Semisynthesis, an anti-inflammatory effect of derivatives of 1β-hydroxy alantolactone from inula britannica

Article abstract of DOI:10.3390/molecules22111835

1β-hydroxy alantolactone, a sesquiterpene lactone mainly isolated from Inula genus plants, exhibits potent anti-inflammatory and anticancer activities. In this work, 1β-hydroxy alantolactone was isolated and five derivatives were prepared through different reactions at the C1-OH and C13-methylene motifs. The structure-activity relationships (SAR) of anti-inflammatory effects against NO production in RAW264.7 cells showed that the α-methylene-butyrolactone motif was essential for NO production suppression and that retaining the C1-OH group can remarkably improve this effect. The NF-B signaling pathway plays a pivotal role in the regulation ofNOexpression. Moreover, the levels of p65 and p50 phosphorylation were investigated and active compound 1 inhibited phosphorylation of p65 and p50 in TNF-α-induced NF-κB signaling. Further molecular docking suggested that 1 may target the p65 of NF-κB.

Full text of DOI:10.3390/molecules22111835

molecules
Article
Semisynthesis, an Anti-Inflammatory Effect of
Derivatives of 1β-Hydroxy Alantolactone from
Inula britannica
Lin Chen 1,2, Jian-Ping Zhang , Xin Liu , Jiang-Jiang Tang , Ping Xiang *
Xing-Ming Ma *
3
2
4
5,
ID
and
1
,
1
Department of Immunology, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, China;
chenlinfree@126.com
2
3
4
Department of Infectious Disease, the First Hospital of Lanzhou University, Lanzhou 730000, China;
liuxin1288@126.com
Department of Pharmacy, the First Hospital of Lanzhou University, Lanzhou 730000, China;
Zhangjianping0306@163.com
College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China;
tangjiang11@nwafu.edu.cn
College of Plant Protection, Northwest A&F University, Yangling 712100, China
Correspondence: xiangp02@163.com (P.X.); maxm@lzu.edu.cn (X.-M.M.); Tel.: +86-298-703-4332 (P.X.)
5
*
Received: 14 September 2017; Accepted: 22 October 2017; Published: 27 October 2017
Abstract:
1
β-hydroxy alantolactone, a sesquiterpene lactone mainly isolated from Inula genus plants,
exhibits potent anti-inflammatory and anticancer activities. In this work, 1
β-hydroxy alantolactone
was isolated and five derivatives were prepared through different reactions at the C1-OH and
C13-methylene motifs. The structure–activity relationships (SAR) of anti-inflammatory effects against
NO production in RAW264.7 cells showed that the
for NO production suppression and that retaining the C1-OH group can remarkably improve this
effect. The NF- B signaling pathway plays a pivotal role in the regulation of NO expression. Moreover,
the levels of p65 and p50 phosphorylation were investigated and active compound inhibited
phosphorylation of p65 and p50 in TNF- -induced NF- B signaling. Further molecular docking
suggested that 1 may target the p65 of NF-κB.
α-methylene-γ-butyrolactone motif was essential
κ
1
α
κ
Keywords: 1β-hydroxy alantolactone; semisynthesis; anti-inflammatory activity
1
. Introduction
Naturally-occurring sesquiterpene lactones (STLs) are plant-derived bioactive constituents
often used in traditional medicines against inflammation, cancer, malaria, and viral and bacterial
infection [ ]. There are about 1500 publications reporting on the anticancer and anti-inflammatory
properties of STLs [ ], in which parthenolide and helenalin are representative STLs of particular
1–7
3,8,9
significance (Figure 1). Parthenolide (with a 10-membered ring), isolated from the medicinal herb
feverfew, is now in cancer clinical trials due to its potential anti-inflammatory, anticancer, and
antiviral properties [3]. Helenalin (a 5/7-fused bicyclic system) found in various Asteraceae such as
Arnica spp., could inhibit the activity of the telomerase, therefore being a potential anti-cancer agent [10].
The mechanisms of action of parthenolide and helenalin on cell functionality were investigated and
found to inhibit transcription factor NF-
signaling pathway [11].
κB, resulting in decreased recruitment of the T- and B-cell
The canonical NF-
and aberrant activation of NF-
chronic inflammatory disease, atherosclerosis, and cancer [12]. One of these STLs structural features
κ
B signaling pathway is a mediator of the cellular inflammatory response,
κ
B is perhaps implicated in a spectrum of human diseases, including
Molecules 2017, 22, 1835; doi:10.3390/molecules22111835
www.mdpi.com/journal/molecules

