 25 mg/kg and 50 mg/kg
administered i.p. When compared with the control group, these two compounds showed
anti-inflammatory activities at a dose of 12.5 mg/kg, with lower inhibition rate, but this was not
statistically significant. Compound 5n exhibited the strongest inhibition of inflammation at 49.26%,
which was higher than ibuprofen (28.13%) and lower than indomethacin (49.36%) at a dose of 50
mg/kg.
Table 1. Anti-inflammatory activity of compounds 5h and 5n administrated i.p.
Compound
Control
Dose (mg/kg)
Edema mean ± S.D. (mg)
10.13 ± 0.62
Inhibition rate (%)
-
-
Ibuprofen
Indometacin
50
7.28 ± 0.65*
28.13
49.36
15.70
29.81
42.94
17.18
34.95
49.26
50
5.13 ± 1.20 ***
8.54 ± 0.44
12.5
25
5h
5n
7.11 ± 0.63*
50
5.78 ± 0.84***
8.39 ± 0.57
12.5
25
6.59 ± 0.73**
5.14 ± 0.72 ***
50
*:0.01 <p< 0.05, **: 0.001<p <*0.01, **: p < 0.001 compared with Control group
The release of pro-inflammatory cytokines (eg: TNF-α and IL-6) and mediators (eg: iNOS and
COX-2) is related to the MAPK pathway in LPS-stimulated macrophages.16-18 MAPKs include ERK,
c-Jun N-terminal kinase (JNK), and p38, and their signaling pathways play important roles in several
biological processes. Accordingly, the effects of compound 5h on the activation of ERK and p38 were
investigated. The results were presented in Figure 4. The phosphorylation levels of ERK and p38
- 6 -

significantly decreased, and the effects of compound 5n were dose-dependent. However, the levels of
ERK and p38 were unaffected after stimulation. These results suggested that compound 5ncould
inhibit pro-inflammatory mediator and cytokine expression via MAPK pathways.
Figure 4. The effect of compound 5n on the protein level of MAPKs in RAW 264.7 cells.
The protein levels of MAPKs were determined using western blot assay.Data were means ± SD; ###: p < 0.001 compared with
Control group; **:0.001 <p< 0.01, ***: p < 0.001compared with LPS group.
NF-κB is an important transcription factor, which can regulate the expression of
pro-inflammatory mediators in activated macrophages.19 The NF-κB protein comprises p65 and p50
subunits. Under normal conditions, a functional NF-κB dimer is exists as a complex with the inhibitor
protein IκB in the cytosol.20 The activation of NF-κB in response to LPS stimulation leads to the
degradation of IκB, followed by the release and nuclear translocation of NF-κB. After nuclear
translocation, p65 and p50 regulate the production of TNF-α, IL-6, and mediators.21-24 Therefore, the
inhibition of NF-κB activation has been regarded as a therapeutic approach for autoimmune and
inflammatory conditions.25,26 As shown in Figure 5, the effects of the extract obtained from
compound 5n on NF-κB activation were investigated in LPS-stimulated RAW264.7 cells. The
phosphorylation levels of p65 reduced significantly, and IκB levels increased dramatically in
- 7 -

LPS-stimulated RAW264.7 cells in a dose-dependent manner. These results indicate that compound
5h inhibits the activation of NF-κB by preventing IκB degradation in LPS-stimulated RAW264.7
cells.
Figure 5. The effect of compound 5n on the protein level of NF-κB in RAW 264.7 cells.
The protein levels of NF-κB were determined using western blot assay. Data were means ± SD; ##: 0.001 <p< 0.01 compared with
Control group; *: 0.05 <p < 0.01, **: 0.001 <p< 0.01, compared with LPS group.
In recent years, a large number of scholars engaged in COX-2 receptor-related research.27-31
Naproxen is a powerful, non-selective non-steroidal anti-inflammatory drug that is extensively used
as a prescription and over-the-counter medication. It interacts with COX as reported by Duggan. et
al.32 The atomic coordinates and structure factors (codes 3NT1) have been deposited in the Protein
Data Bank. Naproxen is a relatively simple molecule with a naphthyl scaffold, similar to compound
5n. Adocking study of compound 5n into the COX-2 binding site was therefore performed. As shown
in Figure 6, there are some cavities around the naproxen molecule in the COX-2 binding pocket.
Compound 5n could shrink the cavity space, thereby enhancing the interaction force. Moreover, the
triazole ring can interact with the upper right hydrogen bond donor, in a manner equivalent to that of
the carbonyl group of naproxen acting as hydrogen bond acceptor. The detailed interaction of
compound 5n and naproxen with COX-2 is shown in Table 2. The results revealed that compound 5n
shows a stronger interaction with COX-2. This may result in a better inhibitory effect on COX-2.
- 8 -

