Synthesis and biological evaluation of parthenolide derivatives with reduced toxicity as potential inhibitors of the NLRP3 inflammasome

Article abstract of DOI:10.1016/j.bmcl.2020.127399

Parthenolide (PTL) can target NLRP3 inflammasome to treat inflammation and its related disease, but its cytotoxicity limits further development as an anti-inflammatory drug. A series of PTL analogs and their Michael-type adducts were designed and synthesi

Full text of DOI:10.1016/j.bmcl.2020.127399

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Synthesis and biological evaluation of parthenolide derivatives with reduced
toxicity as potential inhibitors of the NLRP3 inflammasome
Yitao Ou, Ping Sun, Nannan Wu, Hao Chen, Dan Wu, Wenhui Hu, Zhongjin
Yang
PII:
S0960-894X(20)30510-2
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https://doi.org/10.1016/j.bmcl.2020.127399
BMCL 127399
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29 June 2020
6 July 2020
Please cite this article as: Ou, Y., Sun, P., Wu, N., Chen, H., Wu, D., Hu, W., Yang, Z., Synthesis and biological
evaluation of parthenolide derivatives with reduced toxicity as potential inhibitors of the NLRP3 inflammasome,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127399
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Synthesis and biological evaluation of parthenolide derivatives with
reduced toxicity as potential inhibitors of the NLRP3 inflammasome
Yitao Ou a, †, Ping Sun a, †, Nannan Wu a, †, Hao Chen a, Dan Wu a, Wenhui Hu a, *, and
Zhongjin Yang a, *
aKey Laboratory of Molecular Target & Clinical Pharmacology and State Key
Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & the Fifth
Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong, 511436,
China.
† These authors contributed equally to this work.
* Corresponding authors:
For Zhongjin Yang: phone: +86 20 31100920; E-mail: yzj23@163.com.
For Wenhui Hu: phone: +86 20 31100920; E-mail: huwenhui@gzhmu.edu.cn.
1

Abstract:
Parthenolide (PTL) can target NLRP3 inflammasome to treat inflammation and its
related disease, but its cytotoxicity limits further development as an anti-inflammatory
drug. A series of PTL analogs and their Michael-type adducts were designed and
synthesized, and most of them showed high activities against the NLRP3 inflammasome
pathway. The most potent compound 8b inhibited the release of IL-1β with IC50 values
of 0.3 μM in J774A.1 cell and 1.0 μM in primary glial cells, respectively. Moreover, 8b
showed low toxicity against J774A.1 cell (IC50 = 24.1 μM) and HEK-293T (IC50 = 69.8
μM) with a ~8 folds increase of therapeutic index compared to its parent PTL. The
preliminary mechanism study revealed that 8b mediated anti-inflammation is associated
with the NLRP3 inflammasome signal pathway. Based on these investigations, we
propose that 8b might be a potential drug candidate for ultimate development of the
anti-inflammation drug.
Keywords: Parthenolide; NLRP3 inflammasome inhibitor; NO donor; low cytotoxicity
2

Inflammation is a part of the immune system's defense response triggered by infection
or tissue damage1. However, dysregulated and chronic inflammation can cause
inflammatory tissue reactions, and eventually lead to a diverse range of non-
communicable diseases2. Increasing evidence indicates that over-activation of NLRP3
inflammasome is responsible for various diseases, such as inflammatory diseases —
peritonitis, hyperinflammation following virus infection and inflammatory bowel disease,
and inflammation-related diseases — Alzheimer's disease (AD), stroke, gout and
nonalcoholic steatohepatitis (NASH)3. In particular, knockout of NLRP3 can ameliorate
the pathology in a mice model for these diseases. The findings indicate that NLRP3 is an
attractive drug target for curing NLRP3 inflammasome-driven diseases.
Guaianolides:
Germacranolides:
Eudesmanolides:

X
H
O
O
O
HO

O
O
O
5
Micheliolide ( )
3
-Cyclocostunolide ( )
1
Costunolide ( ): X= single bond
4
-Cyclocostunolide ( )
2
Parthenolide ( ): X = O
Figure 1. Selected naturally occurring SLs.
Parthenolide (PTL, 2), a germacranolide-type sesquiterpene lactone (SL) of a 10,5-ring
structure, was found to have potent anti-inflammatory effects for decades4-6. However,
only recently has it attracted extraordinary research interest due to it targeting NLRP3
inflammasome7. In addition, PTL powerfully alleviates neuroinflammation8 and
ameliorates brain injury in vivo9. PTL of great potency inspires our curiosity about other
types of SL, including eudesmanolides and guaianolides (Figure 1). For another, SLs
suffer from the high cytotoxicity drawback10-12 that limits their further development for
treating inflammatory disease. Structurally, all of SLs contain α-methylene-γ-lactone
moiety with high reactivity and side-chain with lipophilicity12. Indirect evidence links
3

