Discovery of bardoxolone derivatives as novel orally active necroptosis inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.113030

Necroptosis is a form of programmed cell death that contributes to the pathophysiology of cerebral ischemia/reperfusion (I/R) injury. In this study, bardoxolone (CDDO, 7) was an inhibitor of necroptosis identified from an in-house natural product library. Further optimization led to identify a more potent analogue 20. Compound 20 could effectively protect against necroptosis in human and mouse cells. The antinecroptotic effect could also be synergized with other necroptosis inhibitors. It blocked necrosome formation by targeting Hsp90 to inhibit the phosphorylation of RIPK1 and RIPK3 in necroptotic cells. In vivo, this compound was orally active to alleviate TNF-induced systemic inflammatory response syndrome (SIRS) and cerebral I/R injury. Our results suggested that 20 could be a lead compound for discovering necroptosis inhibitors in I/R treatment.

Full text of DOI:10.1016/j.ejmech.2020.113030

Journal Pre-proof
Discovery of bardoxolone derivatives as novel orally active necroptosis inhibitors
Yuanyuan Wang, Hao Ma, Jiaxuan Huang, Zhengguang Yao, Jianqiang Yu, Wannian
Zhang, Lichao Zhang, Zhibin Wang, Chunlin Zhuang
PII:
S0223-5234(20)31002-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.113030
EJMECH 113030
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 September 2020
Revised Date: 15 November 2020
Accepted Date: 15 November 2020
Please cite this article as: Y. Wang, H. Ma, J. Huang, Z. Yao, J. Yu, W. Zhang, L. Zhang, Z. Wang, C.
Zhuang, Discovery of bardoxolone derivatives as novel orally active necroptosis inhibitors, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113030.
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Graphical Abstract

Discovery of bardoxolone derivatives as novel orally active
necroptosis inhibitors
Yuanyuan Wang†§,1, Hao Ma†ʃ,1, Jiaxuan Huang†¶,1, Zhengguang Yao,† Jianqiang Yu¶, Wannian Zhang†¶,
Lichao Zhang,§,* Zhibin Wang†*, Chunlin Zhuang†¶*
† School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
¶ School of Pharmacy, Ningxia Medical University, 1160 Shengli Street, Yinchuan 750004, China
ʃ Chinese Academy of Medical Sciences and Peking Union Medical College, 2A Nan Wei Road, Beijing
100050, China
§ Department of Pharmacy, Shanghai Municipal Hospital of Traditional Chinese Medicine, 274 Middle
Zhijiang Road, Shanghai 200071, China
Supporting Information
AUTHOR INFORMATION
Corresponding Author
* E-mail: zclnathan@163.com (Chunlin Zhuang), methyl@smmu.edu.cn (Zhibin Wang), chang-
haiskin@163.com (Lichao Zhang)
Author Contributions
1 These authors contributed equally to this work.
Notes
The authors declare no competing financial interests.

ABSTRACT
Necroptosis is a form of programmed cell death that contributes to the pathophysiology of cerebral
ischemia/reperfusion (I/R) injury. In this study, bardoxolone (CDDO, 7) was an inhibitor of necroptosis
identified from an in-house natural product library. Further optimization led to identify a more potent
analogue 20. Compound 20 could effectively protect against necroptosis in human and mouse cells. The
antinecroptotic effect could also be synergized with other necroptosis inhibitors. It blocked necrosome
formation by targeting Hsp90 to inhibit the phosphorylation of RIPK1 and RIPK3 in necroptotic cells.
In vivo, this compound was orally active to alleviate TNF-induced systemic inflammatory response
syndrome (SIRS) and cerebral I/R injury. Our results suggested that 20 could be a lead compound for
discovering necroptosis inhibitors in I/R treatment.

¢ INTRODUCTION
Programmed cell death (PCD) has been recognized to mediate normal tissue homeostasis and closely
relate with multiple human diseases.[1] Caspase-dependent apoptosis was the first well-characterized
and was recognized as the only form of PCD in a long period of time.[2, 3] Oppositely, necrosis was
traditionally considered as an uncontrolled accidental cell death (ACD) triggered by physical stresses in
a totally different manner.[4, 5] In the last 30 years, more and more evidences have supported that a
subset of necrosis is an important form of PCD, which has been first named as “necroptosis” by Yuan et
al.[6] Necroptosis is now defined as a caspase-independent PCD that plays a critical role in various le-
thal diseases such as ischemia-reperfusion injury, systemic inflammatory response syndrome (SIRS),
atherosclerosis, etc.[6-9] Thus, targeting the pathologic necroptosis pathway has been deemed as a novel
strategy for treating these diseases.[7]
Necroptosis is triggered by the activation of death receptors and tumor necrosis factor-α (TNF-α) is best
understood to activate the receptor.[10] In response to the activation, downstream receptor-interacting
protein kinase 1 (RIPK1) is recruited and activated to interact with RIPK3 to initiate the formation of
necrosomes and mediate the recruitment and phosphorylation of mixed lineage kinase domain-like pro-
tein (MLKL).[11-14] Oligomers are formed from phosphorylated MLKL, which is then translocated
into the plasma membrane to trigger membrane rupture to mediate necrotic cell death.[15-18] Necropto-
sis is considered to be mediated by RIPK1, RIPK3, and MLKL, which have been identified as critical
therapeutic targets.[19]
In 2005, a pioneering work by Yuan group reported the first necroptosis inhibitor Nec-1,[6] and to date
more than 20 classes of inhibitors have been identified.[20] Compound 1, named GSK2982772 (Figure
1), possessing highly RIPK1 selective inhibitory activity, has been advanced into phase II trials in sev-
eral indications including ulcerative colitis (UC) (NCT02903966), psoriasis (NCT02776033) and rheu-
matoid arthritis (NCT02858492).[21-24] An analogue 2, named GSK3145095, has been advanced to
phase I/II trials in subjects with pancreatic ductal adenocarcinoma cancer (PDAC) and other tumors
(NCT03681951).[25] DNL747 is a RIPK1 inhibitor with undisclosed chemical structure, has been ad-