Figure 6. The docking result of compound 5n (left) and reported naproxen pose (right) into the
COX-2 binding site.
Table 2. The interaction of compound 5n and naproxen with COX-2.
Amino
acid
residues
Distance
(Angstrom)
Distance
(Angstrom)
Types
Types
Carbon
Hydrogen
bond
2.81
SER353
-
-
3.52, 4.41,
4.67
3.93, 4.69,
5.40
4.01, 4.33,
4.46, 5.32
4.54, 4.72,
4.89, 5.24
Pi-Alkyl
VAL523
GLY526
ALA527
LEU352
TYR355
Pi-Alkyl
5.12
4.24, 4.91,
3.35
Amide-Pi
Stacked
Amide-Pi
Stacked
Compound
5h
Pi-Alkyl
Pi-Alkyl
Pi-Alkyl
Pi-Alkyl
Pi-Alkyl
Naproxen
4.64,5.46
4.99
Pi-Pi
T-shaped
Pi-Alkyl
Pi-Alkyl
Pi-Sulfur
-
5.00
5.06
5.11
5.95
-
VAL349
VAL116
MET522
LEU359
Pi-Alkyl
Alkyl
-
462, 4.94
5.24
-
-
4.46
Negative-
Negative
-
-
GLU524
5.28
- 9 -

In order to verify our speculation, the effect of compound 5n on COX-2 was measured. The
result was present in Figure 7. The expression of COX-2 has significant difference between control
group and LPS group. The value of COX-2/GAPDH was significant decrease treatment with
compound 5n. Especially, the fold of COX-2/GAPDH was reduced to 45.19 % at dose of 50 μg/mL.
It suggested that compound 5n was liked to display anti-inflammatory activity related with the site
of COX-2 as a kind of COX-2 inhibitors.
In conclusion, a series of perimidine derivatives containing triazole (5a-s) were synthesized and
their anti-inflammatory activities were evaluated using the MTT assay in a LPS-stimulated
inflammation model. Compounds 5h and 5n reduced the levels of pro-inflammatory cytokines
TNF-α and IL-6 in RAW264.7 cells. Compound 5n showed the most potent anti-inflammatory
activity with an inhibition rate of 49.26% at a dose of 50 mg/kg, which is more potent than that of the
reference drug ibuprofen (28.13%) and slightly less than that of indometacin (49.36%) in a
xylene-induced ear inflammation model. The results of western blotting showed compound 5n
inhibited NF-κB (p65) activation and MAPK (ERK and p38) phosphorylation in a dose-dependent
manner. Moreover, the results of the docking study of compound 5n into the COX-2 binding site and
COX-2 inhibitory test revealed that its mechanism was possibly similar to that of naproxen, a COX-2
inhibitor.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 81360468)
References
1. Alicia Jubert, Ne´stor E. Massa, Leonor Lo´pez Te´vez, Nora Beatriz Okulik. Vibrational and
theoretical
[2-3((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic
[[2-(2,6-dichlorophenyl)amino]-benzeneacetic acid]
studies
of
the
non-steroidal
anti-inflamatorydrugs
acid];
Niflumic
Diclofenac
acids
and
Indometacin
[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid]. Vib. Spectrosc. 2005;37,
161–178.
2. Paprocka R, Wiese M, Eljaszewicz A, Helmin-Basa A, Gzella A, Modzelewska-Banachiewicz
- 10 -

