Synthesis of 6-epi-tuberiferin and the biological activities of tuberiferin, dehydrobrabrachylaenolide, 6-epi-tuberiferin, and their synthetic intermediates

Article abstract of DOI:10.1016/j.bioorg.2021.104642

Tuberiferin, 6-epi-tuberifelin, dehydrobrachylaenolide and two series of eudesmanolides, eudesmane-12,6 α-lactones and eudesmane-12,6β-lactones, were synthesized for the studies of the structure–activity relationships to explore novel anti-inflammatory, a

Full text of DOI:10.1016/j.bioorg.2021.104642

Bioorganic Chemistry 108 (2021) 104642
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 6-epi-tuberiferin and the biological activities of tuberiferin,
dehydrobrabrachylaenolide, 6-epi-tuberiferin, and their
synthetic intermediates
,
*
Dan Lia, Yohsuke Higuchib, Takafumi Kobayashib, Fumito Shimomab, Yuhua Baia
,
,
*
Masayoshi Andoc
a Department of Pharmacy, Daqing Campus of Harbin Medical University, Daqing Hi-Tech Development Zone, Xinyang Road, Daqing 163319, China
b Graduate School of Science and Technology, Faculty of Engineering, Niigata University, Ikarashi, 2-8050, Niigata 950-2181, Japan
c Department of Chemistry and Chemical Engineering, Faculty of Engineering, Niigata University, Ikarashi, 2-8050, Niigata 950-2181, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tuberiferin, 6-epi-tuberifelin, dehydrobrachylaenolide and two series of eudesmanolides, eudesmane-12,6
Tuberiferin
α
-lactones and eudesmane-12,6β-lactones, were synthesized for the studies of the structure–activity relation-
Dehydrobrabrachylaenolide
6-epi-tuberiferin
ICAM-1
ships to explore novel anti-inflammatory, anti-cancer and crop disease prevention agents. The anti-inflammatory
activities were tested by the inhibitory on the induction of inter-cellular adhesion molecule (ICAM-1), the
permeation of leucocyte into inflammatory air pouch of murine, the killing function of cytotoxic T-lymphocytes
(CTL), production of IL-1; The anti-cancer activities were established on the cytotoxic activities to six kinds of cell
lines (P388, CCRF-CEM, VA-13, HepG2, QG-56, and WI-38). Results showed that Dehydrobrachylaenolide (an
CTL
Inflammatory Air pouch of Murine
Cytotoxic Activity
exo-endo cross conjugated dienone and
ICAM-1 (IC50 3.0 M) and the cell line VA-13 (IC50 0.45
embraced the most potent inhibitory activity towards the permeation of leucocyte into inflammatory air pouches
of murine in vivo (inhibitory ratio 54% at 10 mg); Compound 25 with an -bromo-ketone and -methylene trans-
γ-lactone) showed the most significant inhibitory activity on the killing function of CTL (IC50 18 M), as well as
the cell lines of CCRF-CEM (IC50 1.1 M) and P388 (IC50 1.21 M) ; Tuberiferin (an ,β-unsaturated ketone and
methylene γ-lactone) was on the top effective inhibitory on the production of IL-1; Compounds 19 with an
-bromo-ketone and -methylene cis-γ-lactone exhibited the most potent inhibitory of QG-56 (IC50 12.5 M);
Compound 29 with an -bromo- ,β-unsaturated ketone and -methylene γ-lactone) showed significant inhibitory
for HepG2 (IC50 1.23 M) , though potently inhibited WI 38 (IC50 0.31 M) as well. A conclusion may be reached
that the -Methylene γ-lactone moiety, exo-endo cross conjugated dienone moiety, and -bromo-ketone with
α
-methylene γ-lactone) was the most effective compound inhibiting
μ
μ
M); Compound 20 with an -bromo-ketone moiety
α
α
α
μ
μ
μ
α
α-
α
α
μ
α
α
α
μ
μ
α
α
α-
methylene moiety might be essential for eudesmanolides in the expression of their anti-inflammatory, anti-
tumour biological activities. Similarly, the above mentioned key moieties are also responsible for the preventive
activity of crop disease controlling.
1. Introduction
tissue and cellular proliferation at the site of the injured tissue. When
these inflammatory responses become chronic, it has been found to
Inflammation plays an important role in host defence, it is one of the
common events in the majority of acute as well as chronic debilitating
diseases and represent a chief cause of morbidity in today’s era of
modern lifestyle [1].It is the body’s response to tissue damage, caused by
physical injury, ischemic injury, infection, exposure to toxins, or other
types of trauma [2]. The body’s inflammatory response causes cellular
changes and immune responses that result in repair of the damaged
mediate a wide variety of diseases, including cardiovascular diseases,
cancer, diabetes, arthritis, Alzheimer’s disease, pulmonary diseases, and
autoimmune diseases [3]. Cell mutation and proliferation can result,
often creating an environment that is conducive to the development of
cancer. It is only during the past decade that clear evidence has been
obtained that inflammation plays a critical role in tumorigenesis [4].The
lack of specific therapeutic agents has impaired effective treatment for
* Corresponding authors.