vanced into phase I trials to evaluate the safety, tolerability, PK, and PD in patients with amyotrophic
lateral sclerosis (ALS, NCT03757351) and Alzheimer’s disease (AD, NCT03757325).[26, 27] Natural
products, with strong biocompatibility, have been the most successful source of potential drug leads.[28]
A noncanonical Hsp90 inhibitor 3, covalently modifying Cys420 of Hsp90 was reported to block
RIPK3-dependent necroptosis in HT-29 cells (IC50 = 200 nM).[29] Compound 4, named 6E11, selec-
tively targeting RIPK1 (KD = 130 nM), exhibited anti-necroptosis in Jurkat T cells.[30] Compounds 5-6,
named geldanamycins, were reported to decrease RIPK1 and Hsp90 proteins, protecting against neu-
ronal injury.[31]
Figure 1. Disclosed necroptosis inhibitors in the clinical trials and inhibitors from natural sources
Thus, novel chemical structures with valuable biological activities and specificities may be further pro-
vided by analyzing other natural sources. In this study, we found that natural product oleanolic acid de-
rivative, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO, 7) was a novel inhibitor of

necroptosis. Furthermore, highly potent analogues were developed based on the CDDO template and the
in vivo efficacy was also evaluated.
¢ RESULTS AND DISCUSSION
Drug screening identifies CDDO as a necroptosis inhibitor. An in-house library of ~300 natural
products and nature-like derivatives was established and screened for their ability to block TNFα-
induced necroptosis. HT-29 cells were treated with TNFα, a Smac mimetic (SM-164), and a caspase
inhibitor z-VAD-fmk (TSZ) to induce necroptosis and the cell survival ability was measured by a chem-
iluminescence assay for each compound at a concentration of 10 µM (Figure 2A).[32, 33] Among the
natural products investigated, a triterpenoid compound bardoxolone, also named CDDO, was found to
clearly protect HT-29 cells from TSZ-induced necroptosis (viability > 100%, Figure 2B). In addition, a
known inhibitor pazopanib was included as positive control,[34, 35] and was identified (viability >
80%) in our screening, supporting the validity of our experimental protocol. Phase contrast microscopy
further revealed that CDDO prevented TSZ-induced necrotic morphology, including cell swelling and
plasma membrane rupture (Figure 2C).

Figure 2. Identification of CDDO as a potent natural necroptosis inhibitor. (A) Schematic overview of
drug screen workflow; (B) Identification of necroptosis inhibitor by cellular screening with an in-house
natural product library. HT-29 cells were pre-treated with each compound (10 μM) for 30 min and then
stimulated with TSZ for 16 h to induce necroptosis. Cell survival was determined by a CellTiter-Glo
luminescent cell viability assay and normalized to untreated control cells. (C) Representative images
(200×) of HT‐29 cells pretreated with DMSO or CDDO (5 μM) followed by stimulation with TSZ for
12 h.

Figure 3. (A) HT-29 cells were treated with CDDO as indicated concentrations followed by stimulation
with TSZ for 12 h. (B) HT-29 cells were treated with various concentrations of CDDO as indicated by
stimulation with TNF-α (20 ng/mL), cycloheximide (5 μg/mL), and z-VAD-fmk (20 μM) (TCZ) for 16
h. (C) HT-29 cells were treated with CDDO as indicated concentrations followed by stimulation with
TS for 20 h. (D) HT-29 cells were treated with various concentrations of CDDO as indicated by stimula-
tion with TC for 24 h. (E) L929 cells were pretreated with CDDO as indicated concentrations followed
by stimulation with TZ for 12 h. Results shown are means ± S.D. from three independent experiments.