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is the
α
-methylene-
γ
-butyrolactone moiety (Figure 1), which allows STLs to react with proteins via
the Michael addition, especially thiol groups of some proteins, resulting in their alkylation. Moreover,
both STLs (parthenolide and helenalin) were previously shown to target Cys38 in the p65 of NF-
and ablate its DNA-binding ability [13,14].
κB
Figure 1. Chemical structure of representative anti-inflammatory sesquiterpene lactones (STLs) and
relevant active moieties.
1
β
-Hydroxy alantolactone (
Inula genus (Inula britannica, Inula japonica and Inula helenium) plants [
and anti-inflammatory effects [ 17]. -methylene- -butyrolactone has been considered as its active
moiety. However, to our best knowledge, an anti-inflammatory structure–activity relationship (SAR)
for has been not reported with respect to whether or not other functional groups inflect its biological
activity, and its further mode of action remains unknown. In our ongoing efforts in exploring SARs
in anti-inflammatory potency, we herein semisynthesized five derivatives ( , Figure 2) including
oxidized and esterified (with increased lipophilicity) and (with increased hydrophilicity) at the
C1-OH position, which reduced and spiral at the C13-methylene motif. Therein, specific spiro-cycle
natural products have been reported as having potent biological activities [18 22], for example
1
, a 6/6-fused bicyclic system, Figure 2), mainly isolated from the
1
,
15 16], have shown anticancer
,
1
,
α
γ
1
2–6
2
3
4
5
6
–
antitumor activities (artemalogues) [20] and induction of autophagy activity (clionamine D) [22].
Furthermore, their anti-inflammatory activities, including the inhibitory against lipopolysaccharide
(LPS)-induced NO production and TNF-α-induced NF-κB signaling in RAW 264.7 macrophages
were evaluated.
Figure 2. Semisynthetic derivatives of 1β-hydroxy alantolactone (1).
2
. Results and Discussion
-Hydroxy alantolactone (
repeated column chromatography in previous papers by the authors of this paper as well as
others [ 17 23]. Then, 1, as a starting material, was converted to the corresponding derivatives
by various reactions such as oxidation for , esterification for and at C1-OH position,
reduction for , and 1,3-dipolar cycloaddition for at C13-methylene (Scheme 1). The structures
1β
1) was firstly isolated from the dried flowers of I. britannica through
1, ,
2
–
6
2
3
4
5
6