their cytotoxicity to the a-methylene-g-lactone moiety13, 14. Apparently, the α,β-
unsaturated carbonyl system, a Michael acceptor, can increase SL’s toxicity toward cells
via covalently binding to biological nucleophiles12, 15
.
Figure 2. Design of SL derivatives based on reducing cytotoxicity
The key impact of α,β-unsaturated carbonyl moiety drew our attention towards its
modification to reduce cytotoxicity and to improve anti-inflammatory activity. It was also
reported that most Michael adducts of SLs (amino16-18 or thiol19) exhibited less
cytotoxicity compared to its parent SL. Therefore, as shown in Figure 2, the adjunction
of cytoprotective fragment, such as riluzole20 or NO donor21,22, may be a feasible means
to improve the safety of drugs.
Herein, we synthesized SLs of various carbocyclic skeletons and used dialkylamine-
containing cytoprotective agents for conjugation with SL to resolve the aforementioned
potency and cytotoxicity problems.
4

a
H
H
O
O
O
O
O
O
4
-Cyclocostunolide ( )
1
(+)-Costunolide ( )

3
-Cyclocostunolide ( )
b or c
H
O
O
S
O
O
N
R
N
N
N
X =
Y =
Z =
OCF3
N
O
2
O
O
Parthenolide ( )
8a
8b
8c
: R = X
: R = Y
: R = Z
SO2Ph
O
a
N
O
N
N
O
N
O
O
O
b or c
SO2Ph
H
R
O
HO
H
O
O
HO
9a
9b
9c
: R = X
: R = Y
: R = Z
O
5
Micheliolide ( )
O
O
Boc
HN
F3CO
F3CO
N
S
N
S
OH
NH2
NH
d
N
6
Riluzole ( )
10
O
O
PhO2S
SO2Ph
f
e
S
CO2H
S
CO2H
N
N
O
O
11
12
7
NO donor ( )
Scheme 1. Synthesis of diverse types of SLs and their derivatives.
aReagents and conditions: (a) p-TsOH, CH2Cl2, rt, 8 h; (b) 11, TEA, CH2Cl2, rt, 10 h;
(c) 2-(methylamino)ethanol or 3-(methylamino)-1-Propanol, TEA, CH2Cl2, rt, 10 h;
then adding THF, NaH, and 5. (d) HATU, DIEA, DMF, rt, 8h; then HCl in MeOH; (e)
30% H2O2, AcOH, rt, 4 h; (f) fuming HNO3, 90 °C, 4 h.
Firstly, the preparation of sesquiterpene lactones of various carbocyclic skeletons is
illustrated in Scheme 1. Starting with the natural product (+)-costunolide (1), treatment
of 1 with p-toluenesulfonic acid (p-TsOH) in CHCl2 underwent transannular cyclization
5

to generate eudesmanolides 3 and 4 in yields of 36% and 51%, respectively23. Under the
same condition, the guaianolide micheliolide (5) was yielded from parthenolide (2)24.
The intermediates 10 or 7 was readily obtained from compound 6 or 11 according to a
reported procedure25, 26. A Michael addition reaction of PTL or MCL with 10 provided
the riluzole–PTL or MCL conjugates 8a and 9a. Alternatively, the conjugates 8b–c and
9b–c was prepared by the nucleophilic substitution reaction of 7 and Michael adducts of
PTL or MCL with 2-(methylamino)ethanol.
Figure 3. SLs inhibits the IL-1β release in J774A.1 and primary glial cells upon
stimulation with LPS/Nigericin.
Next, we established a cell model using the murine macrophages cell line J774A.127
and primary glial cells28 in which the release of IL-1β is mediated via the NLRP3
inflammasome activation upon priming with lipopolysaccharide (LPS). Firstly, the
natural sesquiterpene lactones 1 – 5 were tested for their abilities to inhibit the release of
IL-1β. As shown in Figure 3, all types of sesquiterpene lactones showed high
6

concentration-dependent inhibition of IL-1β release in both J774A.1 and primary glial
cells. It is obvious that germacranolides of a 5,10-ring skeleton, such as compound 1 and
2, had higher inhibitory potency than eudesmanolides of a 5,6,6-ring skeleton (3 and 4)
and micheliolide (5) of a 5,7,5-ring skeleton.
Figure 4. Compound 8a and 9a inhibit the IL-1β release in J774A.1 cells and primary
glial cells upon stimulation with LPS/Nigericin upon stimulation with LPS/Nigericin.
Table 1. Cytotoxicity and Inhibitory Potency against the IL-1β Production in J774A.1
and Primary Glial Cells of Designed Derivatives
Inhibition of IL-1β
(IC50, μM)
Cytotoxicity
(TC50, μM)
Cmpd
TI a
J774A.1
Glial cells
1.8
J774A.1
HEK-293T
29.9
PTL
7
1.4
0.93
2.3
14.3
11.4
21.9
24.1
66.6
59.5
68.5
10.2
12.2
1.9
22.4
MCL
8b
5.9
51.7
9.5
0.30
0.40
0.50
0.49
1.0
69.8
80.3
8c
2.2
166.1
56.5
166.5
119.0
139.8
9b
1.7
9c
2.3
105.5
a Therapeutic index (TI) calculated for TC50 (J774A.1) versus IC50 (J774A.1)
7