(*p <0.05, ** p <0.01, *** p <0.001 versus TSZ, TCZ, TS, TC or TZ simulation without CDDO treat-
ment).
We then performed a dose-response assay to quantitatively analyze the inhibitory potency of CDDO. It
could dose‐dependently inhibit TSZ (Figure 3A) with an EC50 value of 1.30 ± 0.38 µM (Table 1) or
TNF‐α, cycloheximide, and z-VAD-FMK (TCZ)-induced necroptosis (Figure 3B). However, it showed
cytotoxicity at 10 µM.[36, 37] Remarkably, CDDO did not protect cells from TNF‐α plus Smac mimetic
(TS) or cycloheximide (TC)‐induced apoptosis in HT‐29 cells, suggesting that CDDO specifically in-
hibited the necroptosis pathway (Figure 3C, D). In murine L929 cells, we found that CDDO did not
protect against necroptosis induced by TNF-α and z-VAD-fmk (TZ) (Figure 3E).
Identification of CDDO derivatives as potent antinecroptotic agents. Structurally, CDDO is an
oleanolic acid triterpenoid. Two triterpenoids, Oleanolic acid and Echinocystic acid, showed no appar-
ent protective effect against necroptosis in human HT-29 cells at 10 µM (Table 1). Then, we obtained
four analogues of CDDO with different substituents at the carboxyl group. CDDO-Me with a methyl
ester had a lower antinecroptotic effect than that of CDDO with an EC50 of 4.34 ± 1.00 µM (HT-29).
Similarly, it still had no effect in murine L929 cell as CDDO. The ethyl amide analogue (CDDO-EA)
had a comparable EC50 of 1.38 ± 0.10 µM to CDDO in HT-29 cells and an EC50 of 4.15 ± 0.35 µM in
L929 cells. RTA-408 with a transpositioned amide and difluoro-substitution possessed a similar inhibi-
tory effect (HT-29, EC50 = 1.41 ± 0.16 µM; L929, EC50 = 5.25 ± 0.56 µM) to that of CDDO-EA.
Table 1. Antinecroptotic activity of commercial CDDO derivatives
HT-29, EC50 (µM) L929, EC50 (µM)
Entry Compound name
Chemical structure
a
b

CDDO
1.30 ± 0.38
>10
7
Oleanolic acid
> 10
N.D.
8
Echinocystic ac-
id
> 10
N.D.
9
O
H
OCH3
CDDO-Me
CDDO-EA
H
4.34 ± 1.00
>10
10
11
NC
O
O
H
1.38 ± 0.10
4.15 ± 0.35

RTA-408
1.41 ± 0.16
5.25 ± 0.56
12
aHuman HT-29 cells were pretreated with DMSO or the test compound and then stimulated with TNF-α
(20 ng/mL), Smac mimetic (10 nM), and z-VAD-fmk (20 μM) (TSZ) for 16 h. The inhibition of TSZ-
induced necroptosis in HT-29 cells is presented as the EC50 ± standard deviation (SD). b L929 cells were
pretreated with DMSO or the test compound and then stimulated with mouse TNF-α (20 ng/mL) and z-
VAD-fmk (20 μM) (TZ) for 4 h. The inhibition of TZ-induced necroptosis in L929 cells is presented as
the EC50 ± standard deviation (SD). All experiments were repeated independently at least three times.
Next, we synthesized some analogues based on the CDDO triterpenoid (Table 2). Considering the result
of Table 1, the amide showed good antinecroptotic activity in both human and murine cells. The benzyl
amide (13) was evaluated to show EC50 values of 1.63 ± 0.10 µM (HT-29) and 1.03±0.01 µM (L929).
Compound 14 with aliphatic amine had a less potency against necroptosis. Compound 15 with a cyclo-
hexane group had an EC50 value of 1.30 ± 0.38 µM in human HT-29 cells, however, no apparent protec-
tive effect in murine L929 cells at 10 µM. Then, a tetrahydronaphthalen-1-amino group (16-17) im-
proved the antinecroptotic activity in murine L929 cells with the EC50 values lower than 1 µM and the
activity toward human HT-29 cells was kept. The chirality showed no remarkable influence on the ac-
tivity. Finally, we evaluate a commercially available imidazole CDDO derivative (CDDO-Im, 18). This
compound showed the protective activity at a similar level compared with those of 13-17. Then, if 2-
pyridinyl (19) or 3-pyridinyl (20) group was attached on the imidazole, the antinecroptotic activity was
improved in about 7-fold in HT-29 cells and good protective activity was demonstrated toward L929
cells.
Table 2. Antinecroptotic activity of amide substituted CDDO derivatives

Entry
R
HT-29, EC50 (µM) a
L929, EC50 (µM) b
1.63 ± 0.10
2.81 ± 0.09
1.03 ± 0.01
1.48 ± 0.03
13
14
NHCH2CH2NHBoc
1.30 ± 0.38
1.91 ± 0.20
>10
15
16
0.82 ± 0.03
1.81 ± 0.05
0.62 ± 0.01
17
2.10 ± 0.10
0.29 ± 0.01
0.31 ± 0.02
1.46 ± 0.08
0.61 ± 0.02
0.86 ± 0.11
18
19
20
aHuman HT-29 cells were pretreated with DMSO or the test compound and then stimulated with TNF-α
(20 ng/mL), Smac mimetic (10 nM), and z-VAD-fmk (20 μM) (TSZ) for 16 h. The inhibition of TSZ-
induced necroptosis in HT-29 cells is presented as the EC50 ± standard deviation (SD). b L929 cells were
pretreated with DMSO or the test compound and then stimulated with mouse TNF-α (20 ng/mL) and z-

