Discovery and evaluation of novel synthetic 5-alkyl-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoxaline-1-carbox-amide derivatives as anti-inflammatory agents

Article abstract of DOI:10.1080/14756366.2019.1680658

To develop novel anti-inflammatory agents, a series of 5-alkyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-carboxamide derivatives were designed, synthesised, and evaluated for anti-inflammatory effects using RAW264.7 cells. Structures of the synthesised compounds were determined using ¹H NMR, ¹³C NMR, and HRMS. All the compounds were screened for anti-inflammatory activity based on their inhibitory effects against LPS-induced NO release. Among them, 5-(3,4,5-trimethoxybenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-carboxamide (6p) showed the highest anti-inflammatory activity and inhibited NO release more potently than the lead compound D1. Further studies revealed that compound 6p reduced the levels of NO, TNF-α, and IL-6, and that its anti-inflammatory activity involves the inhibition of COX-2 and iNOS and downregulation of the mitogen-activated protein kinases (MAPK) signal pathway. Notably, compound 6p displayed more prominent anti-inflammatory activity than D1 and the positive control ibuprofen in the in vivo acute inflammatory model. Overall, these findings indicate that compound 6p is a therapeutic candidate for the treatment of inflammation.

Full text of DOI:10.1080/14756366.2019.1680658

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery and evaluation of novel synthetic
5-alkyl-4-oxo-4,5-dihydro-[1,2,4]triazolo[4,3-
a]quinoxaline-1-carbox-amide derivatives as anti-
inflammatory agents
Qing-Kun Shen, Guo-Hua Gong, Gao- Li, Mei- Jin, Li-Hua Cao & Zhe-Shan
Quan
To cite this article: Qing-Kun Shen, Guo-Hua Gong, Gao- Li, Mei- Jin, Li-Hua Cao & Zhe-
Shan Quan (2020) Discovery and evaluation of novel synthetic 5-alkyl-4-oxo-4,5-dihydro-
[1,2,4]triazolo[4,3-a]quinoxaline-1-carbox-amide derivatives as anti-inflammatory agents, Journal of
Enzyme Inhibition and Medicinal Chemistry, 35:1, 85-95, DOI: 10.1080/14756366.2019.1680658
To link to this article: https://doi.org/10.1080/14756366.2019.1680658
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 35, NO. 1, 85–95
https://doi.org/10.1080/14756366.2019.1680658
RESEARCH PAPER
Discovery and evaluation of novel synthetic 5-alkyl-4-oxo-4,5-dihydro-[1,2,4]
triazolo[4,3-a]quinoxaline-1-carbox-amide derivatives as anti-inflammatory agents
a
b,c
a
d
e‡
Qing-Kun Shen , Guo-Hua Gong , Gao- Li , Mei- Jin , Li-Hua Cao and Zhe-Shan Quana‡
ꢀ
ꢀ
aKey Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of
Pharmacy, Yanbian University, Yanji, China; Inner Mongolia Autonomous Region Key Laboratory of Mongolian Medicine Pharmacology for
b
c
Cardio-Cerebral Vascular System, Tongliao, China; Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao, China;
dDepartment of Central Laboratory, Yanbian University Hospital, Yanji, China; College of Medical, Yanbian University, Yanji, China
e
ABSTRACT
ARTICLE HISTORY
Received 22 April 2019
Revised 23 September 2019
Accepted 9 October 2019
To develop novel anti-inflammatory agents, a series of 5-alkyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]qui-
noxaline-1-carboxamide derivatives were designed, synthesised, and evaluated for anti-inflammatory
effects using RAW264.7 cells. Structures of the synthesised compounds were determined using 1H NMR,
13 C NMR, and HRMS. All the compounds were screened for anti-inflammatory activity based on their
inhibitory effects against LPS-induced NO release. Among them, 5-(3,4,5-trimethoxybenzyl)-4-oxo-4,5-dihy-
dro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-carboxamide (6p) showed the highest anti-inflammatory activity
and inhibited NO release more potently than the lead compound D1. Further studies revealed that com-
pound 6p reduced the levels of NO, TNF-a, and IL-6, and that its anti-inflammatory activity involves the
inhibition of COX-2 and iNOS and downregulation of the mitogen-activated protein kinases (MAPK) signal
pathway. Notably, compound 6p displayed more prominent anti-inflammatory activity than D1 and the
positive control ibuprofen in the in vivo acute inflammatory model. Overall, these findings indicate that
compound 6p is a therapeutic candidate for the treatment of inflammation.
KEYWORDS
Synthesis; anti-inflammatory
activity; NO; MAPKs
1. Introduction
mitogen-activated protein kinases (MAPKs) are a family of signal
transduction proteins that include extracellular signal-regulated
kinase (ERK), c-Jun N-terminal kinases (JNK), and the p38 isoform
(p38), which are involved in the classical pathways that modulate
immune-mediated inflammatory responses through the corre-
sponding signalling cascades10,11. Activated MAPKs modify the
phosphorylation of the threonine/tyrosine motifs, accelerating
iNOS, COX-2, and pro-inflammatory cytokine expression (including
NO, TNF-a, and IL-6) in the activated macrophages12–14. Thus, the
pharmacological interference or inhibition of the MAPK signalling
pathway can effectively alleviate the occurrence and development
of inflammation. Development of anti-inflammatory agents that
target the MAPK pathway might be an attractive thera-
peutic approach.
In previous studies, we presented many different derivative ser-
ies with significant anti-inflammatory activities in an in vivo
xylene-induced ear-oedema model15–18. A series of 6-substituted-
[1, 2, 4]triazolo[3,4-a]phthalazine-3-carboxamide derivatives (D)
were found to be especially effective anti-inflammatory agents in
vitro and in vivo18. Among them, compounds A115, B116, C117,
and D118 were shown to be the most active derivatives in each
series, and their structures are shown in Figure 1. Structure ana-
lysis of these derivatives revealed the following: (a) a tricyclic skel-
Inflammation is a protective host immune response to noxious
stimuli, injury, or infection1. Long-term chronic inflammation, how-
ever, can cause increased vascular permeability, fibrosis, and tissue
damage and can lead to the development of metabolic disorders
such as rheumatoid arthritis, atherosclerosis, and even cancer2,3. It
is well known that the use of chemical drugs to inhibit inflamma-
tion is an effective way to alleviate the symptoms of inflamma-
tion-related diseases4,5
. Nonsteroidal anti-inflammatory drugs
(NSAIDs) are still widely used in clinical applications today.
However, they usually cause unexpected side effects, such as pep-
tic ulcers, bleeding, mucosal lesions, and nephrotoxicity6,7. It is,
therefore, better to find novel anti-inflammatory compounds with
fewer adverse reactions.