E-mail addresses: yuhuabai58@yahoo.com (Y. Bai), andomasa@kdp.biglobe.ne.jp (M. Ando).
https://doi.org/10.1016/j.bioorg.2021.104642
Received 2 July 2020; Received in revised form 19 November 2020; Accepted 5 January 2021
Available online 12 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
these inflammatory conditions.Therefore, treating the inflammatory
causes is always essential, and finding a safe and effective treating
approach to control inflammation has been a challenge. In the past few
years, plant-derived molecules known as phytochemicals or phytocon-
stituents or natural products appeared to be a major source of drugs and
evaluated as a drug candidate for anti-inflammatory actions [1].
lymphocyte (CTL), in vitro, respectively, as well as inhibitory activities
against the permeation of leucocyte into inflammatory air pouch of
murine in vivo. Furthermore, the possibility of these synthetic com-
pounds as anti-tumour agents was carried out by screening of the
cytotoxic activity of compounds towards six kinds of cell lines (P388,
CRF-CEM, VA-13, HepG2, QG-56, and WI-38). Also, the preventive or
curative activities of the compounds in controlling crop disease were
tested by pots test.
Sesquiterpenene lactones with an
α-methylene γ-lactone moiety
fused on various skeletons are a rapidly expanding group of natural
products, comprising to date>900 varieties [5]. Some of them has been
shown to possess considerable biological activities including allergenic
agents, cytotoxic and anti-tumour agents, and preventive activity in
controlling crop diseases [6]. However, the research on the activities of
eudesmanolide type sesquiterpene lactones was few reported. Due to the
scarce natural resource of these biological active sesquiterpene lactones,
and with the interests in finding novel promising phytotherapeutics anti-
inflammation and anti-cancer agents, as well as crop diseases preventive
agents, synthesis of these biological compounds are designed and car-
ried out in the present study. Although the synthesis of a naturally
occurring eudesmanolide, (+)-tuberiferin (1) possessing trans-fused
2. Results and discussion
2.1. Synthesis of 6-epi-tuberiferin (2) possessing cis-fused methylene-
lactone, and improved synthesis of 1, dehydrobrachyaenolide (3), and
their related compounds
The starting material of 2 is commercially available l-α-santonin (4)
(Fig. 1). Epimerization of 4 with 5 wt% HCl gas in anhydrous DMF gave
6-epi-santonin (5) in 59% yield [9]. By products of this reaction were
phenolic compounds produced by dienon-phenol rearrangement [9].
Catalytic hydrogenation of 5 over 3% palladium on strontium carbonate
gave 6 in 33% yield. The poor yield of 6 was induced by the hydro-
genolysis of β (ax) γ- lactone oxygen at C-6. Epimerization of the
resulting 6 possessing β(ax)-methyl group at C-4 by 2 M hydrochloric
acid (HCl) in ethanol (EtOH) gave 7 in 96% yield. Acetalization of the
carbonyl group at C-3 under standard condition gave acetal 8 in 94%
yield (Scheme 1). The overall yield of 8 was poor because of the low
yields of 5 and 6. And compound 8 was also prepared from 4 by a
different procedure (Scheme 1). Catalytic hydrogenation of 4 over 2%
palladium on strontium carbonate (Pd-SrCO3) in ethyl acetate (EtOAc)
and successive epimerization of resulting crude product of 9 by 2 M HCl
in EtOH gave 10 in 81% overall yield from 4. Pure 9 was obtained by the
recrystallization of crude 9 from EtOH in 69% yield. Acetalization of the
carbonyl group of 10 under standard conditions gave 11 in 85% yield.
Reduction of lactone 11 with lithium aluminium hydride (LiAlH4) gave
diol 12 in 98% yield. Selective acetylation of the primary hydroxyl
α
-methylene γ-lactone, has already been reported [5,7,8], the studies of
biological activities of 1 and its synthetic intermediates were limited,
such as their cytotoxic activity against murine lymphocytic leukemia
(P388), plant growth inhibitory activities, and preventive activities of
some crop diseases [5]. In the current study, the synthesis of 6-epi-
tuberiferin (2) possessing cis-fused α-methylene γ-lactone, and improved
synthesis of (+)-tuberiferin(1), dehydrobrachyaenolide (3)(Fig. 1), and
their related compounds were designed and developed for detailed study
of their biological activities, as well as the structure–activity
relationship.
The immunomodulatory and anti-inflammatory agents screening
were based on inhibitory activity on the production of IL-1, inhibitory
activity against induction of intercellular adhesion molecule-1 (ICAM-
1), and inhibitory activity against the killing function of cytotoxic T
group of 12 gave acetate 13 in 98% yield. Oxidation of 6α-hydroxyl
group of 13 by Collins reagent (CrO.3Py in CH2Cl2) and successive
reduction of 6-carbonyl group of 14 with LiAlH4 gave 15 in 94% overall
yield. Oxidation of 15 with pyridinium chlorochromate (PCC) gave
desired cis-lactone 8 in 88% yield. The overall yield of 8 by the eight
steps conversion from 4 was 55% (Scheme 1).
Enolization of 8 with lithium diisopropylamide (LDA) and successive
bromination of resulting enolate with carbon tetrabromide (CBr4)[6]
gave the bromide 16 in 92% yield. Dehydrobromination [10] of 16 with
DBU in benzene at refluxing temperature gave an α-methylene γ-lactone
17 in 88% yield. Deacetalization of 17 was achieved by treatment with
20% aqueous acetic acid (aq AcOH) in ethanol (EtOH) at 85 ◦C for 4 h to
give 18 in a quantitative yield. The selective bromination of 18 at C-2
with phenyltrimethylammonium perbromide (PTAB) in tetrahydrofuran
(THF) gave α-bromo ketone 19 in 99% yield. Dehydrobromination of 19
with lithium carbonate (Li2CO3) and lithium bromide (LiBr) in N,N-
dimethyl formamid (DMF) at 125 ◦C for 90 min gave 2 in 71% yield.