VAD-fmk (20 μM) (TZ) for 4 h. The inhibition of TZ-induced necroptosis in L929 cells is presented as
the EC50 ± standard deviation (SD). All experiments were repeated independently at least three times.
Compound 20 inhibit necroptosis and apoptosis. Compounds 19 and 20 showed the best activity at a
similar level. We next randomly selected 20 for further evaluation. It efficiently restored cell viability
from TSZ or TCZ -induced necroptosis in a dose-response manner (Figure 4A, B). The necroptotic
cells could be protected completely at 0.5 and 1 µM. Different from CDDO, this compound could pro-
tect cells from TS or TC-induced apoptosis in HT-29 cells, suggesting that compound 20 inhibits
necroptosis and apoptosis pathways (Figure 4C, D). For murine L929 cells, it dose-dependently pro-
tected against necroptosis induced by TZ (Figure 4E). Thus, our results suggest that compound 20 is an
effective programmed cell death inhibitor better than the parent compound CDDO.

Figure 4. (A) HT-29 cells were treated with 20 as indicated concentrations followed by stimulation with
TSZ for 12 h. (B) HT-29 cells were treated with various concentrations of 20 as indicated by stimulation
with TNF-α (20 ng/mL), cycloheximide (5 μg/mL), and z-VAD-fmk (20 μM ) (TCZ) for 16 h. (C) HT-
29 cells were treated with 20 as indicated concentrations followed by stimulation with TS for 20 h. (D)
HT-29 cells were treated with various concentrations of 20 as indicated by stimulation with TC for 24 h.
(E) L929 cells were pretreated with 20 as indicated concentrations followed by stimulation with TZ for

12 h. Results shown are means ± S.D. from three independent experiments. (*p <0.05, ** p <0.01, *** p
<0.001 versus TSZ, TCZ, TS, TC or TZ simulation without treatment).
Compound 20 targets Hsp90 not Nrf2 to blocks necrosome formation by inhibiting the phos-
phorylation of RIPK1 and RIPK3. CDDO has been reported to target multiple proteins, and Keap1-
Nrf2 and Hsp90 are in the majority.[38] The representative inhibitors of these two targets have been se-
lected to evaluate the relevance of these targets in necroptosis. However, the two Nrf2 modulators
brusatol[39] and dimethyl fumarate[40] showed no protection against necroptosis at 10 µM (Figure
5A). The result, suggesting that Nrf2 was not the common target for CDDO’s antinecroptotic effect, was
consistent with the Yuan’s report, which revealed the protection of necroptosis by CDDO in HT-29 cells
with Nrf2 knockdown.[41] Hsp90 was reported to be associated with RIPK1[42, 43] and the necroptosis
could be effectively blocked by knockdown of Hsp90.[41] Two synthetic chemical Hsp90 inhibitors
(Onalespib[44] and Tanespimycin[45]) exhibited antinecroptotic activity at 10 µM (Figure 5A). Fur-
thermore, compound 20 showed synergistic effects with necroptosis inhibitors including TAK632 (a
RIPK1/3 inhibitor),[33] SZM630 (a RIPK3 inhibitor),[32] Nec-1 (a RIPK1 inhibitor),[6] and NSA (a
MLKL inhibitor)[13] in TSZ-treated HT-29 cells, respectively (Figure 5B). The concentration used for
CDDO was 10 µM.
We next examined if compound 20 could inhibit Hsp90 in HT-29 cells by testing two biomarkers of
Hsp90 inhibition. Consistent with other Hsp90 inhibitor, we found that compound 20 time-dependently
induced reduction in the levels of a well-known Hsp90 client protein, EGFR and remarkable increases
in the levels of Hsp70 (Figure 5C). They were both known to occur upon the inhibition of Hsp90.[46]
Then, we examined phosphorylation of RIPK1, RIPK3, and MLKL in TSZ-treated HT-29 cells with or
without 20. As shown in Figure 5C, 20 could effectively inhibit the phosphorylation RIPK1, RIPK3,
and MLKL. As the phosphorylation of RIPK1 and RIPK3 is required for RIPK1−RIPK3 necrosome
formation,[12] we then explored the formation of necrosome in HT-29 cells after pretreating with 20,
and we found that it blocked TSZ-induced necrosome formation (Figure 5D). These data suggest that

compound 20 potentially targets Hsp90 not Nrf2 to block necrosome formation by inhibiting the phos-
phorylation of RIPK1 and RIPK3.
Figure 5. (A) HT-29 cells were treated with the indicated compounds at 10 µM followed by stimulation
with TSZ for 12 h. (B) HT-29 cells were treated with 20 combined by the indicated compounds fol-
lowed by stimulation with TSZ for 12 h to evaluate the synergistic effects. (C) HT-29 cells were pre-
treated with 20 (10 µM) for 30 min and then treated with TSZ for the indicated periods of time. Cells
were lysed and immunoblotted with the indicated antibodies. (D) HT-29 cells were treated with DMSO
or 20 (10 μM) for 6 h. The cell lysates were immunoprecipitated with an anti-RIPK1 antibody (IP:

RIPK1) and analyzed by immunoblotting with the indicated antibodies. (*** p <0.001 versus TSZ simu-
lation, ### p <0.001 versus TSZ simulation with compound 20 treatment, n.s., not significant).
Compound 20 protects mice from TNF-induced systemic inflammatory response syndrome
(SIRS). To explore whether compound 20 protects against RIP kinase‐driven inflammation in vivo, we
tested it in the TNF‐induced SIRS model.[33] Compound 20 (10 or 20 mg/kg) given by intragastric ga-
vage 2 h before i.v. injection of mTNF‐α, protected mice from hypothermia (Figure 6A) and death
(Figure 6B). Especially, the compound could completely reverse the mice from death at 20 mg/kg
(100%). Furthermore, when these mice were examined at 6 h, the typical serum inflammatory cytokines
including IL-1β (Figure 6C) and IL‐6 (Figure 6D) were markedly decreased after the treatment. Thus,
these results demonstrate that compound 20 protects against TNF‐induced SIRS in vivo.
Figure 6. Compound 20 protects mice from TNF-induced SIRS. (A) C57BL/6 J mice were pre-
treated with/out 20 (10, 20 mg/kg) and then the SIRS model was induced by TNF. The body tempera-

ture (means ± S.E.M.) and (B) survival curve of the vehicle control and 20 treated mice (n=11 for each
group) are shown. (C) After SIRS induction for 6 h, serum levels of IL-1β, IL-6 from vehicle control
and compound 20 (20 mg/kg) treated mice were determined by ELISA. *, p < 0.05; **, p < 0.01; ***, p
< 0.001.
Figure 7. Compound 20 (100 mg/kg) protected against I/R injury in MCAO rats. (A) Neurological defi-
cit scores, n =6 per group. (B) The percentage of infarct volume was detected for each group, n = 6 per
group. (C) Representative TTC staining of the cerebral infarct in brain, n = 6 per group. (D) HE stain-
ing: Ischemic cerebral cortex (magnification: ×200), ischemic cerebral hippocampus CA3 (magnifica-

tion: ×100) and ischemic cerebral hippocampus CA1 (magnification: ×200), n = 6 per group. (*p < 0.05,
** p < 0.01, *** p < 0.001 versus the I/R group.)
Compound 20 protects brain against ischemia/reperfusion (I/R) injury. Extensive evidence sug-
gests that necroptosis is a delayed component of ischemic neuronal injury, representing a promising
therapeutic strategy for treatment of stroke.[6, 26, 47, 48] To examine the effect of potent compound 20
on the ischemic brain injury, we first examined the neurologic scores to determine neurological deficits
after reperfusion for 24 h using middle cerebral artery occlusion (MCAO) rats. The neurological deficit
score of 20-treated group (100 mg/kg, score = 2.72 ± 0.45) was significantly decreased compared with
the I/R group (score = 3.71 ± 0.45) (Figure 7A). The infarct volume of the cerebral ischemic area was
measured by 2,3,5- triphenyltetrazolium chloride (TTC) staining. Pretreatment of 20 reduced infarct
volume (Figure 7B and C). 20 reduced the infarct volume to 22.8 ± 3.7%, comparable to that of ni-
modipine (26.8 ± 1.7 %). Furthermore, the protective effect of 20 against was further determined by HE
staining on sections from ischemic hippocampus and cortex at 24 h after reperfusion (Figure 7D). The
number of cells in I/R group was decreased and arranged irregularly, and karyopyknosis was observed.
In contrast, 20 pretreatment reversed the change of pathological to a certain extent, indicating that the
compound protected brain from I/R injury.
¢
CONCLUSION
On the basis of CDDO scaffold, an inhibitor of necroptosis identified from an in-house natural product
library, we developed a more potent analogue 20 by structural optimization. This compound could ef-
fectively protect against necroptosis in both human HT-29 and murine L929 cells. Compound 20 might
target Hsp90 not Nrf2 to block necrosome formation by inhibiting the phosphorylation of RIPK1 and
RIPK3. The antinecroptotic effect of the compound could also be synergized with necroptosis inhibitors
including TAK632 (a RIPK1/3 in-hibitor), SZM630 (a RIPK3 inhibitor), Nec-1 (a RIPK1 in-hibitor),
and NSA (a MLKL inhibitor). Intragastric administration of this compound alleviates TNF-induced

SIRS by significantly decreasing the serum levels of IL-1β and IL‐6. It was also orally effective toward
cerebral I/R injury in rat models. Our results suggest that compound 20 could be a lead compound for
discovering more necroptosis inhibitors in I/R treatment, providing a unique opportunity to characterize
the role of necroptosis in I/R or other human pathologies.
¢
EXPERIMENTAL SECTION
Chemistry. General Methods. All solvents and starting materials, including anhydrous solvents and
chemicals, were purchased from commercial vendors and used without any further purification. TLC
analysis was carried out on silica gel plates GF254 (Qindao Haiyang Chemical, China). Column chro-
matography was carried out on silica gel 300~400 mesh. Nuclear magnetic resonance (1H NMR, 13C
NMR) spectra were recorded in CDCl3 on a Bruker Avance 600 spectrometer (Bruker Company, Ger-
many). Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respec-
tively, using tetramethylsilane (TMS) as an internal standard; splitting patterns are designated as fol-
lows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; dd, double doublet; dt, double triplet.
MS spectra were recorded on a Mariner Mass Spectrum (ESI) on Agilent Technologies LC/MSD TOF.
1
13
The final structures were fully characterized by H NMR, C NMR, and MS (ESI). Purities of all the
test compounds were >95% determined by high-performance liquid chromatography (HPLC, Agilent
Technologies 1200 Series) analysis using methanol or acetonitrile /water as the mobile phase with a
flow rate of 0.5 or 2.0 mL/min on a C18 column (YMC-Pack ODS-A, 5 μm, 250 × 10 mmI.D.).