B, Michalkiewicz J. Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives.
Bioorg Med Chem Lett. 2015;25(13):2664-2667.
3. García Rodríguez LA, Cattaruzzi C, Troncon MG, Agostinis L Risk of hospitalization for upper
gastrointestinal tract bleeding associated with ketorolac, other nonsteroidal anti-inflammatory
drugs, calcium antagonists, and other antihypertensive drugs. Arch Intern Med.
1998;158(1):33-39.
4. Mekheimer RA, Sayed AA, Ahmed EA, Sadek KU. Synthesis and Characterization of New
1,2,4-Triazolo[1,5-a]pyridines That Extend the Life Span of Caenorhabiditis elegans via Their
Anti-Inflammatory/Antioxidant Effects. Arch Pharm (Weinheim). 2015;348(9):650-665.
5. Liu DC, Gong GH, Wei CX, Jin XJ, Quan ZS. Synthesis and anti-inflammatory activity
evaluation of a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide
derivatives. Bioorg Med Chem Lett. 2016;26(6):1576-1579.
6. Paprocka R, Wiese M, Eljaszewicz A, Helmin-Basa A, Gzella A, Modzelewska-Banachiewicz
B, Michalkiewicz J. Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives.
Bioorg Med Chem Lett. 2015;25(13):2664-2667.
7. Sarigol D, Uzgoren-Baran A, Tel BC, Somuncuoglu EI, Kazkayasi I, Ozadali-Sari K,
Unsal-Tan O, Okay G, Ertan M, Tozkoparan B. Novel thiazolo[3,2-b]-1,2,4-triazoles derived
from naproxen with analgesic/anti-inflammatory properties: Synthesis, biological evaluation
and molecular modeling studies. Bioorg Med Chem. 2015;23(10):2518-2528.
8. Bassyouni FA, Abu-Bakr SM, Hegab KH, El-Eraky W, El Beih, AA. Abdel Rehim ME.
Synthesis of new transition metal complexesof 1H-perimidine derivatives having
antimicrobialand anti-inflammatory activities. Res Chem Intermed. 2012;38:1527–1550.
9. Wilson MR, Choudhury S, Takata M. Pulmonary inflammation induced by high-stretch
ventilation is mediated by tumor necrosis factor signaling in mice. Am J Physiol Lung Cell Mol
Physiol. 2005;288(4):L599-607.
10. Campbell IK, Roberts LJ, Wicks IP. Molecular targets in immune-mediated diseases: the case
of tumour necrosis factor and rheumatoid arthritis. Immunol Cell Biol. 2003;81(5):354-366.
11. Spielmann S, Kerner T, Ahlers O, Keh D, Gerlach M, Gerlach H. Early detection of increased
tumour necrosis factor alpha (TNFalpha) and soluble TNF receptor protein plasma levels after
trauma reveals associations with the clinical course. Acta Anaesthesiol Scand.
- 11 -

2001;45(3):364-370.
12. Pape HC, van Griensven M, Rice J, Gänsslen A, Hildebrand F, Zech S, Winny M,
Lichtinghagen R, Krettek C. Major secondary surgery in blunt trauma patients and
perioperative cytokine liberation: determination of the clinical relevance of biochemical
markers. J Trauma. 2001;50(6):989-1000.
13. Murano M, Maemura K, Hirata I, Toshina K, Nishikawa T, Hamamoto N, Sasaki S, Saitoh O,
Katsu K. Therapeutic effect of intracolonically administered nuclear factor kappa B (p65)
antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp
Immunol. 2000;120(1):51-58.
14. Cheng J, Ma T, Liu W, Wang H, Jiang J, Wei Y, Tian H, Zou N, Zhu Y, Shi H, Cheng X, Wang
C. In in vivo evaluation of the anti-inflammatory and analgesic activities of compound
Muniziqi granule in experimental animal models. BMC Complement Altern Med. 2016;16:20.
15. Eidi A, Oryan S, Zaringhalam J, Rad M. Antinociceptive and anti-inflammatory effects of the
aerial parts of Artemisia dracunculus in mice. Pharm Biol. 2016;54(3):549-554.
16. Coskun M, Olsen J, Seidelin JB, Nielsen OH. MAPkinases in inflammatory bowel disease.
Clinica Chimica Acta, 2011;412(7–8), 513–520.
17. Kim KN, Ko YJ, Yang HM, Ham YM, Roh SW, Jeon YJ, Ahn G, Kang MC, Yoon WJ, Kim D,
Oda T. Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus
medica L. var. sarcodactylis) through blocking JNK, ERK and NF-kappa B signaling pathways
in LPS activated RAW 264.7 cells. Food Chem Toxicol. 2013;57:126–131.
18. Yang CP, Huang GJ, Huang HC, Chen YC, Chang CI, Wang SY, Chen IS, Tseng YH, Chien S C,
Kuo YH. A new butanolide compound from the aerial part of Lindera akoensis with
anti-inflammatory activity. Molecules. 2012;17(6):6585–6592.
19. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth
F R. 2002, 13(4–5);413–421.
20. Schmitz ML, Bacher S, Kracht M. I kappaB-independent control of NF-kappa B activity by
modulatory phosphorylations. Trends in Biochemical Sciences, 2001;26(3):186–190.
21. Liu F, Morris S, Epps J, Carroll R. Demonstration of an activation regulated
NF-kappaB/I-kappaBalpha complex inhuman platelets. Thromb Res. 2002;106(4–5):199–203.
22. Lappas M, Permezel M, Georgiou HM, Rice GE. Nuclear factor kappa B regulation of
- 12 -