Another synthesis of 2 was achieved from 18 in high yield(90%).
Enolsilylation of 18 with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) in the presence of triethylamine (Et3N) in CH2Cl2 gave cor-
responding silyl enol ether. Treatment of the silyl enol ether with
palladium acetate [Pd(OAc)2] in acetonitrile (CH3CN) gave 2 in 90%
overall yield (Scheme 1).
Reference compounds, α-methyl γ-lactone 20 and 21, for the com-
parison of their bioactivity with the corresponding
α-methylene
γ-lactone 19 and 2, were prepared by the following procedure. Bromi-
nation of 6 with bromine (Br2) in the presence of hydrobromic acid
(HBr) in AcOH gave 2α-bromoketone 20 in 87% yield. Dehydrobromi-
nation of 20 with Li2CO3 in DMF gave α,β-unsaturated ketone 21 in 98%
yield (Scheme 1). Phenylselenenylation of 11 by Grieco’s method [11]
gave the corresponding phenyl selenide 22 in 96% yield. Treatment of
Fig. 1. Chemical structures of edesmane, santonin and the target compounds
(1, 2, 3).
2

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
Scheme 1. The synthetic routes of tuberiferin, 6-epi-tuberiferin, dehydrobrachyaenolide, and their related compounds starting from commercially available ma-
terial santonin.
22 with 30% hydrogen peroxide (H2O2) in THF in the presence of AcOH
was achieved in higher yield. Enolsilylation of 24 with TMSOTf in the
presence of Et3N in CH2Cl2 gave the corresponding silyl enol ether.
Treatment of this silyl enol ether with Pd(OAc)2 in CH3CN afforded 1 in
91% yield.
gave α-methylene γ-lactone 23 in 93% yield. Deacetalization of 23 was
achieved by treatment with 20% aq AcOH in EtOH at 85 ◦C for 4 h to
give 24 in a quantitative yield. Compound 24 was also prepared by a
different procedure. Deacetalization of 22 in boiling 50% aq AcOH gave
selenide 28 in 97% yield. Treatment of 28 with 30% H2O2 in THF in the
presence of AcOH gave 24 in 93% yield. The selective bromination at the
Reference compounds,
α
-methyl γ-lactones 26 and 27, for the com-
-methylene
parison of their biological activity with the corresponding
α
γ-lactones 25 and 1, were prepared by the following known procedure
[5]. Bromination of 10 with Br2 in the presence of HBr in CHCl3 gave
bromo ketone 26 in 76% yield. Dehydrobromination of 26 with LiBr,
C-2 position of 24 with PTAB in THF at –6 ◦C for 1 h gave
α-bromo
ketone 25 in 85% yield. Dehydrobromination of 25 with Li2CO3 and LiBr
in DMF at 123–131 ◦C for 90 min gave 1 in 85% yield. Compound 1 was
also obtained in 87% overall yield from 24 without purification of the
crude product of 25. Another new procedure of synthesis of 1 from 24
Li2CO3 in DMF gave α,β-unsaturated ketone 27 in 84% yield.
Finally, from the interest in the influence of cross conjugated exo-
endo dienone moiety to the biological activities of the compound, we
3

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
synthesized dehydrobrachylaenolide (3) from tuberiferin (1). Enolsily-
lation of 1 with TMSOTf and Et3N in CH2Cl2 and successive treatment of
the resulting silyl enol ether with PTAB gave bromide 29 in 79% yield.
Dehydrobromination of 29 with Li2CO3 and LiBr in DMF afforded 3 in
82% yield. On the contrary, treatment of the silyl enol ether with Pd
(OAc)2 gave 3 and 30 in 9% and 56% yields, respectively. The physical
constants and spectral data of 3 were in good agreement with those of
natural dehydrobrachylaenolide (3) in the literature [12] (Scheme 1).
most active compound was trans-
α
-methylene γ-lactone with exo-end
cross conjugated carbonyl moiety (3, IC50 3.0 μM) followed by trans-
α
-methylene γ-lactone with
and it’s cis- counterpart (19, IC50 6.1
with ,β-unsatrated carbonyl moiety (1, IC50 7.1
counterpart (2, IC50 7.3 M). In -methylene γ-lactones, change of
,β-unsaturated carbonyl moiety of 1 to exo-end cross conjugated
carbonyl moiety of 3, and change of 3-carbonyl moiety of 24 to 2
bromo-3-carbonyl moiety of 25 showed 2.5 and 3.3 folds stronger ac-
tivity, respectively. In cis- -methylene γ-lactones, change of 3-carbonyl
moiety of 18 to -bromo-3-carbonyl moiety of 19, and change of
,β-unsatrated 3-carbonyl moiety of 2 to -bromo-3-carbonyl moiety of
19 showed 3 and 1.2 fold stronger activity, respectively. Change of
-methyl γ-lactone moiety of 20 to -methylene γ-lactone moiety of 19,
and Change of -methyl γ-lactone moiety of 27 to -methylene γ-lactone
moiety of 1 showed 3.6 and 7.6 fold stronger activity, respectively.