As a congenital immune cell, macrophages play an important
role in the occurrence and development of inflammation. During
inflammation, macrophages overexpress bioactive inflammatory
mediators such as nitric oxide (NO), interleukin 6 (IL-6), and
tumour necrosis factor-alpha (TNF-a)8. Furthermore, the accumula-
tion of these inflammatory mediators can cause cell and tissue
damage and eventually chronic inflammation9. The inhibition of
cytokine release from activated macrophages is, therefore, an
important mode of action of anti-inflammatory drugs. The eton that contains a triazole ring; and (b) a triazole, 3-amino
CONTACT L.H. Cao
lhcao@ybu.edu.cn
College of Medical, Yanbian University, Yanji, Jilin 133002, China; Z. S. Quan
zsquan@ybu.edu.cn
Key Laboratory
of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin
133002, China
Supplemental data for this article can be accessed here.
ꢀ
Qing-Kun Shen and Guo-Hua Gong contributed equally to this work.
‡Li-Hua Cao and Zhe-Shan Quan are equal correspondence authors.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

86
Q-K SHEN ET AL.
Figure 1. Design of target compounds 6a–6t.
triazole, or 3-amido triazole that was assumed to be the main Yield: 93%, m.p. >300 ꢁC. 1H-NMR (DMSO-d6, 300 MHz) d:
pharmacological moiety conferring the anti-inflammatory effect. In 7.06–7.15 (m, 4H, Ar-H), 11.92 (s, 2H, –CONH–).
addition, functional group transitions and flipping are classic and
effective strategies for the rational optimisation of lead
compounds19.
2.3. Procedure for the synthesis of compound 3
As part of our continuous efforts to develop better novel anti-
A solution of compound 2 (5.0 g, 46 mmol) in hydrazine hydrate
inflammatory agents and based on the above observations, a ser-
(100 ml) was stirred at 100 ꢁC for 2 h. The mixture was cooled and
ies of 5-alkyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-1-
then filtered to obtain 3-hydrazinylquinoxalin-2(1H)-one (3) as a
yellow solid. Yield: 84%, m.p. >300 ꢁC. 1H-NMR (DMSO-d6,
carboxamide derivatives (6a–6t) were designed and synthesised
using the lead compound D and the design principles of skeleton
300 MHz) d: 4.52 (s, 2H, NH2), 7.11–7.37 (m, 4H, Ar-H), 8.37 (s, 1H,
migration and functional group flipping. Moreover, the anti-
–NH–), 12.07 (s, 1H, –CONH–).
inflammatory effects of these target compounds were evaluated
in RAW264.7 cells following lipopolysaccharide (LPS)-induced NO
production. Further, the most potent compound, 6p, with a 3,4,5-
trimethoxybenzyl moiety, was subjected to preliminary studies to
determine the mechanisms of action underlying its anti-inflamma-
tory activity by western blotting. Finally, we confirmed the anti-
inflammatory effects in vivo by using the carrageenan method.
2.4. Procedure for the synthesis of compound 4
To a suspension of compound 3 (1.3 g, 5 mmol) in diethyl oxalate
(15 ml) was stirred at reflux for 3 h. The solvent was removed
under reduced pressure to give light yellow oil. The crude residue
was purified by silica gel column chromatography (EtOAc) and
recrystallized from petroleum ether to give compound 4,
yield: 34%.
2. Materials and methods
2.1. Chemistry
2.5. General procedure for the synthesis of compound (5a–5t)
All chemicals were purchased from commercial source and used
without further purification unless otherwise stated. The reactions
were monitored by TLC using Merck Kieselgel 60 F 254 plates and
visualised under UV light at 254 nm. Column chromatography was
generally performed on silica gel (200 mesh size).1H-NMR and
13C-NMR spectra were measured on an AV-300 (Bruker BioSpin,
Switzerland) and all chemical shifts were given in ppm relative to
tetramethylsilane (TMS). High-resolution mass spectra (HRMS)
were measured with an Thermo Scientific LTQ Orbitrap XL in ESI
mode (Supplementary material).
A mixture of compound 4 (0.52 g, 2 mmol), K2CO3 (0.28 g, 2 mmol)
and all kinds of halogenated alkanes (2.4 mmol) in DMF (25 ml)
was stirred at 50 ꢁC for 3–5 h monitored by TLC and then concen-
trated under reduced pressure. The residue was added water
(50.0 ml) and extracted with dichloromethane. The organic layer
was washed with saturated NaCl and dried over anhydrous
Na2SO4. Then it can be purified by chromatography on silica elut-
ing with a gradient of methanol/dichloromethane (1:50) to obtain
the compounds 5a–5t as white solids.
2.2. Procedure for the synthesis of compound 2
2.5.1. Ethyl 5-benzyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]qui-
noxaline-1-carboxylate (5g)
A mixture of benzene-1,2-diamine (1) (10.0 g, 92 mmol), oxalic acid
(12.50 g, 139 mmol), and 30.00 ml 10% HCl in 30 ml H2O was Yield: 58.9%, m.p. 157–159 ꢁC. 1H-NMR (CDCl3, 300 MHz) d: 1.57
stirred at 100 ꢁC for 2 h. The mixture was cooled and then filtered (t, 3H, J ¼ 7.1 Hz, –CH3), 4.67 (q, 2H, J ¼ 7.1 Hz, –O–CH2–C), 5.63
to obtain 1,4-dihydroquinoxaline-2,3-dione (2) as a white solid. (s, 2H, –CH2–), 7.32–7.46 (m, 8H, Ar-H), 8.61 (d, 1H, J ¼ 8.3 Hz, Ar-H).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
87
2.6 General procedure for the synthesis of compound (6a–6t)
2.6.5. 5-octyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-
1-carboxamide (6e)
To a stirred solution of compound 5a–5t (2 mm0l) in MeOH
(10 ml) and ammonium hydroxide (10 ml) was added. The reaction
mixture was stirred at room temperature for 2 h. The mixture was
filtered and then washed with MeOH to give crude product. The
resulting solid residue was purified by silica column chromatog-
raphy (CH2Cl2/MeOH, 30:1) to give compounds 6a–6t as pale
white solids.
Yield: 30.1%, m.p. 230–232 ꢁC. IR (KBr) cmꢂ1: 3351, 3156, 1682
(C¼O), 1673(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 0.85 (s, 3H,
J ¼ 6.3 Hz, –CH3), 1.24 (s, 8H, –(CH2)4–), 1.40 (s, 2H, –CH2–),
1.58–1.74 (m, 2H, –CH2–) 4.27 (t, 2H, J ¼ 6.6 Hz, –N–CH2–), 7.39 (t,
1H, J ¼ 7.6 Hz, Ar-H), 7.58 (t, 1H, J ¼ 7.6 Hz, Ar-H), 7.69 (d, 1H,
J ¼ 8.2 Hz, Ar-H), 8.47 (t, 1H, J ¼ 8.2 Hz, Ar-H), 8.49, 8.89 (s, 2H,
–NH2). 13 C NMR (75 MHz, DMSO-d6) d: 14.40, 22.54, 26.67, 27.17,
29.09, 29.21, 31.69, 42.03, 117.07, 119.40, 121.12, 123.72, 129.09,
129.93, 144.31, 146.45, 151.78, 160.51. ESI-HRMS calculated for
C18H24N5O2 ([M þ H]þ): 342.1925; found: 342.1922.
