Synthesis and evaluation of ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives as anti-inflammatory agents

Article abstract of DOI:10.1007/s11030-020-10154-7

Here, two series of novel ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives were synthesized and screened for their anti-inflammatory activity by evaluating their inhibition effect of using LPS-induced inflammatory response in RAW 264.7 macrophages in vitro; the effects of different concentrations of the compounds on the secretion of nitric oxide (NO) and inflammatory cytokines including TNF-α and IL-6 were evaluated. Their toxicity was also assessed in vitro. Results showed that the most prominent compound 3 could significantly decrease production of the above inflammatory factors. Docking study was performed for the representative compounds 3, UA, and Celecoxib to explain their interaction with cyclooxygenase-2 (COX-2) receptor active site. In vitro enzyme study implied that compound 3 exerted its anti-inflammatory activity through COX-2 inhibition.

Full text of DOI:10.1007/s11030-020-10154-7

Molecular Diversity
https://doi.org/10.1007/s11030-020-10154-7
ORIGINAL ARTICLE
Synthesis and evaluation of ursolic acid‑based 1,2,4‑triazolo[1,5‑a]
pyrimidines derivatives as anti‑infammatory agents
Tian‑Yi Zhang1 · Chun‑Shi Li2 · Ping Li1 · Xue‑Qian Bai1 · Shu‑Ying Guo1 · Ying Jin1 · Sheng‑Jun Piao3
Received: 4 September 2020 / Accepted: 29 October 2020
© Springer Nature Switzerland AG 2020
Abstract
Here, two series of novel ursolic acid-based 1,2,4-triazolo[1,5-a]pyrimidines derivatives were synthesized and screened for
their anti-infammatory activity by evaluating their inhibition efect of using LPS-induced infammatory response in RAW
264.7 macrophages in vitro; the efects of diferent concentrations of the compounds on the secretion of nitric oxide (NO)
and infammatory cytokines including TNF-α and IL-6 were evaluated. Their toxicity was also assessed in vitro. Results
showed that the most prominent compound 3 could signifcantly decrease production of the above infammatory factors.
Docking study was performed for the representative compounds 3, UA, and Celecoxib to explain their interaction with
cyclooxygenase-2 (COX-2) receptor active site. In vitro enzyme study implied that compound 3 exerted its anti-infammatory
activity through COX-2 inhibition.
Keywords Ursolic acid · Anti-infammatory activity · Molecular modeling · COX-2 inhibition
Introduction
contribute to the pathogenesis of chronic infammatory and
autoimmune diseases such as rheumatoid arthritis [4], car-
diovascular diseases [5], CNS neurological disorders [6],
and ulcerative colitis [7]. Management of infammatory
disorders involves the use of therapeutic agents for reliev-
ing pain and reducing infammation, either decreasing or
neutralizing the levels of pro-infammatory mediators or
inhibiting the recruitment of leukocytes and their activa-
tion. Nonsteroidal anti-infammatory drugs (NSAIDs) are
still widely used in clinical applications today. However,
they usually cause unexpected side efects, such as peptic
ulcers, bleeding, mucosal lesions, and nephrotoxicity [8, 9].
Traditional NSAIDs exhibit pharmacological action through
the inhibition of cyclooxygenase (COX) activity in vivo and
the reduction in the biosynthesis of prostaglandins in local
tissues.
The inflammatory process is an immune response to a
perturbation of the homeostatic equilibrium of the organ-
ism, whether endogenous or exogenous [1–3]. Infamma-
tion can lead to further tissue damage that can eventually
Tian-Yi Zhang and Chun-Shi Li have contributed equally to this
work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10154-7) contains
supplementary material, which is available to authorized users.
* Tian-Yi Zhang
tianyizhang@126.com
* Ying Jin
Pro-infammatory cytokines also play an important role
in the defense of disease [10, 11]. However, uncontrolled
and excess release of pro-infammatory mediators such as
NO, tumor necrosis factor alpha (TNF-α), and interleukin-6
(IL-6) can lead to multiple types of infammatory diseases
such as acute lung injury (ALI) [12–15].
jinying73@126.com
* Sheng-Jun Piao
sjpiao8478@163.com
1
Jilin Medical University, Jilin 132013, Jilin Province,
People’s Republic of China
2
The Third People’s Hospital of Dalian, Dalian 116000,
Ursolic acid (UA) is a lipophilic pentacyclic triterpenoid
found in a wide variety of plants and possesses a wide range
of biological functions, such as anti-infammation [16], anti-
oxidation [17], and anti-fbrosis [18]. On the other hand,
Liaoning Province, People’s Republic of China
3
Department of General Surgery, Afliated Hospital
of Yanbian University, Yanji 133000, Jilin Province,
People’s Republic of China
Vol.:(0123456789)
1 3

Molecular Diversity
1,2,4-triazolo[1,5-a] pyrimidines (TPs), a subtype of purine
analogs, have been widely investigated and identifed to pos-
sess diverse pharmacological properties. Huang et al. [19]
showed that the steroidal containing 1,2,4-triazolo[1,5-a]
pyrimidine moiety resulted in an enhancement of its anti-
cancer activity, as exemplified by compounds A and B
(Fig. 1). In the previous study, our group has designed and
synthesized several efective ursolic acid derivatives bear-
ing a 4-phenyl-1H-1,2,4-triazol-5(4H)-one or aminoguani-
dine moiety. Among them, compounds C and D (Fig. 1),
which displayed the most potent anti-infammatory activity
of all of the compounds prepared, with 69.76% and 81.61%
inhibition after intraperitoneal administration, respectively,
were more potent than the reference drugs. In view of the
pharmacological importance of ursolic acid and triazolo[1,5-
a] pyrimidine functional group and in continuation of our
previous work in developing new anti-infammatory agents,
a total of 23 target compounds were prepared (Fig. 2) and
evaluated for their anti-infammatory activity in vitro. Sub-
sequently, the preliminary action mechanism was also inves-
tigated in the present work.