Scheme 1. Synthetic route for CDDO derivatives.
In tested compounds, 7 (CAS: 218600-44-3), 8 (CAS: 508-02-1) and 9 (CAS: 510-30-5) were purchased
from Tokyo Chemical Industry (TCI) and Aladdin Company. Brusatol (CAS: 14907-98-3) and dimethyl
fumarate (CAS: 624-49-7), Onalespib (CAS: 912999-49-6) and Luminespib (CAS: 747412-49-3) were
purchased from Targetmol (Targetmol, USA). Compound 10 (CAS: 218600-53-4), 11 (CAS: 932730-
51-3), 12 (CAS: 1474034-05-3) and 18 (CAS: 443104-02-7) were purchased from MedChemExpress
company (https://www.medchemexpress.cn/). The HPLC purity of the compounds were >98%. Com-
pounds 13-17, 19-20 were synthesized and the synthetic routes of the target compounds were depicted
in Scheme 1. The first key intermediates (acyl chloride, 21) were prepared using commercially available
bardoxolone 7. Then, the intermediate 21 was reacted with amines using a simple substitution reaction
to provide target compounds.

2-cyano-3,12-dioxooleana-1,9(11)-dien-28-
N-benzylformamide (13).[49] To a solution of bardoxo-
lone (CDDO, 30 mg, 0.06 mmol) in dry dichloromethane, oxalyl chloride (16
µL, 0.18 mmol) and cata-
lytic amount of DMF was added. The mixture was stirred at 23 ± 2 ℃ for 2~5 h before the solvent was
removed. The residue was dried under vacuum, then used to the next step without any purification. The
residue was mixed with trimethylamine (16
µL, 0.11 mmol) in DCM and then cooled to 0 °C for 10
min. Then the benzylamine (20 L, 0.18 mmol) was added dropwise. The mixture was stirred at 0 °C
µ
for 4 h and then extracted with DCM. The organic layer was separated, washed with brine and dried
over sodium sulfate. The combined organic layers were evaporated to dryness to obtain the crude prod-
uct, which was purified by column chromatography on silica gel using PE/EtOAc (5:1) as eluent to ob-
tain product as white solid (25.0 mg, yield: 73.5%), mp 186-188 oC. CAS: 443103-59-1. 1H NMR (600
MHz, CDCl3) δ: 0.90 (s, 3H, CH3), 0.99 (s, 6H, 2×CH3), 1.15 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.24 (s,
3H, CH3), 1.46 (s, 3H, CH3), 1.83-1.88 (m, 1H, CH), 1.96-2.01 (m, 1H, CH), 2.91-2.93 (m, 1H, H-18),
3.01 (d, 1H, J = 4.4 Hz, H-13), 4.43-4.51 (m, 2H, NCH2), 5.96 (s, 1H, H-11), 6.18-6.20 (m, 1H, NH),
13
7.26-7.30 (m, 5H, 5
×
ArH), 8.04 (s, 1H, H-1). C NMR (150 MHz, CDCl3)
δ: 18.2, 21.6, 21.7, 23.1,
24.7, 26.6, 27.0, 27.7, 30.4, 30.6, 31.7, 32.0, 33.3, 34.1, 34.6, 36.1, 42.1, 42.5, 43.7, 45.0, 45.8, 46.5,
47.7, 49.5, 114.4, 114.6, 124.0 (CN), 127.4 (Cp-Ph), 128.0 (Co-Ph), 128.7 (Cm-Ph), 138.8 (Ci-Ph), 165.8,
168.7, 176.9 (C-28), 196.6 (C-3), 199.0 (C-12). MS (ESI): 581.402 [M+H]+, 603.343 [M+Na]+, 619.317
[M+K]+. HPLC purity: 97.7%, Rt = 8.690 min, UV 254 nm, 95% methanol, flowrate: 2.0 mL/min.
Tert-butyl(2-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-amide)ethyl)carbamate (14). White solid
o
1
(58.2 mg, yield: 92%), mp 195-197 C. H NMR (600 MHz, CDCl3)
δ: 0.92 (s, 3H, CH3), 1.02 (s, 3H,
CH3), 1.04 (s, 3H, CH3), 1.10 (s, 3H, CH3), 1.28 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.47 (s, 9H, 3
×CH3),
1.50 (s, 3H, CH3), 1.83-1.86 (m, 1H, CH), 2.01-2.06 (m, 1H, CH), 2.97-2.99 (m, 1H, H-18), 3.10-3.11
(m, 1H, H-13), 3.28-3.34 (m, 2H, CH2N), 3.35-3.42 (m, 2H, NCH2), 6.00 (s, 1H, H-11), 6.85 (s, 1H,
13
NH), 8.08 (s, 1H, H-1). C NMR (150 MHz, CDCl3)
δ: 14.2, 18.2, 21.6, 21.7, 23.0, 23.1, 24.8, 26.6,
27.0, 27.8, 28.4, 30.6, 31.7, 33.3, 34.0, 34.6, 36.0, 42.1, 42.5, 45.0, 45.9, 46.4, 47.7, 49.4, 60.4, 114.4,