þ
2.6.1. 5-butyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-
1-carboxamide (6a)
Yield: 46.4%, m.p. 262–264 ꢁC. IR (KBr) cmꢂ1: 3314, 3180, 1693
(C¼O), 1665 (C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 0.95
2.6.6. 5-nonyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-
(t, J ¼ 7.2 Hz, 3H, –CH3), 1.40–1.47 (m, 2H, –CH2–), 1.61–1.71 (m, 2H, 1-carboxamide (6f)
Yield: 30.1%, m.p. 236–238 ꢁC. IR (KBr) cmꢂ1: 3353, 3155, 1682
–CH2–), 4.29 (t, J ¼ 7.3 Hz, 2H, –N–CH2–), 7.41 (t, J ¼ 7.8 Hz, 1H, Ar-
H), 7.60 (t, J ¼ 7.8 Hz, 1H, Ar-H), 7.70 (d, J ¼ 8.4 Hz, 1H, Ar-H), 8.47 (d,
J ¼ 8.4 Hz, 1H, Ar-H), 8.49, 8.88 (s, 2H, –NH2). 13 C-NMR (75 MHz,
DMSO-d6) d: 14.20, 19.98, 29.28, 41.79, 117.10, 119.38, 121.13,
123.74, 129.09, 129.95, 144.31, 146.44, 151.81, 160.53. ESI-HRMS cal-
culated for C14H16N5O2þ ([M þ H]þ): 286.1299; found: 286.1302.
(C¼O), 1671(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 0.84 (m, 3H,
J ¼ 6.3 Hz, –CH3), 1.24–1.40 (m, 12H, –(CH2)6–), 1.58–1.74 (m, 2H,
–CH2–), 4.26 (t, 2H, J ¼ 6.8 Hz, –N–CH2–), 7.39 (t, 1H, J ¼ 7.6 Hz, Ar-
H), 7.58 (t, 1H, J ¼ 7.6 Hz, Ar-H), 7.69 (d, 1H, J ¼ 8.2 Hz, Ar-H), 8.47
(t, 1H, J ¼ 8.2 Hz, Ar-H), 8.49, 8.89 (s, 2H, –NH2). 13 C NMR (75 MHz,
DMSO-d6) d: 14.41, 22.55, 26.66, 27.17, 29.11, 29.26, 29.36, 31.74,
42.02, 117.07, 119.40, 121.12, 123.72, 129.08, 129.93, 144.31,
þ
146.45, 151.78, 160.51. ESI-HRMS calculated for C19H26N5O2
2.6.2. 5-pentyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxa-
line-1-carboxamide (6b)
([M þ H]þ): 356.2081; found: 356.2078.
Yield: 56.7%, m.p. 232–234 ꢁC. IR (KBr) cmꢂ1: 3354, 3162, 1680
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 0.88 (t, J ¼ 6.5 Hz, 3H,
–CH3), 1.38 (s, 4H, –(CH2)2–), 1.68 (s, 2H, –CH2–), 4.28 (t, J ¼ 6.2 Hz,
2H, –N–CH2–), 7.40 (t, J ¼ 7.8 Hz, 1H, Ar-H), 7.60 (t, J ¼ 7.8 Hz, 1H,
Ar-H), 7.70 (d, J ¼ 8.4 Hz, 1H, Ar-H), 8.47 (d, J ¼ 8.4 Hz, 1H, Ar-H),
8.49, 8.88 (s, 2H, –NH2). 13 C NMR (75 MHz, DMSO-d6) d: 14.35,
22.39, 26.88, 28.81, 42.00, 117.09, 119.39, 121.13, 123.75, 129.11,
129.96, 144.32, 146.45, 151.81, 160.53. ESI-HRMS calculated for
2.6.7. 5-benzyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxa-
line-1-carboxamide (6g)
Yield: 35.8%, m.p. 268–270 ꢁC. IR (KBr) cmꢂ1: 3345, 3157, 1682
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.57 (s, 2H, –N–CH2–),
7.36–7.45 (m, 8H, Ar-H), 8.52 (s, 2H, Ar-H, –NH), 8.92 (s, 1H, –NH).
13 C NMR (75 MHz, DMSO-d6) d: 45.42, 117.56, 119.35, 121.38,
123.96, 127.06 (2 C), 127.77, 128.85, 129.12 (2 C), 130.10, 136.07,
144.59, 146.60, 152.54, 160.53. ESI-HRMS calculated for
þ
C15H18N5O2 ([M þ H]þ): 300.1455; found: 300.1458.
þ
C17H14N5O2 ([M þ H]þ): 320.1142; found: 320.1140.
2.6.3. 5-hexyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxaline-
1-carboxamide (6c)
2.6.8. 5-(2-fluorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carboxamide (6h)
Yield: 36.4%, m.p. 239–241 ꢁC. IR (KBr) cmꢂ1: 3360, 3169, 1725
(C¼O), 1683 (C¼O). 1H-NMR (DMSO-d6, 300 MHz) d: 0.88 (t, 3H,
J ¼ 6.3 Hz, –CH3) 1.30–1.42 (m, 6H, –(CH2)3–), 1.61–1.72 (m, 2H,
–CH2–), 4.27 (t, 2H, J ¼ 7.5 Hz, –N–CH2–), 7.40 (t, J ¼ 7.8 Hz, 1H, Ar-
H), 7.59 (t, J ¼ 7.8 Hz, 1H, Ar-H), 7.71 (d, J ¼ 8.4 Hz, 1H, Ar-H), 8.46
(d, 1H, J ¼ 8.4 Hz, Ar-H), 8.50, 8.89 (s, 2H, –NH2). 13 C NMR (75 MHz,
DMSO-d6) d: 14.35, 22.49, 26.33, 27.14, 31.44, 42.04, 117.03, 119.39,
Yield: 28.2%, m.p. 273–275 ꢁC. IR (KBr) cmꢂ1: 3340, 3165, 1685
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.56 (s, 2H, –N–CH2–),
7.08–7.50 (m, 7H, Ar-H), 8.54 (m, 2H, Ar-H, –NH), 8.95 (s, 1H, –NH).
13 C NMR (75 MHz, DMSO-d6) d: 44.05, 115.90 (d, J ¼ 20.8 Hz),
117.11, 119.42, 121.45, 122.81 (d, J ¼ 14.0 Hz), 124.09, 125.10 (d,
J ¼ 2.5 Hz), 128.60 (d, J ¼ 3.5 Hz), 129.01, 129.88 (d, J ¼ 7.5 Hz),
130.08, 144.58, 146.60, 152.50, 160.33 (d, J ¼ 242.9 Hz), 160.50. ESI-
121.11, 123.71, 129.08, 129.92, 144.29, 146.46, 151.77, 160.50. ESI-
þ
HRMS calculated for C17H13FN5O2
found: 338.1044.
([M þ H]þ): 338.1048;
þ
HRMS calculated for C16H20N5O2
found: 314.1161.