Therefore, a conclusion could be reached that an -methylene
γ-lactone moiety in the molecule is essential for the activity, moreover,
-bromo-3-carbonyl moiety, exo-end cross conjugated carbonyl moiety,
and ,β-unsturated carbonyl moiety are responsible for the increment of
the activity of corresponding -methylene γ-lactones (see Table 1). In
α
-bromoketone moiety (25, IC50 5.2
M), trans- -methylene γ-lactone
M) and it’s cis-
μM)
μ
α
α
μ
μ
α
α
α
-
α
2.2. Immuno-modulating and Anti-inflammatory activities of compounds
α
α
α
2.2.1. Anti-inflammatory Activities of Compounds Based on Inhibitory
Activity on Expression of Intercellular Adhesion Molecule (ICAM-1) Induced
by Interleukin-1 (IL-1)
α
α
α
α
Expression of intercellular adhesion molecule-1 (ICAM-1) is induced
by interleukin-1 (IL-1) on the surface of endothelial cells of blood ves-
sels. ICAM-1 on the activated endothelial cells interacts with lympho-
cyte function-associated antigen-1 (LFA-1) on leucocytes in the blood
stream, and the leucocytes begin rolling, adhere to the surface of
endothelium, and finally migrate from the inside of the blood vessel to
the inflammatory portion by chemotaxis. The attack of leucocyte causes
serious damage to the inflammatory tissue. Expression of excess amount
of ICAM-1 on the surface of endothelial cells of blood vessel plays an
important role in the progress on inflammatory reaction [13]. These
facts suggest that the inhibitors of induction of ICAM-1 may turn out to
yield a new type of anti-inflammatory agent.
α
α
α
α
addition, the activities of eudesmane-12,6α-lactones (trans-lactones) are
higher than those of the corresponding eudesmane-12,6β-lactones (cis-
lactones).
The transcription of the ICAM-1 gene induced by IL-1 is largely
depended on the transcription factor NF-κB. Upon IL-1 stimulation, NF-
κB translocates from the cytoplasm into the nucleus and activates variety
In the present study, two series of eudesmanolides, eudesmane-
of target genes. As reported previously [12,14], trans-α-methyl γ-lactone
12,6α-lactones (trans-lactones, 1, 3, 10, 11, 22–27) and eudesmane-
with
α-bromoketone moiety (26) and the compounds possessing an
12,6β-lactones (cis-lactones, 2, 7, 8, 16–21), were tested for the inhib-
α
-methylene γ-lactone moiety control the signaling pathway upstream of
itory activity of induction of ICAM-1.
the nuclear transcription of NF-κB, which gives a hint of the potential
mechanism of the above tested active compounds, and further research
is under consideration.
As shown in Table 1, all compounds possessing
α-methylene
γ-lactone moiety (1, 3, and 23–25 in trans-lactones and 2, and 17–18 in
cis-lactones) showed significant inhibitory activity on the induction of
ICAM-1. In α-methyl γ-lactones, α-bromoketons 26 with trans-lactone
2.2.2. Inhibitory activity of the test samples against the permeation of
leucocyte into inflammatory air pouch of murine in vivo
structure and 20 with cis-lactone structure showed significant activity.
,β-Unsaturated ketones 27 and 21 with trans- and cis- -methyl
γ-lactone ring showed moderate and weak activity, respectively. Other
trans- -methyl γ-lactones, 10, 11, and 22 and cis- -methyl γ-lactones, 7,
8, and 16 showed no activity. The order of the inhibitory activity of
α
α
The leucocytes migrate from the inside of the blood vessel to the
inflammatory portion by chemotaxis in the final stage of inflammatory
reaction. Since the attack of leucocyte causes serious damage to the
inflammatory tissue in this stage, the compounds possessing the inhib-
itory activity of permeation of leucocyte may be anti-inflammatory
agents [15]. Thus, we examined the assay of anti-inflammatory activ-
ity of compounds using inflammatory air pouch of murine in vivo. As
α
α
eudesm-12,6α-lactones (trans-lactones) on induction of ICAM-1 was 3 >
25 > 1 > 26 > 23 > 24 > 27≫>≫10, 11, 22, and the order of the
inhibitory activity of 3-oxoeudesm-12,6β-lactones (cis-lactones) on in-
duction of ICAM-1 was 19 > 2 > 18 > 17 > 20 > 21>>>7, 8, 16. The
Table 1
Inhibitory Activity on Induction of ICAM-1 of trans- and cis-lactonesa
Compounds
IC50
(μM)
SDEV (%)
Compounds
IC50
(
μM)
SDEV(%)
trans-α-methyl γ–lactone
cis-α-methyl γ-lactone
10
11
22
26
27
>1000
>316
>1000
11.1
7
>1000
>1000
>1000
22.5
8
16
20
21
1.9
4.8
1.2
2.2
59.0
164.7
trans-
α
-methylene γ-lactone
cis-
α-methylene γ-lactone
23
24
25
3
7.7
4.3
4.1
1.1
4.8
4.2
3.4
2.9
4.6
2.7
17
18
19
2
19.1
18.3
5.4
0.9
8.4
3.0
2.4
7.2
1
a
A549 cells (3 × 104 cells/well) were pretreated with various concentrations of compounds for 1 h and then incubated in the presence of IL-1β for 6 h. Absorbance of
415 nm was assayed after treatment of the cells with primary and secondary antibodies and addition of the enzyme substrate as described in the Materials and Methods
for Bioassays. The experiments were carried out in triplicate cultures. IC50 was caluculated by using the formula in Materials and Method for Bioassay.