Molecules 2017, 22, 1835
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of all compounds were well characterized by NMR, (HR)ESI-MS and HPLC purity analysis referred to
in the Supplementary Material.
Scheme 1. Semisynthetic route of 1
β-hydroxy alantolactone (
1) derivatives. Conditions and reagents:
(a
) Dess–Martin periodinane (DMP), CH Cl , r.t., 85%; (b
) a different anhydride, Et N, DMAP, r.t., 76%
2
2
3
for
3 and 85% for 4; (c) NaBH , THF, 2 h, r.t., 67%; (d) (i) p-methylbenzaldehyde and 50% NH OH in
4 2
H O, Et O, 89%; (ii) DMF, NCS, r.t., 98%; (iii) CH Cl , Et N, r.t., 76%.
2
2
2
2
3
The potent anti-inflammatory properties of STLs have been reported to be chemically mediated
-methylene- -butyrolactone and other functional groups such as the -unsaturated carbonyl
motif [24]. To investigate the SAR of 1 -hydroxy alantolactone, we investigated the anti-inflammatory
effects of through inhibition of the LPS-induced NO production in RAW 264.7 macrophages.
by
α
γ
α,β
β
1–6
Aminoguanidine (AG) was used as the positive control. Meanwhile, a cytotoxicity test was performed
at the same time in order to explore the influence of cytotoxicity on the inhibition of NO production
assay. The IC50 values of NO production suppression and the cytotoxicity of
2
1
–
6
in LPS-induced RAW
64.7 macrophages are summarized in Table 1. In the compound treatment periods, retaining
-methylene moiety exhibited inhibitory effects against NO production with IC50 values of 5.61, 36.1,
M, respectively. However, and with C13 reduction and cycloaddition showed
no inhibitory activity (>1000 M), suggesting that -methylene- -butyrolactone is essential to the
anti-inflammatory potency, which is consistent with previous reports [ ]. Among these tested
compounds, bearing the C1-OH group displayed the highest potency against NO production, at a
level 6.5–8.4 fold greater than that of , and it is more active than aminoguanidine (positive control).
This result hinted the importance of the C1-OH functionality in terms of anti-inflammatory potency.
1–4
α
46.5, and 39.6
µ
5
6
µ
α
γ
3,8
1
2–4
Table 1. Inhibitory activities against NO production and cytotoxicities of 1β-hydroxy alantolactone (1)
and its derivatives in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.
IC50 (µM)
NO Inhibition 1
Compound
Cytotoxicity 2
1
2
3
4
5.61 ± 0.34
36.1 ± 3.8
46.5 ± 5.6
39.6 ± 5.7
>1000
>50
34.5 ± 5.8
>50
>50
4
5
ND
6
>1000
10.9 ± 1.5
ND
ND
2
3
Aminoguanidine
1
Inhibitory effects of samples against LPS-induced NO production in RAW264.7 cells. Inhibition of cell viability
during the periods of samples-treated RAW264.7 cells. Positive control. Not determined.
3
4

Molecules 2017, 22, 1835
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In cytotoxicity test,
not notably cytotoxic at the concentration (IC50 > 50
suppression of NO production (Table 1) but weak cytotoxicity (IC50 > 50
functionality is different between the cytotoxicity and anti-inflammatory effects.
NF- B is a transcription factor that controls immune responses and plays a pivotal role in the
regulation of NO expression [25]. In order to investigate the inflection of the active compound on
-induced RAW264.7
2
showed cytotoxicity with IC50 of 34.5
µM, yet other derivatives were
µM). However, precursor
1 exhibited stronger
µM), indicating that C1-OH
κ
1
NF-
κ
B signaling, we next conducted an NF-
κ
B luciferase reporter assay in TNF-
α
cells to evaluate the impact of
1
on the transcriptional activity of NF-
κ
B. Parthenolide (P) was utilized
as a positive control (benchmark). It could be seen in Figure 3, after this 8 h treatment test, that
displayed dose-dependent inhibition (such as 75% inhibition at 20 M) towards the NF- B pathway.
The observation that and parthenolide were comparably potent in this assay implies the possibility
that may have similar properties, like parthenolide in cells. When treatment with compound at
the concentration of 20 M, the NF- B-luciferase inhibitory percentage was 55% (data not shown),
which further indicated the importance of the C1-OH in NF- B-luciferase inhibition in RAW264.7 cells.
Moreover, the levels of p65 and p50 phosphorylation were investigated by western blotting analysis.
As shown in Figure 4, dose-dependently suppressed TNF- -induced phosphorylation of the NF-
p65 and p50 subunit, demonstrating inhibition potency of 1 in NF-κB signaling.
1
µ
κ
1
1
2
µ
κ
κ
1
α
κB
100
50
0
1
5 10 20 50
P
1
5 10 20 50
1
(μM)
Figure 3. Compound
in RAW264.7 cells. and parthenolide (
stably transfected NF- B luciferase reporter and stimulated with TNF-
a non-induced control, Non-Ind). Luminescence was normalized to the non-compound induced (Ind)
control and plotted as NF- B luciferase activity (%). Mean standard deviation values are shown with
more than three replicates.
1
inhibited TNF-
α
-induced NF-
, positive control) were dosed to RAW264.7 cells containing a
(50 ng/mL) for 8 h (except for
κB activation by NF-κB-luciferase inhibition assay
1
P
κ
α
κ
±
Figure 4. Compound
in RAW264.7 cells. After treatment with
total cell lysates were tested by western blot experiments.
1
blocks TNF-
α
-induced NF-
κ
B activation through p65 and p50 phosphorylation
1
and parthenolide (
P) at the indicated concentrations for 8 h,