([M þ H]þ): 314.1612;
2.6.9. 5–(3-fluorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1- carboxamide (6i)
2.6.4. 5-heptyl-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-a]quinoxa-
line-1-carboxamide (6d)
Yield: 32.4%, m.p. 276–278 ꢁC. IR (KBr) cmꢂ1: 3342, 3155, 1685
Yield: 32.2%, m.p. 231–233 ꢁC. IR (KBr) cmꢂ1: 3349, 3156, 1682
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 0.85 (s, 3H, J ¼ 6.3 Hz,
–CH3), 1.26–1.38 (m, 8H, –(CH2)4–), 1.67 (s, 2H, –CH2–), 4.27 (s, 2H,
–N–CH2–), 7.39–7.70 (m, 3H, Ar-H), 8.46 (s, 1H, Ar-H), 8.49, 8.89 (s,
2H, –NH2). 13 C-NMR (75 MHz, DMSO-d6) d: 14.41, 22.51, 26.64,
27.19, 28.92, 31.68, 42.03, 117.06, 119.39, 121.10, 123.72, 129.08,
129.92, 144.31, 146.45, 151.78, 160.51. ESI-HRMS calculated for
1
(C¼O). H-NMR (300 MHz, DMSO-d6) d: 5.57 (s, 2H, –N–CH2–), 7.10
(t, J ¼ 8.0 Hz, 1H, Ar-H), 7.25 (t, J ¼ 7.1 Hz, 2H, Ar-H), 7.35–7.49 (m,
4H, Ar-H), 8.51 (s, 1H, Ar-H), 8.54, 8.93 (s, 2H, –NH2). 13 C-NMR
(75 MHz, DMSO-d6) d: 45.09, 114.17 (d, J ¼ 38.6 Hz), 114.46 (d,
J ¼ 37.3 Hz), 117.37, 119.38, 121.53, 123.15, 123.97, 128.84, 130.10,
131.1 (d, J ¼ 8.2 Hz), 139.12 (d, J ¼ 7.2 Hz), 144.76, 146.55, 152.65,
160.54, 162.93 (d, J ¼ 242.1 Hz). ESI-HRMS calculated for
þ
þ
C17H22N5O2 ([M þ H]þ): 328.1768; found: 328.1765.
C17H13FN5O2 ([M þ H]þ): 338.1048; found: 338.1047.

88
Q-K SHEN ET AL.
2.6.10. 5-(4-fluorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3- –N–CH2–), 6.89 (s, 2H, Ar-H), 7.33–7.48 (m, 5H, Ar-H), 8.51 (s, 2H,
Ar-H, –NH), 8.91 (s, 1H, –NH). 13 C NMR (75 MHz, DMSO-d6) d:
44.85, 55.51, 114.51 (2 C), 117.60, 119.33, 121.33, 123.91, 127.86,
a]quinoxaline-1-carboxamide (6j)
Yield: 25.7%, m.p. 274–276 ꢁC. IR (KBr) cmꢂ1: 3342, 1685 (C¼O).
1H-NMR (300 MHz, DMSO-d6) d: 5.54 (s, 2H, –N–CH2–), 7.16 (t,
128.55 (2 C), 128.82, 130.04, 144.55, 146.59, 152.49, 158.96, 160.53.
ESI-HRMS calculated for C18H16N5O3 ([M þ H]þ): 350.1248;
J ¼ 8.8 Hz, 2H, Ar-H), 7.35-8.46 (m, 5H, Ar-H), 8.50 (s, 1H, Ar-H),
8.52, 8.91 (s, 2H, –NH2). 13 C-NMR (75 MHz, DMSO-d6) d: 44.79,
found: 350.1251.
115.88 (d, J ¼ 21.4 Hz, 2 C), 117.46, 119.37, 121.44, 123.97, 128.86,
129.27 (d, J ¼ 8.1 Hz, 2 C), 130.04, 132.24 (d, J ¼ 2.7 Hz), 144.64,
2.6.16. 5-(3,4,5-trimethoxybenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]tria-
146.57, 152.57, 160.52. 161.87 (d, J ¼ 241.7 Hz), ESI-HRMS calcu-
lated for C17H13FN5O2 ([M þ H]þ): 338.1048; found: 338.1045.
þ
zolo[4,3-a]quinoxali- ne-1-carboxamide (6p)
Yield: 50.6%, m.p. 286–288 ꢁC. IR (KBr) cmꢂ1: 3317, 1688 (C¼O),
1665(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 3.62 (s, 3H, –O–CH3),
2.6.11. 5-(2-chlorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3- 3.71 (s, 6H, –O–CH3), 5.46 (s, 2H, –N–CH2–), 6.73 (s, 2H, Ar-H),
7.39–8.50 (m, 3H, Ar-H), 8.52 (s, 1H, Ar-H), 8.54, 8.92 (s, 2H, –NH2).
13 C NMR (75 MHz, DMSO-d6) d: 45.96, 56.49 (2 C), 60.44, 104.79
(2 C), 117.57, 119.28, 121.40, 123.90, 128.84, 130.39, 131.91, 137.16,
144.77, 146.57, 152.69, 153.61 (2 C), 160.55. ESI-HRMS calculated
for C20H20N5O5 ([M þ H]þ): 410.1459; found: 410.1461.
a]quinoxaline-1-carboxamide (6k)
Yield: 31.6%, m.p. 278–280 ꢁC. IR (KBr) cmꢂ1: 3334, 3164, 1685
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.51 (s, 2H, –N–CH2–),
7.14–7.23 (m, 3H, Ar-H), 7.32–7.51 (m, 3H, Ar-H), 7.59 (d, J ¼ 7.9,
1H, Ar-H), 8.53 (s, 1H, Ar-H), 8.56, 8.95 (s, 2H, –NH2). 13 C-NMR
(75 MHz, DMSO-d6) d: 44.24, 117.11, 119.45, 121.55, 124.11, 127.81,
127.94, 129.07, 129.58, 130.00, 130.16, 132.09, 132.81, 144.70,
þ
2.6.17. 5-(2,4-dichlorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]tria-
zolo[4,3-a]quinoxaline- 1-carboxamide (6q)
146.60, 152.53, 160.51. ESI-HRMS calculated for C17H13ClN5O2
([M þ H]þ): 354.0752; found: 354.0749.
Yield: 26.3%, m.p. 266–268 ꢁC. IR (KBr) cmꢂ1: 3342, 3160, 1682
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.47 (s, 2H, –N–CH2–),
7.21–7.27 (m, 3H, Ar-H), 7.40–7.48 (m, 2H, Ar-H), 7.77 (s, 1H, Ar-H),
8.53 (s, 1H, Ar-H), 8.56, 8.96 (s, 2H, –NH2). 13 C-NMR (75 MHz,
DMSO-d6) d: 44.01, 117.06, 119.46, 121.59, 124.15, 127.98, 129.08,
129.29, 129.50, 130.09, 132.17, 133.13, 133.26, 144.73, 146.59,
152.55, 160.49. ESI-HRMS calculated for C17H12Cl2N5O2 ([M þ H]þ):
388.0363; found: 388.0367.