Results and discussion
Chemistry
The synthetic pathway used for preparation of the target
compounds 1–23 is outlined in Scheme 1. Intermediate Z1
was prepared by reacting UA with Jones reagent in acetone.
Compound Z2 was synthesized by Claisen–Schmidt conden-
sations between compound Z1 and diferent aldehydes. Prep-
aration of Z3 was done according to a previously described
method [20]. The target compounds 1–23 were subjected to
aza-Michael addition reaction and intramolecular cycliza-
tion reaction with 3-amino-1,2,4-triazole subsequently in
Fig. 1 Previously reported UA
A
B
and TPs derivatives
C
D
Fig. 2 Synthesis of target compounds
1 3

Molecular Diversity
Scheme 1 Synthesis of the target compounds 1–23. a Jones reagent, 0 °C, 5 h, 90%. b Aldehydes, 5% NaOH, ethanol, r.t, 2 h, 40–70%. c
3-amino-1,2,4-triazole, n-BuOH, refux, 50–70%. d Iodoethane, K2CO3, DMF, r.t., 6 h, 40–60%
the presence of t-BuOK [19]. The fnal compounds were
was conducted to avoid inaccuracies arising from this fac-
tor. To investigate the relation between anti-infammatory
activity and cell viability for the compounds 1–23 and 24
(UA), the cytotoxic efects of these compounds were evalu-
ated by MTT assay in RAW 264.7 cells. As presented in
Fig. 3, most of the compounds showed low toxicity at
30 μM. Compounds 1–6, 9, 10, 12, 15, and 18–23 show
weak cytotoxicity, which were lower than that of ursolic
acid. Therefore, these compounds were found to be suitable
for further evaluation.
1
characterized by H NMR, 13C NMR, mass spectroscopy,
and HRMS.
Anti‑infammatory activity
The anti-infammatory activity of the title compounds was
tested applying a LPS-stimulated inflammation model.
Because induced macrophages’ cells death, if any occurs,
would lead to a reduction in the measured production of
cytokines (TNF-α and IL-6) in addition to nitric oxide
regardless of the inhibition of activity, cell viability assay
Nitric oxide (NO) is a cell-signaling molecule that
produces anti-inflammatory effects under normal
Fig. 3 Cellular viability of compounds 1–24. Data are shown as the mean SD (n=3) of three independent experiments
1 3

Molecular Diversity
physiological conditions. High levels of NO are produced
in response to LPS in the activated RAW 264.7 mac-
rophages [21]. Therefore, NO inhibitors have been identi-
fed as potential agents for the treatment of infammatory
diseases [22]. In view of this, the inhibitory efects of
compounds 1–6, 9, 10, 12, 15, and 18–23 on LPS-induced
production of the infammatory mediators NO in RAW
264.7 cells were examined. Three diferent concentrations
(3 μM, 10 μM, and 30 μM) of these compounds were used
for the study. Dexamethasone (DXMS) was used as a posi-
tive control. One-way ANOVA followed by Dunnett’s mul-
tiple comparison test was used to detect the signifcance.
As shown in Fig. 4, the results demonstrated that com-
pounds 2 and 3 could signifcantly inhibit NO production
at 3 μM, 10 μM, and 30 μM concentration levels in a dose-
dependent manner. Compounds 2 and 3 showed signifcant
NO production inhibitory efects at 30 μM.
indicate the potential anti-infammatory properties of the
prepared compounds.
These studies revealed that the incorporation of a
1,2,4-triazolo[1,5-a]pyrimidines moiety to UA could provide
unexpected improvement in the anti-infammatory activity of
UA derivatives. The previous study [20] showed that signif-
cant further improvement in the anti-infammatory activity
was observed while retaining the carboxylic acid functional
group at the 17 position. Similar results were seen in our
study, where compounds 3, 5, 9, and 15 had more potent
IL-6 inhibitory efects than compounds 18–23, which had
an ethyl group that was introduced at the 17-COOH posi-
tion. However, there was no consistent trend observed for
TNF-α inhibition by this class of compounds. The introduc-
tion of diferent substituents (diferently substituted benzene
rings, a naphthalene ring, and a heterocycle) to the C-2 of
UA increased the anti-infammatory activity. Furthermore,
the replacement of the benzene rings derivatives at the
p-position displayed higher potency, as exemplifed by the
compounds 2, 3, 5, and 15. No clear pattern was found for
the structure/activity relationship between the anti-infam-
matory activity and physicochemical properties of diferent
substituents on the phenyl ring, indicating that the electronic
efect of the substituent on the benzene ring is not critical.
Based on the above, compound 3 was found to be the most
prominent one. Thus, compound 3 was used as the main
compound for mechanism exploration of action.