4

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
Table 2
active compounds was trans-
carbonyl moiety (25, IC50 18 M), followed by it’s cis- counterpart(19,
IC50 20 M), and trans- -Methylene γ-lactone with ,β-unsaturated
carbonyl moiety (1, IC50 23 M). In trans- -methylene γ-lactones,
change of 3-carbonyl moiety of 24 to 2 -bromo-3-carbonyl moiety of 25
and change of 3-carbonyl moiety of 24 to ,β-unsaturated-carbonyl
moiety of 1, the activity was increased by 0.5 and 0.2 folds, respectively;
In cis- -methylene γ-lactones, change of 3-carbonyl moiety of 18 to
-bromo-3-carbonyl moiety of 19 and change of ,β-unsatrated 3-
carbonyl moiety of 2 to -bromo-3-carbonyl moiety of 19 showed 2.5
and 2.3 folds stronger activity, respectively. In addition, Change of
-methyl γ-lactone moiety of 27 to -methylene γ-lactone moiety of 1
and change of -methyl γ-lactone moiety of 21 to -methylene γ-lactone
moiety of 2 showed 15 and 7.6 fold stronger activity, resoectively.
Hence, a conclusion could be reached that -methylene γ-lactone moiety
in the molecule must be essential for the inhibitory activity; and 2
α-methylene γ-lactone with α-bromo-
Inhibitory Activity of Compounds against the Permeation of Leucocyte into In-
flammatory Air Pouch in vivo
μ
μ
α
α
Inhibitory ratioa
%
μ
α
α
Additional
Quantity of wetability
Number of all
α
compound
amounts (mg)
solution (mL)
leucocyte
10
20
10
10
10
–15.5
5.4
35.6
83.6
7.7
26
α
α
α
27
20
21
9.1
2.5
54
α
–7.8
16.4
a
α
α
Inhibitory ratio (%) = (1–T/C) × 100 where T = leucocytes count after wet
with compound and carrageenin, C = Leucocytes count after wet with
carrageenan.
α
α
α
α
-
shown in Table 2, cis- and trans-lactones 20 and 26 with bromo-ketone
structure showed significant inhibitory activity against the permeation
of leucocyte into inflammatory air pouch of murine in vivo.
bromo-3-carbonyl moiety and α,β-unsturated carbonyl moiety play a
key role in the increment of the activity of corresponding α-methylene
γ-lactones.
2.2.3. Inhibitory activity of killing function of cytotoxic T Lymphocytes
Cytotoxic T lymphocytes (CTL) plays a vital role in the elimination of
virus-infected cells , tumors and graft rejection in the human body. On
the other hand, the attack of CTL causes sever damage to the inflam-
matory tissue; as a result, autoimmune disease are finally induced [16].
It suggests the inhibitors of the killing function of the CTL may turn out
to be a new type of immuno-modulating and anti-inflammatory agent.
As shown in our previous study [6,15], Dehydrocostus lactone and its
2.2.4. Inhibitory activity on production of IL-1.
IL-1 is a potent enhancer of immune responses but also very potent
inducers of acute- phase responses and inflammation [17]. IL-1 has also
played a role in the biology of cancer cells and solid tumors. The biologic
effects and function of IL-1 involve synthetic and local effects that have
influence immunologic properties including T-cell and leucocyte [18].
The attack of IL-1 and leucocyte to own tissue causes serious damage to
the inflammatory tissue and autoimmune disease are finally induced.
Thus, the compounds with inhibitory activity on over-production of IL-1
may be useful agents for allergenic diseases. As shown in Table 4,
Tuberiferin (1) showed the highest inhibitory activity and other com-
related guaiane type α-methylene γ-lactones exerted efficient inhibitory
activity toward the killing function of CTL, which prompted us to
examine the structure–activity relationships of tuberiferin (1), 6-epi-
tuberifelin (2), and their 16 kinds of synthetic intermediates. The
compounds were screened for inhibitors of cytotoxic activity of CTL
clone OE4.
pounds 2, 17–19, 25, 27 with α-methylene γ-lactone structure, showed
Two series of eudesmanolides, namely, eudesmane-12,6α-lactones
Table 4
(trans-lactones, 1, 10, 11, 22–27) and eudesmane-12,6β-lactones (cis-
lactones, 2, 7, 8, 16–21) were tested the inhibitory activity of on the
killing function of CTL. As shown in Table 3, all compounds possessing
Inhibitory Activity of Compounds on Production of IL-1
Compound
IC50(µM)
SDEV(%)
an α-methylene γ-lactone moiety, such as 1 and 23–25 in trans-lactones
Trans-lactone
4
NEa
and 2, and 17–19 in cis-lactones, showed significant inhibitory activity
on killing function of CTL. -bromo ketone 26 with -methyl trans-
γ-lactone ring showed moderate activity and ,β-unsaturated ketone 27
with -methyl trans-γ-lactone ring showed weak activity. Other -methyl
trans-γ-lactones, 10, 11, and 22 and -methyl cis-γ-lactones, 7, 8, and 16
showed no activity. The order of the inhibitory activity of 3-oxoeudesm-
α
α
27
10.6
2.29
0.75
4.7
2.8
3.1
25
α
1
α
α
Cis-lactone
α
17
18
19
2
4.22
7.79
1.60
2.97
1.2
2.3
6.6
6.2
12,6α-lactones (trans-lactones) on killing function of CTL was 25 > 1 >
23 > 24 > 26 > 27≫>≫10, 11, 22 and that of 3-oxoeudesm-12,6β-
lactones (cis-lactones) was 19 > 2, 17 > 18>>21>>>7, 8, 16. The most
a
Not effective.