2.6.12. 5-(3-chlorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carboxamide (6 l)
Yield: 25.8%, m.p. 269–271 ꢁC. IR (KBr) cmꢂ1: 3338, 3158, 1685
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.56 (s, 2H, –N–CH2–),
7.36–7.51 (m, 7H, Ar-H), 8.52 (s, 1H, Ar-H), 8.54, 8.92 (s, 2H, –NH2).
13 C-NMR (75 MHz, DMSO-d6) d: 45.07, 117.36, 119.38, 121.55,
123.98, 125.82, 126.94, 127.80, 128.87, 130.14, 130.93, 133.91,
138.77, 146þ.54, 152.69, 160.53. ESI-HRMS calculated for
C17H13ClN5O2 ([M þ H]þ): 354.0752; found: 354.0748.
2.6.18. 5-(3-(Trifluoromethyl)benzyl)-4-oxo-4,5-dihydro-[1, 2, 4]tria-
zolo[4,3-a]quinox- aline-1-carboxamide (6r)
Yield: 36.7%, m.p. 262–264 ꢁC. IR (KBr) cmꢂ1: 3331, 3165, 1685
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.65 (s, 2H, –N–CH2–),
7.35–7.50 (m, 3H, Ar-H), 7.56 (d, J ¼ 7.6 Hz, 1H, Ar-H), 7.66 (t,
J ¼ 7.0 Hz, 2H, Ar-H), 7.85 (s, 1H, Ar-H), 8.52 (s, 1H, Ar-H), 8.55, 8.93
(s, 2H, –NH2). 13 C-NMR (75 MHz, DMSO-d6) d: 45.28, 117.29, 119.42,
121.55, 122.82, 124.01, 124.14, 124.57 (d, J ¼ 4.4 Hz), 128.03 (d,
J ¼ 240.5 Hz), 128.89, 129.84 (d, J ¼ 31.7 Hz), 130.14 (d, J ¼ 6.1 Hz),
131.14, 137.76, 144.81, 146.56, 152.76, 160.54. ESI-HRMS calculated
for C18H13F3N5O2 ([M þ H]þ): 388.1016; found: 388.1018.
2.6.13. 5-(4-chlorobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carboxamide (6m)
Yield: 20.3%, m.p. 282–284 ꢁC. IR (KBr) cmꢂ1: 3334, 3158, 1685
1
(C¼O). H-NMR (300 MHz, DMSO-d6) d: 5.55 (s, 2H, –N–CH2–), 7.40
(s, 7H, Ar-H), 8.52 (s, 2H, Ar-H, –NH), 8.93 (s, 1H, –NH). 13 C-NMR
(75 MHz, DMSO-d6) d: 44.88, 117.43, 119.38, 121.44, 124.01, 128.88,
129.03 (2 C), 129.10 (2 C), 130.02, 132.38, 135.17, 14þ4.64, 146.57,
152.57, 160.52. ESI-HRMS calculated for C17H13ClN5O2 ([M þ H]þ):
354.0752; found: 354.0750.
2.6.19. 5-(4-nitrobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carb- oxamide (6 s)
2.6.14. 5-(3-methoxybenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carboxamide (6n)
Yield: 25.1%, m.p. 273–275 ꢁC. IR (KBr) cmꢂ1: 3465, 3311, 1674
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.70 (s, 2H, –N–CH2–),
7.36–7.48 (m, 3H, Ar-H), 7.68 (d, J ¼ 8.4 Hz, 2H, Ar-H), 8.18 (d,
J ¼ 8.5 Hz, 2H, Ar-H), 8.52 (s, 1H, Ar-H), 8.55, 8.96 (s, 2H, –NH2).
13 C-NMR (75 MHz, DMSO-d6) d: 45.27, 117.29, 119.47, 121.54,
124.15, 128.37 (3 C), 128.94 (2 C), 130.02, 144.18, 144.71, 146.60,
Yield: 38.3%, m.p. 266–268 ꢁC. IR (KBr) cmꢂ1: 3314, 3191, 1691
(C¼O), 1668(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 3.72 (s, 3H,
–O–CH3), 5.52 (s, 2H, –N–CH2–), 6.82–6.97 (m, 3H, Ar-H), 7.23 (t,
J ¼ 8.8 Hz, 1H, Ar-H), 7.35-7.47 (m, 3H, Ar-H), 8.50 (s, 1H, Ar-H),
8.52, 8.91 (s, 2H, –NH2). 13 C-NMR (75 MHz, DMSO-d6) d: 45.43,
55.51, 112.87, 113.16, 117.56, 119.03, 119.30, 121.39, 123.94, 147.28, 152.65, 160.50. ESI-HRMS calculated for C17H13N6O4
128.84, 130.18, 130.26, 137.71, 144.62, 146.58, 152.55, 160.02, ([M þ H]þ): 365.0993; found: 365.0996.
160.53. ESI-HRMS calculated for C18H16N5O3 ([M þ H]þ): 350.1248;
found: 350.1249.
2.6.20. 5-(4-cyanobenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1-carboxamide (6t)
Yield: 23.6%, m.p. 298–300 ꢁC. IR (KBr) cmꢂ1: 3440, 3194, 1680
2.6.15. 5-(4-methoxybenzyl)-4-oxo-4,5-dihydro-[1, 2, 4]triazolo[4,3-
a]quinoxaline-1- carboamide (6o)
(C¼O). 1H-NMR (300 MHz, DMSO-d6) d: 5.65 (s, 2H, –N–CH2–),
Yield: 42.5%, m.p. 264–266 ꢁC. IR (KBr) cmꢂ1: 3340, 1682 (C¼O). 7.38–7.46 (m, 3H, Ar-H), 7.60 (d, J ¼ 7. 3 Hz, 2H, Ar-H), 7.82 (d,
1H-NMR (300 MHz, DMSO-d6) d: 3.71 (s, 3H, –O–CH3), 5.49 (s, 2H, J ¼ 7.3 Hz, 2H, Ar-H), 8.52 (s, 1H, Ar-H), 8.54, 8.94 (s, 2H, –NH2).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
89
13 C-NMR (75 MHz, DMSO-d6) d: 45.38, 110.60, 117.28, 119.19, 2.11. Western blot analysis
119.45, 121.55, 124.07, 128.10 (2 C), 128.91, 130.05, 132.99 (2 C),
142.05, 144.74, 146.58, 152.66, 160.51. ESI-HRMS calculated for
C18H13N6O2 ([M þ H]þ): 345.1095; found: 345.1097.
RAW 264.7 cells (5ꢃ 105 cells/well) plated onto 6-well plates were
incubated for 24 h and treated with 7.5, 15.0 or 30.0mM of com-
pound 6p for 30min and then stimulated with 1 mg/L of LPS for
24 h. The cells were collected and washed three times with ice-cold
PBS. The cells were treated with a cell lysis buffer [50mM Tris (pH
7.6), 150 mM NaCl, 5 mM EDTA (pH 8.0), 0.6% NP-40, 1 mM Na3VO4,
20 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride,
2 mM p-nitrophenyl phosphate, and 1:25 Complete Mini Protease
Inhibitor cocktail (Boehringer, Mannheim,Germany)] and kept on ice
for 30min. The cell lysates were centrifuged (12,000g at 4 ꢁC) for
5 min to obtain a cytosolic fraction. The protein concentration was
determined by BCA protein assay kit (Beyotime, Haimen, China).