TNF-α has a wide range of biological activities, par-
ticipates in infammation and anti-tumor activities, and
engages in the pathological process of endotoxin shock
[23]. IL-6 is produced after TNF-α stimulation and is
closely related to the pathological and physiological pro-
cesses of various diseases [24]. Compounds 1–6, 9, 10,
12, 15, and 18–23 were also evaluated for their activity
against LPS-induced production of other infammatory
mediators, TNF-α and IL-6 in RAW 264.7 cells. Three dif-
ferent concentrations (3 μM, 10 μM, and 30 μM) of these
compounds were measured. Dexamethasone (DXMS) was
used as a positive control. As shown in Fig. 5, the results
demonstrated that compounds 3, 5, 10, 18, 19, 21, and 22
showed signifcant TNF-α production inhibitory efects
at 30 μM. Compounds 3, 5, 9, and 15 showed signifcant
IL-6 production inhibitory efects at 30 μM. These results
Molecular docking
Wei et al. [25] reported that the UA derivatives exhibited
high afnity for the COX-2 active site and possibly exhibit
their anti-inflammatory potency via inhibiting COX-2
Fig. 4 The NO inhibitory efects of compounds 1–6, 9, 10, 12, 15, and 18–23. Data are shown as the mean SD (n=3) of three independent
experiments. *p<0.05, **p<0.01, ***p<0.001, signifcant with respect to the control
1 3

Molecular Diversity
Fig. 5 The TNF-α and IL-6 inhibitory efects of compounds 1–6, 9, 10, 12, 15, and 18–23. Data are shown as the mean SD (n=3) of three
independent experiments. *p<0.05, **p<0.01, ***p<0.001, signifcant with respect to the control
enzymes. The molecular docking analysis of compound
3, UA, and Celecoxib was carried out to elucidate their
anti-infammatory activity in vitro. The scoring functions
and hydrogen bonds formed with the surrounding amino
acids were used to explore the binding modes, binding
affinities, and orientations of the docked compounds at
the active site of COX-2 enzyme. The crystal structure of
the COX-2 enzyme obtained from the protein data bank
(PDB ID: 5FDQ) was used for the molecular docking
studies. The binding mode and the protein–ligand interac-
tions of compound 3, UA, and Celecoxib are depicted in
Fig. 6. The binding mode of the most active compound 3
simulates certain key interactions with the reference drug
Celecoxib. The result shows that compound 3 may bind
well to the COX-2 protein and while interacting with
Val444 (4.33 Å and 3.95 Å), Val447 (5.35 Å and 4.51 Å),
Ala202 (6.48 Å), Tyr385 (4.58 Å), and His207 (5.19 Å and
5.35 Å), respectively. UA showed interaction with Val447
(5.43 Å and 4.20 Å), Trp387 (2.01 Å), Tyr385 (4.49 Å),
His388 (4.46 Å), His386 (4.64 Å) and His207 (5.28 Å and
4.39 Å), respectively. Furthermore, compound 3 presented
better anti-infammatory activity and showed higher dock-
ing scores (145.52 kcal/mol) compared to UA (106.12 kcal/
mol). The results of preliminary docking studies suggested
that compound 3 possibly shows its anti-infammatory activ-
ity through the interaction with COX-2 protein by targeting
residues in the active cavities of COX-2.
1 3

Molecular Diversity
Fig. 6 The docked poses
of compounds 3, UA, and
Celecoxib into COX-2 enzyme
(PDB ID: 5FDQ) binding site.
a Compound 3 in binding site
of 5FDQ. b Docking study
of compound 3 with COX-2
complex (3D plot). c Dock-
ing study of compound 3 with
COX-2 complex (2D plot). d
Docked conformation of the
most active compound 3 in
COX-2. e Compound UA in
binding site of 5FDQ. f Dock-
ing study of compound UA with
COX-2 complex (3D plot). g
Docking study of compound 3
with COX-2 complex (2D plot).
(H) Docked conformation of the
most active compound UA in
COX-2. i Celecoxib in binding
site of 5FDQ. j Docking study
of Celecoxib with COX-2 com-
plex (2D plot)
1 3

Molecular Diversity
anti-infammatory potency via inhibiting COX-2 enzyme.
The COX-2 inhibitory activity of compound 3 was slightly
lower in magnitude compared to the reference drug
Celecoxib. Combination of all the results obtained in the
present study establishes the therapeutic potential of com-
pound 3 and presents it as a potential anti-infammatory lead.
COX‑2 enzymatic activity
According to the docking study, the inhibitory efect on
COX-2 enzyme produced by the compound 3 represents a
possible mechanism of action for their strong anti-infamma-
tory activity. In order to verify our speculation, the efect of
compound 3 on COX-2 enzyme was evaluated. The produc-
tion of COX-2 was determined by enzyme-linked immu-
nosorbent assay (ELISA) [26]. Celecoxib and UA were
used as a positive control. Five diferent concentrations
(0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM) of compounds 3,
Celecoxib, and UA were measured. As shown in Fig. 7, the
results showed a dose-dependent decrease in COX-2 pro-
duction in the presence of compounds 3, Celecoxib, and
UA. The 10 µM concentration of compound 3 decreased the
COX-2 activity by 54% when compared with the negative
control. The COX-2 inhibitory activity of compound 3 was
slightly lower than the activity of reference drug Celecoxib.
To sum up, in vitro enzyme study implied that compound
3 exerted its anti-infammatory activity through COX-2
enzyme inhibition.
Experimental section
Silica gel thin-layer chromatography (TLC) plates (Qing
Dao Marine Chemical Factory, Qingdao, China) were
used to monitor the reaction progress. Developed plates
were examined with UV lamps at wavelengths of 254 nm.