Table 3
Inhibitory Activity on Killing Function of CTL of trans- and cis-lactonesa
Compounds
IC50
(
μM)
SDEV (%)
Compound
cis-α-methyl γ-lactone
IC50
(μ
M)
SDEV (%)
trans-
α-methyl γ-lactone
10
11
22
26
27
724
2.7
7
8
>1000
>1000
>1000
>1000
630
>1000
>1000
55
16
20
21
2.5
2.4
165
6.9
trans-
α
-methylene γ-lactone
cis-α-methylene γ-lactone
23
24
25
1
23
28.9
18
4.8
1.7
8.7
3.6
17
18
19
2
46
53
29
46
3.9
1
3.7
3.9
21
a
To examine the dose response of the compounds, OE4 cells (1 × 104 cells/well) were preincubated with various concentrations of the compounds for 2 h, mixed
with P815 (1× 103 cells/well) labeled with thymidine, and incubated for 4 h. The experiments were carried out in triplicate cultures. IC50 was deduced from the 50%
inhibition of the specific release, which was calculated by using the formula descrived in materials and Methods for Bioassay.
5

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
significant inhibitory activity on production of IL-1 (Table 4). Therefore,
α
-methylene γ-lactone structure in the molecule was the key for the
inhibitory activity; and 2
carbonyl moiety play a vital role in the increment of the activity of
corresponding -methylene γ-lactones.
α-bromo-3-carbonyl moiety and α,β-unsturated
α
2.3. Cell growth inhibitory activity of the test samples to QG-56, CCRF-
CEM, P388, WI-38, VA-13, and HepG2 cells in vitro
Two series of eudesmanolides, eudesmane-12,6α-lactones (trans-
lactones, 1, 3, 9–11, 22–27, 29) and eudesmane-12,6β-lactones (cis-
lactones, 2, 6–8, 16–21), were tested the cell growth inhibitory activities
against six cell lines, results were shown in Table 5. Those compounds
with a value of WI-38/name of a cell line(fold) > 1 are with excellent
safety and are within the expectation of becoming candidate leading
drugs. Otherwise, they are excluded due to low security.
As we can see from Table 5, almost all compounds exhibited mod-
erate to significant inhibitory activity towards CCRF-CEM, as well as the
P388 cell line, and with excellent safety. Among those compounds,
trans-
cis-
showed top inhibitory effect, followed by trans-
α
-bromoketone with
α
-methylene γ-lactone(25, IC50, 1.1
-methylene γ-lactone(19, IC50, 1.6
,β-unsaturated ketone
1.8 M), and cis-
μ
M)) and
α
-bromoketone with
α
μ
M))
α
,
with
α
-methylene γ-lactone moiety (1, IC50
,β-unsaturated ketone with -methylene γ-lactone moiety (2, IC50, 2.1
M). Besides, trans- ,β-unsaturated ketone with -methylene γ-lactone
M) exhibited most strongest inhibition against
,β-unsaturated ketone with
M), and cis- ,β-unsaturated
-methylene γ-lactone moiety (2, IC50, 2.8 M). -bromo-
-methylene γ-lactone moiety (19, IC50, 12.4 M) and
-methyl γ-lactone moiety (26, IC50, 19.1 M)
μ
α
α
μ
α
α
moiety (3, IC50, 0.45
μ
VA-13, followed by followed by trans-
α
μ
α
-methylene γ-lactone moiety (1, IC50, 2.8
α
μ
ketone with
α
α
α
μ
ketons with
α
-bromoketone with
α
μ
showed moderate inhibition against QG-56.
Interestingly, trans- ,β-unsaturated ketone with
α
α
-methylene
γ-lactone moiety (29) exhibited most potent inhibition against HepG2
(IC50, 1.33 M) , but also significantly inhibited WI-38 (IC50, 0.33 M),
which means it is toxic. And the same trend could be found in -bro-
moketons (3, IC50, 2.5, 2.29 M) with -methylene γ-lactone moiety and
Exo-endo cross conjugated ketone (25, IC50, 8.8, 1.78 M) with
-methylene γ-lactone; and those exhibited moderate inhibition against
WI-38, also showed moderate inhibitory activity towards HepG2, such
μ
μ
α
μ
α
μ
α
as
α
-methylene γ-lactone 23(IC50, 22.5, 12.7
μ
M, respectively), and
α
-Bromoketons with
M).