Aliquots of the lysates were separated on 10% sodium dodecyl sul-
phate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then
electro blotted onto a polyvinylidene difluoride (PVDF) membrane.
The blots were blocked with 5% (w/v) non-fat dry milk for 2 h at
37 ꢁC, followed by incubation with specific primary antibody at 4 ꢁC
overnight. Blots were washed with Tween 20/Tris-buffered saline
[TTBS, 20 mM Tris–HCl buffer, pH 7.6, containing 137 mM NaCl and
0.05% (vol/vol) Tween 20] and incubated with a peroxidase-conju-
gated secondary antibody for 1 h. Blots were again washed with
TTBS and the immune active proteins were detected using ECL plus
(Thermo, USA). All Western blot analyses were carried out at least
three times. Results are expressed as the relative ratio of the spe-
cific band compared with the internal reference.
2.7. Cell culture and sample treatment
The RAW 264.7 mouse macrophage cell line was obtained from the
China Cell Line Bank (Beijing, China). Cells were cultured in complete
medium (DMEM supplemented with 10% heat-inactivated FBS, 3 mM
glutamine and antibiotics (100 U/mL penicillin and 100 U/mL strepto-
mycin)) at 37 ꢁC in a humidified incubator containing 5% CO2 and
95% air. Cells were treated with various concentrations of synthesised
compounds for 30 min followed by stimulation with LPS (1 mg/L).
2.8. MTT assay for cell viability
Cell viability studies induced by synthesised compounds were eval-
uated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. RAW264.7 macrophages were seeded in 96-well plates
at a density of 5 ꢃ 105 cells/mL in complete medium and incubated
for 24 h (100 mL/well). Then the cells were treated with different con-
centrations of synthesised compounds for 24 h. 150 mL MTT (5g/L in
PBS) was added to each well and the cells were further incubated
for 4 h. The supernatant was removed and the cells were lysed with
150 mL/well DMSO. The optical density was measured at 570 nm on a
microplate reader (Thermo Scientific, MA, USA).
2.12. In vivo anti-inflammatory activities determination
2.9. Assay for NO production
The animal procedures were in strict accordance with the National
Institutes of Healthy Guidelines for the Care and Use of Laboratory
Animals (NIH Publication No. 85-23, revised 1996) and were
approved by the Institutional Animal Care and Utilisation Committee
of Yanbian University. Male albino rats weighing 280–300 gwere
obtained from Animal Department of Yanbian University . They were
kept in the animal house under standard condition of light and tem-
perature with free access to food and water. The animals were ran-
domly divided into five groups (control, D1, 6p, ibuprofen and
celecoxib group) The groups with D1, 6p, ibuprofen and celecoxib
were administered oral administration (p.o.) 25 mg/kg in a vehicle of
0.5% methylcellulose, respectively. The negative control group was
treated with the same vehicle (0.5% methylcellulose). A solution of
carrageenan (0.1 ml, 1.0% w/v in 0.9% of normal saline) was injected
into the subplanter region of the left hind paw under light ether
anaesthesia 1 h after oral administration (p.o.) of the test compound
(at a dose level of 25 mg/kg body weight). The paw volume was
measured at hourly interval for 5 h (0, 1, 3 and 5 h) and the percent
inhibition of oedema was calculated using formula:
Nitrite, a stable product of nitric oxide, was used to assess NO pro-
duction. To study the effects of the isolated compounds on NO pro-
duction, RAW264.7 cells were plated at 1 ꢃ 105 cells/well in a 96-well
microplate and incubated overnight. The cells were pre-treated with
various concentrations of the test compounds (3, 7.5, 10, 15, 30 or
100 mM) and further co-cultured with 1 mg/mL LPS for 24 h. Un-pre-
treated and un-stimulated RAW 264.7 cells were conducted as the
blank control group. At different time points, the supernatant (50 mL)
was assayed with NO assay kit. The culture supernatant (50 mL) was
mixed with an equal volume of the Griess reagent (1% sulphanila-
mide, 0.1% N-[1-naphthy] ethylenediamine dihydrochloride) and NO
production were determined at 540 nm with an ELISAplate reader.
2.10. Cytokine production assay
RAW264.7 cells were seeded at 3 ꢃ 105 cells/well in a 48-well
microplate and incubated overnight.The cells were pre-treated
with the different concentrations of the test compunds (3, 7.5, 10
or 30 mM) for 30 min before the stimulation with LPS (1 mg/mL).
According to the protocol of the manufacturer, supernatants were
harvested at the 24th hour for the assay of TNF-a or IL-6 with
ELISA kits (BD Biosciences, San Diego, CA, USA). Briefly, 100 mL of
biotinylated antibody reagent and the culture supernatants were
added to anti-mouse TNF-a or IL-6 precoated 96-well plates, and
the plates were incubated for 2 h at RT. The plate was washed
with a washing buffer and subsequently incubated with 100 mL of
the streptavidin-HRP solution for 1 h at RT. The plate was washed,
and incubated with 100 mL of TMB substrate solution for 30 min at
RT in the dark. The reaction was stopped by adding 50 mL of stop
solution, and then the absorbance was measured at 450 nm by a
%Inhibition ¼ ðVt ꢂ VoÞcontrol ꢂ ðVt ꢂ VoÞtested compound
=ðVt ꢂ VoÞcontrol ꢃ 100
where,
Vo ¼ volume of oedema at zero time interval.
Vt ¼ volume of oedema at specific time interval.
2.13. Ulcerogenic effect
The lead compound D1 and the newly synthesised compound 6p
were subjected to in vivo testing to measure their ulcerogenic
plate reader. A standard curve was produced for each assay plate effect in comparison to celecoxib and ibuprofen. Male albino rats
using serially diluted recombinant IL-6. (220–250 g) divided into 5 groups (control, D1, 6p, ibuprofen and

90
Q-K SHEN ET AL.
celecoxib group) of five rats each. The groups with D1, 6p, ibu- 6a–6t18. The synthesised compounds were analysed by 1H-NMR,
13 C-NMR, and HRMS.
profen, and celecoxib were administered oral administration (p.o.)