Nuclear magnetic resonance spectra were measured on an
AV-300 or AV-500 spectrometer (Bruker, Zurich, Switzer-
1
land) operating at 300 MHz for H and 126 MHz for 13C
NMR while using TMS as the internal standard. Mass spec-
tra were measured on an MALDI-TOF (Shimadzu, Japan).
HRMS was measured on a Thermo Scientifc LTQ Orbitrap
XL spectrometer. All commercial chemicals were used as
supplied unless otherwise indicated.
Conclusions
General synthetic procedure for the intermediates
Z1, Z2, Z3, and Z4
In this report, two series of novel ursolic acid-based
1,2,4-triazolo[1,5-a]pyrimidines derivatives were synthe-
sized and evaluated for their anti-infammatory activity.
Compound 3 exerted anti-infammatory activity through
suppressing of the expression of pro-inflammatory fac-
tors, including NO, TNF-α, and IL-6 in LPS-stimulated
RAW 264.7 macrophages. The molecular docking study
results indicated that the compound 3 exhibited high afn-
ity for the COX-2 active site and possibly exhibited its
Intermediates Z1, Z2, Z3, and Z4 were synthesized using the
reported procedure [20].
General synthetic procedure for the target
compounds 1–23
The intermediate Z2 or Z4 (1.0 mmol) was dissolved in
n-BuOH (20 mL). To the solution was added 3-amino-1,2,4-
triazole (4.0 mmol) and t-BuOK (4.0 mmol). The resulting
mixture was refuxed for about 24 h. After removal of the
solvent, the residue was purifed by silica gel chromatogra-
phy using dichloromethane/methanol (15/1) as the eluent to
give the target compounds 1–23.
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑Bromophenyl)‑1,2,6a,6b,9
,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17
,18b‑hexadecahydrochryseno[1,2‑g][1, 2, 4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (1) White solid. Yield
1
48%, m.p. 185–187 °C. H NMR (300 MHz, CDCl3) δ
9.97 (s, 1H), 7.54 (d, J=4.2 Hz, 1H), 7.35 (s, 1H), 7.07 (d,
J=7.1 Hz, 1H), 6.91 (s, 1H), 5.55 (d, J=14.2 Hz, 1H), 5.30
(s, 1H), 2.19–0.64 (m, 42H, protons in UA skeleton). 13
C
NMR (126 MHz, CDCl3) δ 175.67, 149.58, 148.58, 142.19,
138.16, 134.90, 131.61, 130.90, 130.16, 125.36, 122.84,
63.18, 53.42, 50.86, 48.01, 42.07, 39.43, 35.72, 29.71,
Fig. 7 Inhibition of COX-2 activities of compounds 3, UA, and
Celecoxib. Data are shown as the mean SD (n=3) of three inde-
pendent experiments. *p<0.05, **p<0.01, signifcant with respect to
the control
1 3

Molecular Diversity
27.81, 23.20, 20.60, 18.67, 16.64, 14.96. MS (MALDI-TOF)
(d, J=8.2 Hz, 1H), 5.59 (s, 1H), 5.19 (s, 1H), 2.73–0.57 (m,
42H, protons in UA skeleton). 13C NMR (126 MHz, CDCl3)
δ 172.30, 138.96, 138.10, 134.42, 132.42, 131.75, 131.13,
130.97, 129.27, 125.34, 122.52, 40.62, 40.52, 39.39, 38.84,
35.99, 35.68, 32.21, 29.71, 28.61, 23.96, 23.55, 23.18,
21.15, 20.48, 16.93, 16.65, 14.97. MS (MALDI-TOF) m/z
685 (M+ +H).
m/z 685 (M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(4‑Fluorophenyl)‑1,2,6a,6b,9
,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (2) White solid. Yield
52%, m.p. 140–142 °C. 1H NMR (300 MHz, CDCl3) δ 8.39
(d, J=12.0 Hz, 1H), 7.56 (s, 1H), 7.28 (s, 1H), 7.17 (s, 1H),
6.95 (s, 1H), 5.62 (s, 1H), 5.22 (d, J=10.2 Hz, 1H), 2.71–
0.64 (m, 42H, protons in UA skeleton). 13C NMR (126 MHz,
CDCl3) δ 170.27, 129.36, 125.37, 125.25, 125.12, 124.90,
116.56, 116.25, 115.74, 115.64, 115.46, 48.00, 42.42, 42.14,
39.49, 39.19, 38.87, 36.77, 35.95, 35.74, 32.39, 30.70,
29.72, 29.41,29.26, 29.08, 28.61, 28.42, 28.09, 24.25, 23.48,
23.17, 21.16, 20.45, 18.67, 15.02, 14.18. MS (MALDI-TOF)
m/z 625 (M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(2‑Bromophenyl)‑1,2,6a,6b,9
,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (6) White solid. Yield
42%, m.p. 177–179 °C. 1H NMR (300 MHz, CDCl3) δ 10.38
(s, 1H), 7.75 (s, 1H), 7.57 (s, 1H), 7.26 (s, 1H), 6.96 (d,
J = 6.9 Hz, 1H), 6.36 (s, 1H), 5.19 (s, 1H), 2.69–0.55 (m,
42H, protons in UA skeleton). 13C NMR (126 MHz, CDCl3)
δ 178.60, 150.16, 149.86, 148.77, 148.42, 139.45, 139.33,
138.08, 138.03, 134.74, 134.55, 132.60, 132.53, 129.83,
129.72, 125.52, 125.43, 53.41, 50.85, 48.01, 45.80, 39.04,
38.87, 36.00, 35.62, 29.71, 28.73, 27.81, 27.68, 14.91. MS
(MALDI-TOF) m/z 686 (M+ +2).