α-methylene γ-lactone moiety(19, IC50, 14.9, 13.4
μ
In conclusion,
α
-methylene γ-lactone moiety in the molecule are
-bromo-3-carbonyl moiety,
,β-unsaturated carbonyl moiety and exo-endo cross conjugated
essential for the anti-cancer activity, and
α
α
carbonyl moiety increase the activity of corresponding
α-methylene
γ-lactone, significantly, for the inhibitory activity against CCRF-CEM,
P388 and VA-13cell lines.
2.4. Control of crop disease
The preventive and curative activities of a-methyl trans-γ-lactones
9–11, 26, 27, and
α-methylene trans-γ-lactones, tuberiferin (1) and
23–25 in controlling crop diseases were examined by pot test.
ylene trans-γ-lactones 1, 24, and 25 showed significant preventive ac-
tivity in downy mildew of grape and late blight of tomato. -Methyl
trans-γ-lactone 10 showed significant preventive activity in leaf rust of
wheat and leaf biotech of barley. -Methyl trans-γ-lactone 11 showed
moderate preventive activity in sheath blight of rice. -Methylene trans-
-Methyl trans-γ-lactones 26 and 27 showed
α-Meth-
α
α
α
γ-lactones 23 and 25, and
α
moderate preventive activity in blast of rice, damping off of cucumber,
scab of apple and leaf rust of wheat, respectively (see Table 6 in sup-
porting information).
Then, α-methyl cis-γ-lactones 5, 7, 8, 16, α-methylene cis-γ-lactones,
6-epi-tuberiferin (2), 18, and 21 in controlling crop diseases were
6

D. Li et al.
Bioorganic Chemistry 108 (2021) 104642
examined by pot test. epi-Tuberiferin (2) showed excellent preventive
activity on killing function of CTL, Dr. Y. Yanagi (Sumitomo Pharma-
ceuticals Co. Ltd.) for assay of cell growth inhibitory activities against
QG-56, CCRF-CEM, and P-388 cells, Dr. T. Hasegawa (Mitubishi Gas
Chemical company inc.) for assay of cell growth inhibitory activities
against WI-38, VA-13, and HepG2 cells, Dr. Y Tamai (Kuraray Co. Ltd.
NIC Division) for assay of inhibitory activity on production of IL-1, T.
Kurihara (Sapporo Breweries Ltd., Research and development labora-
tories) for the assay of inhibitory activities of compounds against the
permeation of leucocyte into inflammatory air pouch of murine in vivo,
and Dr. N. Hirata and Dr. H. Yamazaki (Sumitomo Chemical Co. Ltd) for
preventive or curative activity in controlling crop disease, thanks Mrs.
H. Ando (the Instrumental Analysis Center for Chemistry, Tohoku Uni-
versity) for microanalysis. Finally, we thanks the scientific research fund
(Grand No. JFXN202001) supported by Harbin Medical University-
Daqing.
activity in leaf rust of wheat and weak preventive activity in glume
biotech of wheat. α-methyl cis-γ-lactones, 5 and 7, and α-methylene cis-
γ-lactones, 18 and 21 showed moderate preventive activity in leaf rust of
wheat. α-methylene cis-γ-lactones 2 and 18 showed weak preventive
activity in glume biotech of wheat (see Table 7 in supporting
information).
The pot test procedures of compounds in controlling crop disease are
shown in see Table 8 and Table 9 in supporting information.
3. Experimental Section
3.1. General Experimental procedures
All melting points are uncorrected. 1H NMR and 13C NMR spectra
were recorded at 500 (200) MHz and at 125 (50) MHz respectively in
CDCl3. The assignments of 1H NMR spectra were determined by
Appendix A. Supplementary material
decoupling and H H COSY experiments. The assignments of 13C NMR
–
–
spectra were determined by DEPT, C H COSY, HMQC, and HMBC ex-
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104642.
periments. All reactions were under an atmosphere of N2. CHCl3 was
dried over CaCl2 and distilled from CaH2. CH2Cl2, benzene, toluene,
DMF, pyridine, diisopropylamine, and triethylamine were distilled from
CaH2. MeOH was distilled from Mg(OMe)2⋅THF and ether were distilled
from sodium benzophenone ketyl. Water in Ethylene glycol was elimi-
nated as the benzene azeotrope. To describe HPLC conditions, the col-
umn, solvent, flow rate (mL/min), and retention time (tR min) are
designated in this order. The column code are as follows: A, 25- × 0.46-
References
[1] Patil KR, Mahajan UB, Unger BS, Goyal SN, Belemkar S, Surana SJ, Ojha S, Patil
CR. Animal Models of Inflammation for Screening of Anti-inflammatory Drugs:
Implications for the Discovery and Development of Phytopharmaceuticals. Int J
Mol Sci. 20 (2019) 4367. https://doi: 10.3390/ijms20184367. PMID: 31491986;
PMCID: PMC6770891.
[2] Singh N, Baby D, Rajguru JP, Patil PB, Thakkannavar SS, Pujari VB. Inflammation
and cancer. Ann Afr Med. 18 (2019) 121-126. https://doi: 10.4103/aam.aam_56_
18. PMID: 31417011; PMCID: PMC6704802.
cm i.d. stainless column packed with 10
column packed with 10 m silicagel; C, 15- × 0.46-cm i.d. stainless
column packed with 5 m silicagel; D 25- × 1.0-cm i.d. stainless column
packed with 10 m silica gel. Silica gel (230–400 mesh) was employed
μm silica gel; B, 25- × 0.8-cm
μ
[3] B.B. Aggarwal, Nuclear factor-kappaB: The enemy within, Cancer Cell. 6 (2004)
203–208, https://doi.org/10.1016/j.ccr.2004.09.003.