Molecules 2017, 22, 1835
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The p65 of NF-
κ
B is a main target of parthenolide [14]. To further understand the possible
binding mode of
p65 of NF- B by the Surflex–Dock protocol using the Sybyl-X software package (PDB: 1VKX) [
The docking result (Figure 5) showed that could superimpose well with parthenolide in a p65 binding
site. The best matching interaction between and p65 site exhibited that the spatial distance between
1
with p65, we simulated the molecular modeling of
1
and parthenolide (P) with
κ
5,
13].
1
1
the exocyclic methylene (C13) and -SH of Cys38 is about 3.5 Å, similar to that of parthenolide, implying
that 1 may form covalent protein adducts with Cys38 of p65 [5].
Figure 5. The docking mode of parthenolide (
P, left, green) and
1 (right, gray) through binding to
NF- B/p65 (PDB: 1VKX). can superimposed well with P
κ
1
in the p65 binding site and the spatial
distance between both the α-methylene and SH group of C38 is about 3.5 Å.
3
. Materials and Methods
3
.1. General
Analytical HPLC was performed on a Waters 1525 series with an Agilent TC-C18 column and UV
(
PDA) detection at the max wavelength of compounds. Column chromatography (CC) was performed
over silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China). The progress of all
reactions was monitored by TLC on 2 cm 5 cm precoated silica gel GF254 plates with a thickness of
.25 mm (Qingdao Marine Chemical Group, Co., Qingdao, China). NMR spectra were recorded on a
00 MHz Bruker spectrometer. HRESI-MS spectra were obtained on a Thermo Scientific LTQ Orbitrap
×
0
5
(Thermo Scientific, Waltham, MA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum (FBS) were purchased from Invitrogen. Lipopolysaccharide (LPS) and Sulforhodamine
B (SRB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A nitric oxide assay kit was
purchased from Beyotime (Shanghai, China). All commercially available solvents and reagents were
freshly purified and dried by standard techniques prior to use.
3
.2. Extraction and Isolation from Plant Material
Air-dried flowers of I. britannica were extracted with 95% EtOH under reflux. 1β-hydroxy
alantolactone (
1) was isolated through repeated chromatography on a silica gel column according to a
1
13
previous method and H, C-NMR, specific rotation, and ESI-MS were reported in our literature [17].
3
.3. Synthesis of Derivatives
Using as a starting material, derivative
Dess–Martin periodinane (DMP) in 85% yield;
with acetic anhydride or succinic anhydride in 76% and 85% yield, respectively;
through NaBH reduction. Furthermore, was converted to spiro[lactone-isoxazol]
1
2
3
was prepared through oxidation reaction with
and was synthesized by esterification reaction
was obtained
with new-made
4
5
2
6
4
aldoxime chloride by 1,3-dipolar cycloaddition reaction. Detailed semisynthetic methods and spectra
data of 2–6 are referred to in the Supplementary Material.