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑(p‑tolyl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17,18b
‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]quina‑
zoline‑4a(2H)‑carboxylic acid (3) White solid. Yield 52%,
m.p. 194–196 °C. 1H NMR (300 MHz, CDCl3) δ 10.99 (s,
1H), 8.39 (s, 1H), 7.42 (s, 2H), 7.17 (d, J=8.1 Hz, 1H), 7.11
(d, J=8.0 Hz, 1H), 5.58 (s, 1H), 2.50 (s, 3H), 2.77–0.80 (m,
42H, protons in UA skeleton). 13C NMR (126 MHz, CDCl3)
δ 183.01, 174.39, 154.09, 145.79, 141.13, 138.33, 129.81,
129.19, 125.87, 125.11, 117.32, 53.09, 52.71, 47.98, 45.29,
42.21, 41.85, 41.47, 40.27, 39.44, 39.07, 38.82, 36.67, 36.12,
32.32, 32.22, 27.96, 25.12, 23.49, 23.29, 21.66, 20.27. MS
(MALDI-TOF) m/z 621 (M+ +H). HRMS (ESI) m/z calcd
for C40H53N4O2+ (M+H)+ 621.41630, found 621.41589.
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑(thiophen‑2‑yl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b
,17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (7) White solid. Yield
54%, m.p. 211–213 °C. 1H NMR (300 MHz, CDCl3) δ 9.79
(s, 1H), 7.54 (s, 1H), 7.14 (s, 1H), 7.04 (d, J=3.0 Hz, 1H),
5.94 (s, 1H), 5.20 (s, 1H), 2.46–0.75 (m, 42H, protons in UA
skeleton). 13C NMR (126 MHz, CDCl3) δ 172.40, 148.04,
147.82, 143.59, 138.13, 134.55, 133.11, 130.56, 127.20,
127.01, 126.55, 126.19, 125.45, 53.41, 50.82, 48.02, 45.98,
42.15, 40.86, 39.48, 38.88, 36.01, 35.82, 32.31, 30.67,
28.64, 19.64, 16.95, 15.33. MS (MALDI-TOF) m/z 613
(M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(2‑Fluorophenyl)‑1,2,6a,6b,9
,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (4) White solid. Yield
46%, m.p. 164–166 °C. 1H NMR (300 MHz, CDCl3) δ 10.33
(s, 1H), 7.57 (s, 1H), 7.26 (s, 1H), 7.12 (s, 1H), 6.91 (s, 1H),
6.06 (s, 1H), 5.18 (s, 1H), 2.72–0.59 (m, 42H, protons in UA
skeleton). 13C NMR (126 MHz, CDCl3) δ 176.65, 150.15,
148.51, 138.08, 134.85, 130.92, 130.01, 129.19, 128.85,
126.83, 125.44, 124.34, 115.51, 99.35, 65.58, 50.83, 48.02,
45.75, 42.02, 39.39, 39.02, 38.85, 35.63, 30.59, 28.68,
27.74, 21.15, 20.30, 19.66, 19.20, 16.95, 16.51, 14.87. MS
(MALDI-TOF) m/z 625 (M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑Fluorophenyl)‑1,2,6a,6b,9
,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (8) White solid. Yield
1
52%, m.p. 198–200 °C. H NMR (300 MHz, CDCl3) δ
9.67 (s, 1H), 8.36 (s, 1H), 7.62 (s, 1H), 7.26 (s, 1H), 7.06–
6.84 (m, 2H), 5.20 (s, 1H), 2.65–0.65 (m, 42H, protons
in UA skeleton). 13C NMR (126 MHz, CDCl3) δ 182.13,
173.51, 155.64, 138.48, 138.11, 131.05, 130.94, 125.00,
123.51, 123.41, 117.92, 117.66, 116.76, 115.88, 115.56,
114.78, 114.50, 42.28, 42.12, 41.44, 39.45, 39.12, 36.70,
36.15, 35.98, 35.73, 30.66, 28.59, 28.04, 25.15, 23.50,
20.35, 19.59, 17.10, 16.94, 15.03. MS (MALDI-TOF) m/z
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(4‑Bromophenyl)‑1,2,6a,6b,9,9
,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17,18
b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]quina‑
zoline‑4a(2H)‑carboxylic acid (5) White solid. Yield 48%,
+
625 (M+ + H). HRMS (ESI) m/z calcd for C39H50FN4O2
(M+H)+ 625.39123, found 625.39124.
1
m.p. 172–174 °C. H NMR (300 MHz, CDCl3) δ 9.98 (s,
1H), 7.60 (s, 1H), 7.32 (d, J=7.5 Hz, 1H), 7.26 (s, 1H), 7.03
1 3

Molecular Diversity
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑(naphthalen‑2‑yl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16,
16a,16b,17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]
triazolo[5,1‑b]quinazoline‑4a(2H)‑carboxylic acid (9) White
19.43, 18.58, 15.95, 15.83, 13.99. MS (MALDI-TOF) m/z
607 (M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(2‑Methoxyphenyl)‑1,2,6a,6b
,9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (13) White solid. Yield
50%, m.p. 220–221 °C. 1H NMR (300 MHz, CDCl3) δ 9.26
(s, 1H), 8.32 (s, 1H), 7.52 (s, 1H), 7.20 (d, J=7.2 Hz, 1H),
7.05 (s, 1H), 6.84 (d, J = 8.2 Hz, 1H), 5.18 (s, 1H), 3.74
(s, 3H), 2.62–0.58 (m, 42H, protons in UA skeleton). MS
(MALDI-TOF) m/z 637 (M+ +H).