25 mg/kg in a vehicle of 0.5% methylcellulose, respectively. The
negative control group was treated with the same vehicle (0.5%
methylcellulose). All groups were orally administered once a day
3.2. Cytotoxicity of the target compounds 6a–6t
To investigate whether the anti-inflammatory activities of the tar-
get compounds 6a–6t were related to cell viability, their cytotoxic
effects were evaluated by MTT assay in RAW264.7 cells22. As
for three consecutive days. Animals were sacrificed by diethyl
ether 6 h after the last dose and the stomach was removed. An
opening at the greater curvature was made and the stomach was
cleaned by washing with cold saline and inspected with a three- shown in Table 1, except for compounds 6i and 6j, the other
time magnifying lens for any evidence of hyperaemia, haemor-
rhage, definite haemorrhagic erosion, or ulcer. An arbitrary scale
was used to calculate the ulcer index which indicates the severity
of the stomach lesions The % ulceration for each group was calcu-
lated as follows: % Ulceration ¼ Number of animals bearing ulcer
in a group/Total number of animals in the same group ꢃ100.
compounds at concentrations of 10 or 30 mM showed no obvious
Table 1. Effect of compounds 6a–6t on the viability of RAW264.7 cells.
Cell viability (%)
Compound
R
30 lM
10 lM
blank
LPS
D1
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
–
–
–
100
100
98.60 2.12a
96.30 1.72
92.33 1.53
97.95 1.1
NTb
NT
NT
NT
NT
NT
3. Results and discussion
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
–CH2C6H5
3.1. Chemistry
99.11 0.69
81.00 1.10
99.51 2.30
60.00 3.40
85.02 1.00
66.67 1.33
59.33 2.52
48.00 3.00
46.33 2.89
51.67 2.12
44.67 3.06
93.33 0.58
80.12 1.00
83.67 1.53
56.67 1.34
99.30 0.60
80.10 1.34
90.33 1.21
The steps involved in the preparation of the target compounds
6a–6t are outlined in Scheme 1. The starting material, o-phenyl-
enediamine (1), was reacted with oxalic acid in 10% hydrochloric
acid to obtain quinoxaline-2,3(1H,4H)-dione (2) (a white needle
solid). Unlike in the previous synthesis method20, diethyl oxalate
was replaced with hydrochloric acid solution in oxalic acid, and
the white needle-like solid was directly precipitated after the reac-
tion, which not only shortened the reaction time but also made
the post-treatment easier. Compound 2 was then reacted with
hydrazine hydrate to obtain compound 3 (3-hydrazinylquinoxalin-
2(1H)-one) with only one carbonyl group substituted21, and com-
pound 4 was prepared by cyclizing compound 3 with diethyl oxal-
NT
99.15 1.65
NT
–CH2C6H4(o-F)
–CH2C6H4(m-F)
–CH2C6H4(p-F)
–CH2C6H4(o-Cl)
–CH2C6H4(m-Cl)
–CH2C6H4(p-Cl)
–CH2C6H4(m-OCH3)
–CH2C6H4(p-OCH3)
–CH2C6H2(3,4,5-(OCH3)3)
–CH2C6H3(2,4-Cl2)
–CH2C6H4(m-CF3)
–CH2C6H4(p-NO2)
–CH2C6H4(p-CN)
98.49 1.76
99.46 4.17
73.45 2.27
74.56 1.38
87.92 5.18
82.18 1.74
NT
NT
NT
84.69 6.49
NT
ate. Finally, compound
4
was reacted with appropriate
6s
6t
NT
NT
brominated alkanes and substituted with chlorobenzyl in the pres-
ence of K2CO3 to yield compounds 5a–5t, which were reacted fur-
ther with NH3ꢄH2O in methanol to obtain the target compounds
aLPS (1 lg/mL).
bNo tested.
6a:n-C4H9
6b:n-C5H11
6k:-CH2C6H4(o-Cl)
6l:-CH2C6H4(m-Cl)
6p:-CH2C6H2(3,4,5-(OCH3)3)
6f:n-C9H19
6g:-CH2C6H5
6q:-CH2C6H3(2,4-Cl2)
6r:-CH2C6H4(m-CF3)
6s:-CH2C6H4(p-NO2)
6t:-CH2C6H4(p-CN)
6c:n-C6H13
6d:n-C7H15
6e:n-C8H17
6h:-CH2C6H4(o-F)
6i:-CH2C6H4(m-F)
6j:-CH2C6H4(p-F)
6m:-CH2C6H4(p-Cl)
6n:-CH2C6H4(m-OCH3)
6o:-CH2C6H4(p-OCH3)
Scheme 1. Reagents and conditions: (a) oxalic acid, HCl/H2O, 100 ꢁC, 2 h; (b) Hydrazine hydrate, 100ꢁC, 2 h; (c) Diethyl oxalate, reflux, 3 h; (d) RX, K2CO3, DMF, 60 ꢁC,
3 h; (e) NH3ꢄH2O, methanol, r.t., 2 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
91
cytotoxic effects in RAW264.7 cells, and the relative cell viabilities compounds 6a–6t were tested based on their ability to inhibit
LPS-induced NO production in RAW264.7 macrophages. As shown
in Figure 1(A), LPS treatment caused a significant increase in NO
release compared to that in untreated controls, which was inhib-
ited after treatment with the synthetic derivatives in RAW264.7
cells. Among them, compounds 6b, 6d, 6e, 6f, 6i, 6j, 6n, 6o, 6p,
6r, and 6s exhibited similar or better NO inhibitory activity than
the lead compound D1. Compound 6p (10 mM) exhibited the
most potent inhibitory activity (38.82%) and was more potent
than D1 (a 10.31% inhibition). When the concentration of com-
pound 6p was reduced to 3 mM, it was still able to significantly
of the treated cells were more than 80%. Thus, 10 mM concentra-
tion was chosen for subsequent experiments.
3.3. Inhibition of NO production in (LPS)-stimulated RAW264.7
cells and SAR studies
High levels of NO are produced in response to LPS (1 mg/mL) in
the activated RAW264.7 macrophages23. Therefore, NO inhibitors
have been identified as good options for the treatment of inflam-
matory diseases24. The anti-inflammatory activities of the target inhibit NO production (p < .001) (Figure 2(A)).
Figure 2. RAW264.7 cells were pre-cultured for 24 h, the cells were then treated with the indicated concentrations of compounds for 30 min, and then exposed to
1 lg/mL LPS for 24h. The levels of NO in the culture medium were measured with NO assay kit. (A) Cells were treated with 10 lM compounds. Cells were pre-treated
with different concentrations of compound 6p. NO, TNF-a and IL-6 levels in the medium were determined with an ELISA kit. (B, C and D). ### p < .01, vs. Control.
ꢀ
ꢀꢀ
ꢀꢀꢀ
p < .001 vs. LPS alone.
p < .05, p < .01,

92
Q-K SHEN ET AL.