Molecules 2017, 22, 1835
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3
.4. Cell Culture
RAW 264.7 cell line was originally obtained from ATCC (American type culture collection).
The cell line was grown in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) thermally
inactivated fetal bovine serum (FBS, Invitrogen), penicillin (100 KU/L) and streptomycin (100 KU/L)
◦
at 37 C in a 5% CO humidified incubator.
2
3
.5. Measurement of NO Production in RAW264.7 Macrophages
Then, 100- L aliquots of the exponentially growing RAW 264.7 cells were seeded in 96-well culture
µ
4
◦
plates at 1
medium was replaced by fresh free-FBS medium and cells were pretreated with different concentrations
0, 1, 3, 10, 30, 100, 300, 1000 M) of samples for 4 h. Thereafter, the medium with samples was
removed and then incubated for 48 h with or without 2 g/mL LPS 10% FBS DMEM. The nitrite
×
10 cells/well at 37 C for overnight in DMEM medium with 0.1% FBS. The culture
(
µ
µ
concentration (NO production) in the culture supernatant was measured using Nitric Oxide assay
kit mainly containing Griess reagent. The absorbance was measured at 540 nm using a microplate
reader (M5). The amount of NO was calculated from a standard curve created using sodium nitrite
with known concentrations. The cell viability was evaluated by SRB colorimetric assay [26].
3
.6. Luciferase NF-κB Reporter Assay
RAW 264.7 cells (10,000 cells/well) were placed in a 96-well plate and the cells were then
transfected with pNF-
κ
B-Luc expression plasmid using a mixture of plasmid and lipofectamin
®
2000 PLUS in the Opti-MEM I Reduced Serum Medium according to manufacturer’s instructions
(
Life Technologies, Carlsbad, CA, USA). After 12 h, the cells were co-incubated with the active samples
1, 5, 10, 20, 50 M) and TNF- . Parthenolide ( ) was used as positive control. NF- B was induced
(50 ng/mL, delivered in PBS) to the treated wells and the induced control wells.
After an additional 8 h, Bright-Glo luciferase reagent (Beyotime) was added to each well (100 L) and
the plate was allowed to stand for two minutes. Luminescence measurements with a relative light unit
RLU) were then obtained using a Fluoroskan Ascent multiple microplate reader (Molecular Device,
(
µ
α
P
κ
by adding TNF-
α
µ
(
Sunnyvale, CA, USA). Each experiment was performed in biological triplicate (at minimum) with
three technical replicates per experiment.
3
.7. Western Blotting Analysis
4
RAW 264.7 cells were seeded in a 24-well plate at 5
×
10 cells/well. After cells adherence for
M) and TNF- (50 ng/mL)
overnight, the test compound
1
(50, 10 and 1
µ
M) or parthenolide (10
µ
α
were added to the wells and incubated for 8 h. Then, cell pellets were collected and lysed with
RIPA lysis buffer containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor
cocktail. The protein concentration of cell samples was analyzed by using the BCA method. Equal
amount proteins of each sample were electrophoresed on 12% separating gel and 4% stacking gel and
electrotransferred onto an NC membrane. After incubation with appropriate primary and secondary
antibodies of p65 and p50, protein blots were detected by using ECL solution and ChemiDoc XRS+
imaging system (Bio-Rad, Hercules, CA, USA). β-actin was used as loading control.
3
.8. Molecular Modeling
Computational docking of the interaction of
1 with p65/NF-κB was performed with the
Surflex–Dock protocol using Sybyl-X 2.1.1 software [27]. The original parameters of blind docking
were used in combination with an evaluation scheme based on binding free energy ( G). The structure
of NF-κB p50–p65 heterodimer bound to DNA was obtained from the Protein Data Bank (1VKX).
∆

Molecules 2017, 22, 1835
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3
.9. Statistics Analysis
All the data were the mean
-tailed Student t-test using GraphPad software.
±
standard deviation (SD). Statistical analysis was performed by the
2
4
. Conclusions
In conclusion, we semisynthesized five 1β-hydroxy alantolactone derivatives by four different
reactions at the C1 and C13 positions and evaluated their anti-inflammatory effects in vitro. Among the
derivatives, compound 1, containing a C1-OH group, displayed significant inhibitory activity against
NO production and a suppressive effect against TNF-α-induced NF-κB without strong cytotoxicity.
Predicted binding modes showed that may have the ability to form covalent adducts with Cys38 of
1
p65. Further target identification will soon be underway.
Supplementary Materials: The supplementary materials are available online.
Acknowledgments: This work was supported by the National Natural Science Foundation of China
(No. 31200254).
Author Contributions: L.C. and P.X. conceived and designed the experiments; L.C. and X.L. performed the
experiments; P.X. and J.-P.Z. analyzed the data; J.-J.T. and X.-M.M. contributed reagents/materials/analysis tools;
and P.X. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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