1
solid. Yield 52%, m.p. 110–112 °C. H NMR (300 MHz,
CDCl3) δ 9.77 (s, 1H), 8.35 (s, 1H), 8.09 (d, J=8.9 Hz, 1H),
7.98 (d, J=6.4 Hz, 1H), 7.88–7.74 (m, 2H), 7.66–7.57 (m,
1H), 7.52 (d, J=3.9 Hz, 1H), 7.23 (d, J=8.4 Hz, 1H), 5.11
(s, 1H), 2.77–0.63 (m, 42H, protons in UA skeleton). 13
C
NMR (126 MHz, CDCl3) δ 173.51, 138.07, 138.01, 133.48,
133.44, 132.98, 132.95, 130.94, 130.90, 128.77, 128.71,
128.23, 128.12, 127.82, 127.74, 127.57, 127.49, 127.03,
126.96, 125.38, 125.31, 124.46, 124.42, 42.17, 41.44, 39.43,
39.11, 38.84, 36.00, 35.77, 30.68, 29.11, 23.25, 23.17,
20.56, 20.48, 19.60, 19.56, 16.75. MS (MALDI-TOF) m/z
657 (M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑(4‑(trifluoromethyl)phenyl)‑1,3,4,5,6,6a,6b,7,8,
8a,9,16,16a,16b,17,18b‑hexadecahydrochryseno[1,2‑g]
[1,2,4]triazolo[5,1‑b]quinazoline‑4a(2H)‑carboxylic acid
(14) White solid. Yield 50%, m.p. 203–205 °C. 1H NMR
(300 MHz, CDCl3) δ 10.01 (s, 1H), 7.58 (s, 2H), 7.49 (s,
2H), 5.69 (s, 1H), 5.18 (s, 1H), 2.67–0.56 (m, 42H, protons
in UA skeleton). 13C NMR (126 MHz, CDCl3) δ 175.90,
143.71, 143.45, 138.20, 134.69, 130.03, 128.02, 126.26,
126.09, 125.68, 125.22, 125.00, 124.84, 45.83, 42.13, 40.62,
39.40, 39.06, 38.84, 36.74, 36.01, 35.71, 30.65, 28.52,
27.87, 24.11, 23.94, 23.47, 23.12, 21.13, 20.44, 19.54. MS
(MALDI-TOF) m/z 675 (M+ +H). HRMS (ESI) m/z calcd
for C40H50F3N4O2+ (M+H)+ 675.38804, found 675.38721.
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑Methoxyphenyl)‑1,2,6a,6b
,9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (10) White solid. Yield
48%, m.p. 178–180 °C. 1H NMR (300 MHz, CDCl3) δ 9.48
(s, 1H), 7.54 (s, 1H), 7.17 (s, 1H), 6.88 (s, 1H), 6.79 (s,
1H), 5.54 (s, 1H), 5.20 (s, 1H), 3.88 (s, 3H), 3.77–0.75 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 637
(M+ +H).
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(Furan‑2‑yl)‑1,2,6a,6b,9,9,
16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17,
18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (11) White solid. Yield
(1S,2R,4aS,6aS,6bR,16aR)‑15‑(4‑Carboxyphenyl)‑1,2,6a,6b,
9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (15) White solid. Yield
48%, m.p. 227–229 °C. 1H NMR (300 MHz, DMSO) δ 12.17
(s, 2H), 9.36 (s, 1H), 8.26–8.04 (m, 1H), 7.91 (d, J=8.2 Hz,
1H), 7.52 (s, 1H), 7.29 (s, 1H), 5.13 (s, 1H), 2.09–0.77 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 651
(M+ +H).
1
48%, m.p. 185–187 °C. H NMR (300 MHz, CDCl3) δ
8.76 (s, 1H), 7.57 (s, 1H), 7.31 (d, J=5.5 Hz, 1H), 6.39 (d,
J = 2.8 Hz, 1H), 5.74 (s, 1H), 5.23 (s, 1H), 2.20–0.76 (m,
42H, protons in UA skeleton). 13C NMR (126 MHz, CDCl3)
δ 180.94, 154.94, 151.03, 145.69, 143.05, 138.23, 120.74,
112.32, 110.46, 109.76, 45.90, 45.72, 42.40, 41.38, 40.99,
39.54, 39.45, 38.96, 36.85, 36.00, 35.88, 32.77, 32.32,
28.43, 24.89, 23.24, 23.20. MS (MALDI-TOF) m/z 597
(M+ +H). HRMS (ESI) m/z calcd for C37H49N4O3+ (M+H)+
597.37992, found 597.37927.
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑(pyridin‑3‑yl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylic acid (16) White solid. Yield
1
(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑Heptame‑
thyl‑15‑phenyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17,18b
‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]quina‑
zoline‑4a(2H)‑carboxylic acid (12) White solid. Yield 50%,
m.p. 216–218 °C. 1H NMR (300 MHz, CDCl3) δ 10.13 (s,
1H), 7.56 (s, 1H), 7.26 (s, 1H), 7.14 (s, 3H), 5.60 (s, 1H),
5.16 (s, 1H), 2.72–0.60 (m, 42H, protons in UA skeleton).