After the evaluation of inhibitory activity on IL-1β release in J774A.1 and primary glial
cells for the sesquiterpene lactones, the two highly potent and readily available
compounds, PTL and MCL, were selected for further research. The preliminary screening
results showed that riluzole (6) had no inhibition of IL-1β release in J774A.1 cell and
slight activity in primary glial cells at 10 μM (Figure 4). Unsurprisingly, the conjugate of
PTL or MCL with 6, 8a or 9a, led to a significant drop in the inhibition of IL-1β release.
Despite this, no complete loss of activity by modification of α, β-unsaturated double bond
indicates the design is workable if the linked molecule possesses good activity against
NLRP3. The notion is supported by the results of 8b–c and 9b–c, as a conjugation of PTL
or MCL with NO donor 7. As shown in Table 1, PTL, MCL and 7 exhibited good potency
on inhibiting IL-1β release in J774A.1 with IC50 values of 1.4 μM, 2.3 μM and 0.93 μM,
respectively, but with relatively high cytotoxicity (PTL: 14.3 μM; MCL: 21.9 μM; 7: 11.4
μM). As expected, all the four synthesized conjugates 8b–c and 9b–c significantly
reduced cytotoxicity against J774A.1 and HEK-293T, and resulted in a remarkable
increase of inhibitory potency. Notably, their conjugates 8b showed the highest inhibitory
potency in J774A.1 (IC50 = 0.30 μM) and primary glial cells. The cytotoxicity data clearly
demonstrated a 1.5-folds and 2.3-folds decrease of cytotoxicity against J774A.1 and
HEK-293T, respectively, as compared to that of PTL. The therapeutic index of 8b is up
to 80.3, which is important for a wide therapeutic window. Although the chain extension
to aminopropanol as in 8c led to lower toxicity, a slightly decreased potency was
observed. A similar case was found in the conjugates 9b and 9c. Further tests in primary
glial cells confirmed 8b as the most potent inhibitor against NLRP3 inflammasomes (IC50
= 1.0 μM).
8

Figure 5. Compound 8b reduces NLRP3-inflammasome-derived IL-1β secretion.
Medium SN and cell extracts were analyzed by immunoblotting, as indicated
Taking account that compound 8b showed the most potent inhibition of IL-1β release,
it was further selected for investigation of its preliminary mechanism. Western blots
showed that treatment with 8b dose-dependently decreased the amount of caspase-1 p20
and IL-1β in supernatants (Fig. 5). Compound 8b did not affect the expression levels of
pro-IL-1β, pro-caspase-1, NLRP3, or ASC in cell lysates (Fig. 5). These findings suggest
compound 8b suppresses NLRP3 Inflammasome-mediated caspase-1 activation and IL-
1β secretion.
9

Figure 6. Compound 8b ameliorates peritoneal inflammation in vivo.
Finally, we investigated whether 8b could inhibit the NLRP3-driven inflammation in
animal models of LPS-induced inflammatory responses in vivo29. mice were injected
intraperitoneally with LPS and showed significantly increased IL-1β in lavage fluid. As
shown in Figure 6, parthenolide was tested as a positive control. The level of IL-1β in
both serum and peritoneal fluid was obviously reduced by the treatment of 8b
(30mg/kg/d). Notably, compounds 8b exhibited activity comparable to that of PTL in the
LPS-challenged mice. This could be due to different molecular weight as well as
pharmacokinetic properties.
In conclusion, the rapid formation of eudesmanolides and guaianolides can be achieved
via germacranolides. SLs of the various skeleton, especially PTL and MCL, showed great
inhibitory potency on NLRP3-mediated IL-1β release. Design of the linkage between
PTL or MCL with NO donor 7 leads to four conjugates with high potency and low
cytotoxicity. It is worth noting that compound 8b was the most potent against the release
of IL-1β with a high TI value of 80. These in-vitro results encouraged us to further
evaluate its in-vivo efficacy on NLRP3-driven inflammation. Indeed, the treatment with
8b improved the high IL-1β levels caused by peritoneal inflammation in mice.
Collectively, the results of 8b together with that of 9b prove the effectiveness of our
10

design for lowering Michael receptor-induced toxicity. These data also warrant further
development of 8b and analogs as potential agents for the treatment of peritoneal
inflammation as well as other NLRP3 inflammasome-driven diseases.
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.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (NSFC)
(NO. 81803364 to Z. Yang and NO. 81872743 to W. Hu). The authors especially thank
Prof. Chen Yue from the Nankai University for providing us with raw materials of
parthenolide and costunolide free of charge.
Appendix A. Supplementary data
The following are the Supplementary data to this article. Supporting data to this article
can be found online.
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Graphical Abstract
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