μ
μ
[4] M. Karin, Nuclear factor-kappaB in cancer development and progression, Nature.
441 (2006) 431–436, https://doi.org/10.1038/nature04870.
for flash chromatography and 70– 230 mesh silica gel was employed for
column chromatography. To describe the conditions of column and flash
chromatographies, the weight of silicagel, column i.d. and solvent are
designated in this order.
[5] M. Ando, T. Wada, H. Kusaka, K. Takase, N. Hirata, Y. Yanagi, Studies on the
syntheses of sesquiterpene lactones. 10. Improved syntheses of (+)-tuberiferin and
the related alpha-methylene-gamma-lactones and their biological activities, J. Org.
Chem. 52 (1987) 4792–4798, https://doi.org/10.1021/jo00230a028.
[6] Y. Higuchi, F. Shimoma, M. Ando, Synthetic method and biological activities of cis-
fused alpha-methylene gamma-lactones, J. Nat. Prod. 66 (2003) 810–817, https://
doi:10.1021/np020586y. PMID: 12828467.
Chemistry Section (Detailed please refer to supporting information)
Biological Activity Experimental Section(Detailed please refer sup-
porting information)
[7] K. Yamakawa, K. Nishitani, T. Tominaga, Chemical transformation of α-santonin
into sesquiterpene α-methylene-γ-lactones, tuberiferine and artecalin, Tetrahedron
Lett. 33 (1975) 2829–2832, https://doi.org/10.1016/S0040-4039(00)75007-6.
[8] P.A. Grieco, M. Nishizawa, S. Lactones, Total Synthesis of (+/-)-vernolepin and (
+/-)-vernomenin, J Am Chem Soc. 99 (1977) 5773–5780, https://doi.org/
10.1021/ja00459a039.
4. Conclusion
Tuberiferin, 6-epi-tuberifelin, dehydrobrachylaenolide and two se-
ries of eudesmanolides, eudesmane-12,6 a-lactones and eudesmane-
12,6b-lactones, were successfully synthesized. The structures of the
compounds were determined by IR, 1H NMR, 13C NMR, and 2D-NMR.
The anti-inflammatory, anti-cancer and crop disease prevention activ-
ity were tested, respectively. Moreover, the structure–activity relation-
[9] R.T. Arnold, S.T. Kulenovic, J. Org. Chem. 43 (1978) 3687–3689, https://doi.org/
10.1021/jo00413a010.
[10] P.A. Grieco, M. Miyashita, J. Org. Chem. 39 (1974) 120–122, https://doi.org/
10.1021/jo00915a036.
[11] F. Bohlmann, C. Zdero, Phytochemistry. 21 (1982) 647–651, https://doi.org/
10.1016/0031-9422(82)83157-9.
[12] S. Kawai, T. Kataoka, H. Sugimoto, A. Nakamura, T. Kobayashi, K. Arao,
Y. Higuchi, M. Ando, K. Nagai, Immunopharmacology. 48 (2000) 129–135,
https://doi.org/10.1016/S0162-3109(00)00196-X.
ship was analyzed. A conclusion could be drawn that the
α-methylene
γ-lactones moiety, exo-endo cross conjugated dienone moiety, and
[13] J.S. Poter, R.S. Cotran, Cytokines and endothelial cell biology, Physiol Rev. 70
(1990) 427–451, https://doi.org/10.1152/physrev.1990.70.2.427.
[14] S. Yuuya, H. Hagiwara, T. Suzuki, M. Ando, A. Yamada, K. Suda, T. Kataoka,
K. Nagai, Guaianolides as Immunomodulators. Synthesis and Biological Activities
of Dehydrocostus Lactone, Mokko Lactone, Eremanthin, and Their Derivatives,
J. Nat. Prod. 62 (1999) 22–30, https://doi.org/10.1021/np980092u.
[15] R.R. Medzhitov, Origin and physiological roles of inflammation, Nature. 454
(2008) 428–435, https://doi.org/10.1038/nature07201. PMID: 18650913.
[16] T. Berke, G. Cytotoxic T-lymphocytes, How do they function? Immunol Rev. 72
(1983) 5–42, https://doi.org/10.1111/j.1600-065x.1983.tb01071.x. PMID:
6347868.
α
-bromo-ketone with a-methylene moiety might be essential for eudes-
manolides in the expression of their anti-inflammatory, anti-tumour
biological activities as well as the preventive activity of crop disease
control.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[17] A.F. Suffredini, G. Fantuzzi, R. Badolato, et al., New Insights into the Biology of the
Acute Phase Response, J. Clin. Immunol. 19 (1999) 203–214, https://doi.org/
10.1023/A:1020563913045.
[18] Mantovani A, Dinarello CA, Molgora M, Garlanda C. Interleukin-1 and Related
Cytokines in the Regulation of Inflammation and Immunity. Immunity. 50 (2019)
778-795. https://doi: 10.1016/j.immuni.2019.03.012. PMID: 30995499; PMCID:
PMC7174020.
Acknowledgment
We thank Prof. K. Nagai and Prof. T. Kataoka (Tokyo Institute of
Technology) for assay of inhibitory activity on ICAM-1 and inhibitory
7