13C NMR (126 MHz, CDCl3) δ 180.34, 154.50, 137.07,
129.57, 128.31, 128.05, 127.62, 126.63, 124.37, 51.78,
49.79, 46.96, 41.10, 38.43, 38.08, 37.84, 35.75, 35.08,
31.35, 29.65, 27.58, 26.96, 23.10, 22.48, 22.20, 20.13,
46%, m.p. 221–223 °C. H NMR (300 MHz, CDCl3) δ
8.57 (d, J=12.0 Hz, 1H), 7.62 (s, 1H), 7.55 (d, J=8.4 Hz,
1H), 5.67 (s, 1H), 5.21 (d, J=14.0 Hz, 1H), 2.38–0.62 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 608
(M+ +H).
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑methoxyphenyl)‑1,2,6a
,6b,9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b
,17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylate (17) White solid. Yield
1
52%, m.p. 109–110 °C. H NMR (300 MHz, DMSO) δ
1 3

Molecular Diversity
7.45 (d, J=7.8 Hz, 1H), 7.19–7.05 (m, 3H), 5.76 (s, 1H),
5.15 (s, 1H), 4.12 (q, J = 9.4 Hz, 2H), 3.80 (s, 3H), 3.51
(s, 3H), 2.46–0.70 (m, 42H, protons in UA skeleton). MS
(MALDI-TOF) m/z 665 (M+ +H). HRMS (ESI) m/z calcd
for C42H57N4O3+ (M+H)+ 665.44252, found 665.44183.
,16a,16b,17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]
triazolo[5,1‑b]quinazoline‑4a(2H)‑carboxylate (22) White
1
solid. Yield 54%, m.p. 134–136 °C. H NMR (300 MHz,
DMSO) δ 8.16 (s, 1H), 8.04 (d, J = 8.9 Hz, 3H), 7.72 (d,
J=8.4 Hz, 1H), 7.63 (d, J=5.1 Hz, 2H), 5.77 (s, 1H), 5.08
(s, 1H), 4.12 (q, J=9.4 Hz, 2H), 3.49 (s, 3H), 2.60–0.70 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 685
(M+ +H). HRMS (ESI) m/z calcd for C45H57N4O2+ (M+H)+
685.44760, found 685.44733.
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑hepta‑
methyl‑15‑phenyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17
,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylate (18) White solid. Yield
1
48%, m.p. 112–114 °C. H NMR (300 MHz, DMSO) δ
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑heptame‑
thyl‑15‑(thiophen‑2‑yl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,
17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylate (23) White solid. Yield
48%, m.p. 158–160 °C. 1H NMR (300 MHz, DMSO) δ 7.82
(d, J=4.4 Hz, 2H), 7.36–7.25 (m, 1H), 5.76 (s, 1H), 5.24
(s, 1H), 4.10 (q, J = 9.2 Hz, 2H), 3.53 (s, 3H), 2.59–0.75
(m, 42H, protons in UA skeleton). MS (MALDI-TOF) m/z
641 (M+ +H).
7.59 (d, J=1.6 Hz, 2H), 7.53 (m, 3H), 5.76 (s, 1H), 5.14 (s,
1H), 4.14 (q, J=9.8 Hz, 2H), 3.51 (s, 3H), 2.53–0.70 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 635
(M+ +H). HRMS (ESI) m/z calcd for C41H55N4O2+ (M+H)+
635.43195, found 635.43176.
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑fuorophenyl)‑1,2,6a,6
b,9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,1
7,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylate (19) White solid. Yield
50%, m.p. 130–132 °C. 1H NMR (300 MHz, CDCl3) δ 7.50
(s, 1H), 7.32 (d, J=5.1 Hz, 2H), 7.21 (s, 1H), 5.74 (s, 1H),
5.24 (s, 1H), 4.10 (q, J=9.2 Hz, 2H), 3.60 (s, 3H), 2.70–0.75
(m, 42H, protons in UA skeleton). MS (MALDI-TOF) m/z
653 (M+ +H).
TNF‑α, IL‑6, and NO inhibitory activity
RAW264.7 cells were plated at 3 × 105 cells/well in a
48-well microplate and incubated overnight. The cells were
divided into six groups: normal group, positive group (DEX,
10μM), LPS group, and the test compounds groups (3, 10, or
30 μM). Un-pretreated and un-stimulated RAW 264.7 cells
were conducted as normal group. The positive group, LPS
groups, and the test compounds groups were pretreated with
LPS (1 μg/mL) for 1 h. Then, the diferent concentrations of
compounds were treated for 24 h. The culture supernatants
were recovered and assayed using an ELISA kit (BD Bio-
sciences, San Diego, CA, USA) to analyze the TNF-α pro-
ductions according to the manufacturer’s instruction. One-
way ANOVA followed by Dunnett’s multiple comparison
test (*p<0.05, **p<0.01, ***p<0.001) was used to detect
the signifcance. Same to the TNF-α inhibitory activity, the
efects of compounds 1–6, 9, 10, 12, 15, and 18–23 on LPS-
induced production of the infammatory mediators NO and
IL-6 by RAW 264.7 cells were evaluated.
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑15‑(3‑bromophenyl)‑1,2,6a,
6b,9,9,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b
,17,18b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]
quinazoline‑4a(2H)‑carboxylate (20) White solid. Yield
52%, m.p. 150–152 °C. 1H NMR (300 MHz, CDCl3) δ 7.71
(t, J = 1.6 Hz, 1H), 7.64–7.58 (m, 1H), 7.45 (dt, J = 7.7,
1.3 Hz, 1H), 7.37 (d, J=7.8 Hz, 1H), 5.77 (s, 1H), 5.24 (s,
1H), 4.12 (q, J=9.4 Hz, 2H), 3.60 (s, 3H), 2.68–0.77 (m,
42H, protons in UA skeleton). MS (MALDI-TOF) m/z 713
(M+ +H).