proinflammatory cytokines in human gestational tissues in vitro. Biol Reprod.
2002;67(2):668–673.
23. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NF-kappa B/I
kappa
B
family: Intimate tales of association and dissociation. Genes Dev.
1995;9(22):2723–2735.
24. Song SM, Ham YM, Ko YJ, Ko EY, Oh DJ, Kim CS, Kim D, Kim KN, Yoon WJ.
Anti-inflammatory activities of the products of supercritical fluid extraction from Litsea
japonicafruit in RAW 264.7 cells. J Funct Foods. 2016;22:44–51.
25. Bufalo MC, Ferreira I, Costa G, Francisco V, Liberal J, Cruz MT, Lopes MC, Batista MT,
Sforcin JM. Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory
response by blocking NF-kappaB and MAPK activation in macrophages. J Ethnopharmacol.
2013;149(1):84–92.
26. Lee Y, Shin DH, Kim JH, Hong S, Choi D, Kim YJ, Kwak, MK, Jung Y. Caffeic acid
phenethyl ester-mediated Nrf2 activation and IkappaB kinase inhibition are involved in
NF-kappaB inhibitory effect: Structural analysis for NFkappaB inhibition. Eur J Pharmacol.
2010;643(1):21–28.
27. Abdel-Aziz AA, Abou-Zeid LA, ElTahir KE, Ayyad RR, El-Sayed MA, El-Azab AS. Synthesis,
anti-inflammatory, analgesic, COX-1/2 inhibitory activities and molecular docking studies of
substituted 2-mercapto-4(3H)-quinazolinones. Eur J Med Chem. 2016;121:410-421.
28. Chen L, Teng H, Fang T, Xiao J. Agrimonolide from Agrimonia pilosa suppresses inflammatory
responses through down-regulation of COX-2/iNOS and inactivation of NF-κB in
lipopolysaccharide-stimulated macrophages. Phytomedicine. 2016;23(8):846-855.
29. Cha SM, Cha JD, Jang EJ, Kim GU, Lee KY. Sophoraflavanone G prevents Streptococcus
mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting
MAPK-mediated pathways in RAW 264.7 macrophages. Arch Oral Biol. 2016;68:97-104.
30. Park JM, Han YM, Lee JS, Ko KH, Hong SP, Kim EH, Hahm KB. Nrf2-mediated
mucoprotective and anti-inflammatory actions of Artemisia extracts led to attenuate stress
related mucosal damages. J Clin Biochem Nutr. 2015;56(2):132-142.
31. Niu X, Mu Q, Li W, Yao H, Li H, Huang H. Esculentic acid, a novel and selective COX-2
inhibitor with anti-inflammatory effect in vivo and in vitro. Eur J Pharmacol.
- 13 -

2014;740:532-538.
32. Duggan KC, Walters MJ, Musee J, Harp JM, Kiefer JR, Oates JA, Marnett LJ. Molecular basis
for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen. J Biol
Chem. 2010;285:34950-34959.
- 14 -

R1 = n-C3H7
R2 = n-C4H9
R3 = n-C5H11
R5 = n-C8H17
R6 = n-C9H19
R7 = n-C10H21
R9 = -CH2C6H4(p-CH3)
R10 = -CH2C6H5
R11 = -CH2C6H4(o-Cl)
R12 = -CH2C6H4(m-Cl)
R13 = -CH2C6H4(p-Cl)
R14 = -CH2C6H4(o-F)
R15 = -CH2C6H4(m-F)
R16 = -CH2C6H4(p-F)
R17 = -CH2C6H3(2,4-Cl2)
R18 = -CH2C6H3(2,6-Cl2)
R19 = -CH2C6H4(m-CF3)
R4 = n-C6H13 R8 = -CH2C6H4(m-CH3)
33.
- 15 -