Figure 3. Compound 6p inhibited LPS-induced inflammatory response in RAW 264.7 cells. Cells were pre-treated with different concentrations of 6p (7.5–30 mM) and
Celecoxib (7.5 mM) for 24 h. iNOS, COX-2 and GAPDH proteins expression were detected by Western blot analysis. Total cellular proteins were prepared and analysed
ꢀ
ꢀꢀ
ꢀꢀꢀ
p < .001 vs. LPS alone.
by Western blotting. Results are the mean SD, n ¼ 3. ### p < .001, vs. Control. p < .05, p < .01,
The following structure-activity relationships (SAR) of the syn- As shown in Figure 2(B,C), LPS stimulation significantly elevated
thetic compounds were determined: the length of the alkyl chain the production of TNF-a and IL-6. After treatment with compound
6p, the LPS-induced increase in IL-6 and TNF-a levels was inhib-
ited in a concentration-dependent manner. Even at low concentra-
tions (3 mM), 6p significantly inhibited the production of these two
inflammatory factors (p < .001).
appeared to have a direct impact on the anti-inflammatory activity
of the 5-alkyl derivatives 6a–6f. As the alkyl chain length of the
derivatives 6a–6e increased, their anti-inflammatory activities
increased, with compound 6e (n-octyl substitution) being the
most active. When the length of the linear alkyl group reached 9
carbon atoms (compound 6f), the anti-inflammatory activity
began to decrease. We hypothesised that the n-octyl-substituted
compound, 6e, has the most favourable exponential partition
coefficient and easily passes through the cell membrane. For the
benzyloxy-substituted derivatives 6g–6t, the type and position of
the substituent on the benzene ring had a significant effect on
anti-inflammatory activity. The activity order was 3,4,5-OCH3>m-
OCH3>p-OCH3>m-F>p-F>m-CF3>p-NO2>p-CN, indicating that
the electron-donating group exerts a greater effect than the elec-
tron-withdrawing group in increasing the anti-inflammatory activ-
ities of these derivatives.
3.5. Compound 6p inhibited the expression of iNOS and COX-2
The pro-inflammatory mediator NO plays an important role in
inflammation-related diseases and its production is closely related
to the modulation of iNOS and COX-2 expression2. Our results
show that compound 6p can significantly inhibit the production
of NO. To establish whether this inhibitory effect was associated
with the reduced expression of iNOS and COX-2 in LPS-induced
RAW 264.7 cells, the levels of these proteins were measured by
western blotting after exposure to LPS for 24 h in the presence or
absence of compound 6p (7.5, 15, and 30 mM). Celecoxib (7.5 mM)
was used as a positive control. As shown in Figure 3, LPS (1 mg/
mL) stimulation markedly increased COX-2 and iNOS protein
expression and compound 6p inhibited this response (p < .001) by
53.44% (COX-2) at 15 mM and 39.89% (iNOS) at 30 mM, respectively.
Moreover, compound 6p inhibited iNOS protein expression in
LPS-induced cells in a dose-dependent manner. These results sug-
gest that 6p exerts its anti-inflammatory activity through inhibit-
ing the expression of iNOS, and COX-2 by LPS in macrophages.
3.4. Compound 6p inhibited IL-6 and TNF-a production in
RAW264.7 cells
Based on the initial screening results of these synthetic deriva-
tives, compound 6p was chosen for further assessment of its anti-
inflammatory activity in LPS-induced macrophages. It is well
known that the pro-inflammatory mediators IL-6 and TNF-a play
important roles in the development of inflammation-related dis-
eases25. To measure the effects of compound 6p on LPS-induced
IL-6 and TNF-a production, RAW264.7 cells were cultured with LPS
3.6. Compound 6p inhibited MAPK signalling activation
(1 mg/mL) in the presence of compound 6p for 24 h, and the levels As upstream proteins, phosphorylation of the MAPK family of pro-
of IL-6 and TNF-a in the supernatant were determined by ELISA. teins is closely related to and plays an important role in the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
93
Figure 4. Compound 6p inhibited LPS-induced inflammatory response in RAW 264.7 cells. Cells were pre-treated with different concentrations of 6p (7.5–30 mM) and
Celecoxib (7.5 mM) for 24 h. The levels of p-p38/P38, p-ERK/ERK, p-JNK/JNK and GAPDH proteins, and their phosphorylated forms were analysed using Western blotting.
ꢀ
ꢀꢀ
ꢀꢀꢀ
p < .001
Total cellular proteins were prepared and analysed by Western blotting. Results are the mean SD, n ¼ 3. ### p < .001, vs. Control. p < .05, p < .01,
vs. LPS alone.
expression of iNOS and COX-2, and the release of inflammatory 3.7. In vivo anti-inflammatory activity
factors in LPS-induced inflammatory responses13,14. To find the
To evaluate the anti-inflammatory activity of compound 6p in
vivo, a carrageenan-induced mouse paw oedema model was
used10,26. Each test compound (lead compound D1, 6p, and the
positive controls ibuprofen and celecoxib) was administered orally
at a dose of 25 mg/kg body weight prior to the induction of
inflammation by carrageenan (0.1 ml) injection. The paw volume
was measured over 5 h (at 0, 1, 3, and 5 h) and the percent inhib-
ition of oedema was calculated (Table 2). Inhibition of paw swel-
ling was the highest in mice 3 h after carrageenan administration
by all the tested compounds. It is noteworthy that compound 6p
displayed anti-inflammatory effects (50.83%) that were significantly
greater than those of the lead compound D1 (42.81%, p < .01)
and ibuprofen (39.30%, p < .01), but slightly lesser than those of
celecoxib (58.34%, p < .05) (Figure 5).
molecular target of compound 6p further upstream of the MAPK
signalling pathway, the effects of compound 6p on the LPS-
induced phosphorylation of ERK, JNK, and p38 MAPKs in RAW
264.7 cells were assessed by western blotting. Celecoxib (7.5 mM)
was used as positive control. As expected (Figure 4), phosphoryl-
ation of p38, JNK, and ERK increased after LPS stimulation.
Compound 6p markedly diminished the phosphorylation of JNK
(p < .001), ERK (p < .001) and p38 (p < .01) MAPKs at 30 mM (Figure
4(A–C)). Furthermore, compound 6p (7.5, 15, and 30 mM) inhibited
the LPS-induced phosphorylation of JNK and ERK in a concentra-
tion-dependent manner. These results suggest that the anti-
inflammatory effect of compound 6p might be associated with
the inhibitory effects on the MAPK signalling pathways.

94
Q-K SHEN ET AL.
Table 2. The anti-inflammatory activity and ulcerogenic activity in vivo.
Funding
% of oedema inhibition (% mean SD)a
This work was supported by the National Natural Science
Foundation of China (No. 21662036, 81660837, 81760627,
81660579) and Science and Technology Planning Projects from
the Education Commission of Jilin Province of China
(JJKH20191136KJ).
Compounds
1 h
3 h
5 h
% ulceration
D1
6p
Ibuprofen
Celecoxib
38.26 2.82
37.01 8.45
31.92 5.76
48.09 5.33
42.81 2.11
50.83 3.47
39.30 4.86
58.34 4.08
28.71 2.92
38.24 6.04
25.29 3.43
41.92 2.05
20
20
100
0
aValues are expressed as the mean S.D (n ¼ 4).
Control: 0.5% sodium CMC solution in distilled water (25 ml/kg, p.o.).
Compounds and positive drugs were administered at a dose of 25 mg/kg, p.o. in
0.5% sodium CMC solution.
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Disclosure statement
15. Pan FJ, Wang SB, Liu DA, et al. Synthesis of 4-phenyl-
thieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidine-5(4H)-one
The authors declare no conflict of interest.

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