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑15‑(furan‑2‑yl)‑1,2,6a,6b,9,9
,16a‑heptamethyl‑1,3,4,5,6,6a,6b,7,8,8a,9,16,16a,16b,17,18
b‑hexadecahydrochryseno[1,2‑g][1,2,4]triazolo[5,1‑b]quina‑
zoline‑4a(2H)‑carboxylate (21) White solid. Yield 52%,
m.p. 140–142 °C. 1H NMR (300 MHz, DMSO) δ 8.07 (d,
J=1.1 Hz, 1H), 7.31 (d, J=3.5 Hz, 1H), 6.76 (dd, J=3.5,
1.8 Hz, 1H), 5.74 (s, 1H), 5.25 (s, 1H), 4.10 (q, J=9.2 Hz,
2H), 3.53 (s, 3H), 2.45–0.76 (m, 42H, protons in UA skel-
eton). MS (MALDI-TOF) m/z 625 (M+ +H). HRMS (ESI)
Molecular docking
All the calculations were performed using MOE 2008.10
software form Discovery studio 3.1 installed on 2.4G Core
i5. To gain insight into the interaction between compound
3 and COX-2 enzyme, a docking simulation was performed
using COX-2 (PDB ID: 5FDQ). The structures of compound
3, UA, and Celecoxib were sketched in 2D and converted
into 3D using the DS molecule editor. For the docking simu-
lation, default values of quaternation, translation, and tor-
sion steps were applied. The identifcation of ligand binding
+
m/z calcd for C39H53N4O3 (M + H)+ 625.41122, found
625.41071.
Ethyl(1S,2R,4aS,6aS,6bR,16aR)‑1,2,6a,6b,9,9,16a‑hepta‑
methyl‑15‑(naphthalen‑2‑yl)‑1,3,4,5,6,6a,6b,7,8,8a,9,16
1 3

Molecular Diversity
9. Wang W, Wu YL, Chen XX, Zhang P, Li H, Chen LX (2019)
Synthesis of new ent-labdane diterpene derivatives from andro-
grapholide and evaluation of their anti-infammatory activities.
Eur J Med Chem 162:70–79. https://doi.org/10.1016/j.ejmec
h.2018.11.002
modes was done iteratively by evaluating ligand conforma-
tions and estimating the binding energy of their interactions
with these binding pockets. The binding pose with the top
5% highest scores was returned for further visual inspection
[27]. The output poses of the ligands generated were ana-
lyzed based on the LibDock score function. Enzyme struc-
tures were checked for missing atoms, bonds, and contacts.
Hydrogen atoms were added to the enzyme structure. Water
molecules and bound ligands were manually deleted. The
most stable molecular-docked model was selected accord-
ing to the best scored conformation predicted by the MOE
scoring function.
10. Liu ZG, Tang LG, Zhu HP, Xu TT, Qiu CY, Zheng SQ, Gu YG,
Feng JP, Zhang YL, Liang G (2016) Design, synthesis, and struc-
ture-activity relationship study of novel indole-2-carboxamide
derivatives as anti-infammatory agents for the treatment of sep-
sis. J Med Chem 59:4637–4650. https://doi.org/10.1021/acs.jmedc
hem.5b02006
11. Chen GZ, Zhang YL, Liu X, Fang QL, Wang Z, Fu LL, Liu ZG,
Wang Y, Zhao YJ, Li XK, Liang G (2016) Discovery of a new
inhibitor of myeloid diferentiation 2 from cinnamamide deriva-
tives with anti-Infammatory activity in sepsis and acute lung
injury. J Med Chem 59:2436–2451. https://doi.org/10.1021/acs.
jmedchem.5b01574
Acknowledgements This research was supported by the Natural Sci-
ence Foundation of Jilin Province (No. 20190201077JC); The Health
Department of Jilin Province (No. 2019ZC007); The Doctoral Foun-
dation of Jilin Medical University (No. JYBS2018007); and The Jilin
Province Students’ Program for Innovation and Entrepreneurship Train-
ing (No. 202013706011).
12. Albrecht W, Unger A, Bauer SM, Laufer SA (2017) Discovery
of N-{4-[5-(4-Fluorophenyl)-3-methyl-2-methylsulfanyl-3H-im-
idazol-4-yl]-pyridin-2-yl}-acetamide (CBS-3595), a dual p38α
MAPK/PDE-4 inhibitor with activity against TNFα-related dis-
eases. J Med Chem 60:5290–5305. https://doi.org/10.1021/acs.
jmedchem.6b01647
13. Imam F, Al-Harbi NO, Al-Harbi MM, Ansari MA, Zoheir
KMA, Iqbal M, Anwer MK, Hoshani ARA, Attia SM, Ahmad
SF (2015) Diosmin downregulates the expression of T cell
receptors, pro-infammatory cytokines and NF-κB activation
against LPS-induced acute lung injury in mice. Pharmacol Res
102:1–11. https://doi.org/10.1016/j.phrs.2015.09.001
14. Cohen J (2002) The immunopathogenesis of sepsis. Nature
420:885–891. https://doi.org/10.1038/nature01326
Compliance with ethical standards
Conflict of interest The authors state no confict of interest.
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