114.6, 124.0 (CN), 157.2, 165.8, 171.2 (NCOO), 178.1 (C-28), 196.6 (C-3), 199.0 (C-12). MS (ESI):
656.428 [M+Na]+, 672.394 [M+K]+. HPLC purity: 97.5%, Rt = 8.726 min, UV 254 nm, 95% methanol,
flowrate: 2.0 mL/min.
2-cyano-3,12-dioxooleana-1,9(11)-dien-28-
N-cyclohexylformamide (15). White solid (43 mg, yield:
o
74%), mp 187-189 C. CAS: 1788897-54-0. 1H NMR (600 MHz, CDCl3)
δ: 0.90 (s, 3H, CH3), 0.99 (s,
3H, CH3), 1.01(s, 3H, CH3), 1.18 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.34-1.39 (m, 4H,
2
×CH2), 1.48 (s, 3H, CH3), 1.94-1.99 (m, 1H, CH), 2.85-2.87 (m, 1H, H-18), 3.06 (d, 1H, J = 4.6 Hz,
H-13), 3.80-3.82 (m, 1H, NCH), 5.58 (d, 1H, J = 7.8 Hz, NH), 5.97 (s, 1H, H-11), 8.04 (s, 1H, H-1). 13C
NMR (150 MHz, CDCl3) δ: 18.2, 21.6, 21.7, 23.0, 23.1, 24.9, 25.0, 25.5, 26.6, 27.0, 27.8, 30.6, 31.7,
32.0, 33.2, 33.3, 33.3, 34.2, 34.6, 36.1, 42.1, 42.5, 45.0, 46.0, 46.3, 47.7, 48.0, 49.5, 114.4, 114.6, 124.0
(CN), 165.7, 168.6, 176.0 (C-28), 196.6 (C-3), 199.0 (C-12). MS (ESI): 573.467 [M+H]+, 595.398
[M+Na]+. HPLC purity: > 99%, Rt = 9.718 min, UV 254 nm, 95% methanol, flowrate: 2.0 mL/min.
(
R
)-1-
White solid (52 mg, yield: 83.8%), mp 205-207 C. CAS: 2490323-21-0. H NMR (600 MHz, CDCl3)
: 0.90 (s, 3H, CH3), 0.96 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.18 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.29-
N
-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-amide)-1,2,3,4-tetrahydronaphthalene
(16).
o
1
δ
1.36 (m, 2H, CH2), 1.40 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.94-2.04 (m, 2H, CH2), 2.73-2.85 (m, 3H,
CH2, H-18), 3.07 (d, 1H, J = 4.7 Hz, H-13), 5.20-5.23 (m, 1H, NCH), 5.95 (d, 1H, J = 8.2 Hz, NH), 5.97
(s, 1H, H-11), 7.12-7.26 (m, 4H, 4
× δ: 18.3,
ArH), 8.03 (s, 1H, H-1). 13C NMR (150 MHz, CDCl3)
20.1, 21.6, 21.8, 22.9, 23.1, 25.0, 26.7, 27.0, 27.8, 29.3, 30.4, 30.6, 31.7, 32.1, 33.2, 34.2, 34.6, 36.2,
42.2, 42.6, 45.0, 46.0, 46.6, 47.3, 47.7, 49.6, 114.7, 114.4, 124.0 (CN), 126.4, 127.4, 128.4, 129.4,
136.8, 137.8, 165.6, 168.8, 176.2 (C-28), 196.5 (C-3), 198.8 (C-12). MS (ESI): 621.416 [M+H]+,
643.386 [M+Na]+, 659.359 [M+K]+. HPLC purity: 95.0%, Rt = 8.796 min, UV 254 nm, 90% methanol,
flowrate: 2.0 mL/min.
(S)-1-
N-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-amide)-1,2,3,4-tetrahydronaphthalene
(17).
1
White solid (45 mg, yield: 73%), mp 185-186 oC. CAS: 2490323-22-1. H NMR (600 MHz, CDCl3)
δ:

0.90 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.02(s, 3H, CH3), 1.17 (s, 3H, CH3), 1.26 (s, 3H, CH3), 1.17-1.33
(m, 2H, CH2), 1.39 (s, 3H, CH3), 1.49 (s, 3H, CH3), 2.05-2.08 (m, 2H, CH2), 2.75-2.86 (m, 2H, CH2),
3.01-3.03 (m, 1H, H-18), 3.16 (d, 1H, J = 4.7 Hz, H-13), 5.20-5.23 (m, 1H, NCH), 5.91 (d, 1H, J = 8.0
Hz, NH), 5.96 (s, 1H, H-11), 7.10-7.27 (m, 4H, 4
CDCl3) : 18.2, 20.2, 21.6, 21.6, 23.1, 23.4, 24.9, 26.7, 27.0, 27.8, 29.3, 30.4, 30.6, 31.7, 32.2, 33.3,
×
ArH), 8.04 (s, 1H, H-1). 13C NMR (150 MHz,
δ
34.2, 34.7, 36.0, 42.2, 42.5, 45.0, 46.0, 46.4, 47.4, 47.7, 49.4, 114.4, 114.6 124.1 (CN), 126.3, 127.3,
128.4, 129.3, 136.7, 137.7, 165.8, 168.1, 176.1 (C-28), 196.6 (C-3), 198.8 (C-12). MS (ESI): 621.393
[M+H]+. HPLC purity: 98.7%, Rt = 10.135 min, UV 254 nm, 95% methanol, flowrate: 2.0 mL/min.
1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-4-(pyridin-2-yl)-1
H-imidazole (19). White solid
o
1
(25 mg, yield: 89%), mp 197-199 C. CAS: 1883650-96-1. H NMR (400 MHz, DMSO-d6) : 0.93 (s,
δ
3H, CH3), 0.95 (s, 3H, CH3), 0.99(s, 3H, CH3), 1.04 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.21 (s, 3H, CH3),
1.42 (s, 3H, CH3), 2.99-3.03 (m, 1H, H-18), 3.12 (d, 1H, J = 4.4 Hz, H-13), 6.26 (s, 1H, H-11), 7.31-
7.34 (m, 1H, Ar-H), 7.88-7.97 (m, 2H, Ar-H), 8.26 (s, 1H, H-1), 8.58 (m, 1H, Ar-H), 8.68 (s, 1H, Ar-H),
13
8.74 (s, 1H, Ar-H). C NMR (150 MHz, CDCl3)
δ: 18.2, 21.4, 21.5, 23.2, 23.5, 24.4, 26.6, 26.9, 28.5,
30.3, 31.6, 32.6, 32.8, 34.3, 35.6, 42.4, 42.5, 45.0, 45.8, 47.8, 48.6, 50.0, 113.4, 114.3, 114.6, 122.9,
124.1 (CN), 128.9, 133.4, 138.2, 139.5, 146.2, 148.1, 165.5, 168.0, 175.2 (C-28), 196.4 (C-3), 198.3 (C-
12). MS (ESI): 619.365 [M+H]+. HPLC purity: 98.1%, Rt = 6.538 min, UV 210 nm, 90% acetonitrile,
flowrate: 1.0 mL/min
1-(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl)-4-(pyridin-3-yl)-1
H-imidazole (20). White solid
(37 mg, yield: 92%), mp 193-195 oC. CAS NO. 1883650-95-0. 1H NMR (400 MHz, DMSO-d6)
δ: 0.95
(s, 6H, 2
×CH3), 1.00 (s, 3H, CH3), 1.08 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.20 (s, 3H, CH3), 1.43 (s, 3H,
CH3), 3.16-3.18 (m, 1H, H-18), 3.35 (d, 1H, J = 4.0 Hz, H-13), 6.26 (s, 1H, H-11), 7.44 (dd, 1H, J = 4.0
Hz, 7.9 Hz, Ar-H), 8.28 (d, 1H, J = 8.0 Hz, Ar-H), 8.50 (d, 1H, J = 4.8 Hz, Ar-H), 8.59 (s, 1H, H-1),
8.68 (s, 1H, Ar-H), 8.72 (s, 1H, Ar-H), 9.16 (s, 1H, Ar-H). 13C NMR (150 MHz, CDCl3)
δ: 18.2, 21.6,
23.6, 23.7, 24.5, 26.6, 26.9, 28.4, 30.4, 31.6, 31.9, 32.7, 32.8, 34.3, 35.6, 42.3, 42.5, 45.0, 45.8, 47.8,

48.7, 50.0, 113.4, 114.3, 114.7, 123.9, 124.0 (CN), 128.9, 133.4, 137.8, 139.4, 146.1, 148.1, 165.4,
168.1, 174.8 (C-28), 196.4 (C-3), 198.1 (C-12). MS (ESI): 619.366 [M+H]+. HPLC purity: > 99%, Rt =
12.983 min, UV 210 nm, 30~90% acetonitrile, flowrate: 0.5 mL/min
Biology
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin−streptomycin liq-
uid (100×), phosphate buffer saline (PBS). The 2,3,5-triphenyl tetrazolium chloride (TTC) was obtained
from BIO BASICINC (Shanghai, China). Recombinant mouse/human TNF-α were purchased from No-
voprotein (Shanghai, China).
Cell culture and Transfection
HT-29 (NCI-DTP Cat# HT-29), L929 (ECACC Cat# 14112101) cells were cultured in DMEM with
10% FBS (v/v), 1% L-glutamine and 100 U/mL penicillin/streptomycin(v/v). Cells were grown at 37 °C
in a humidified atmosphere with 5 % CO2 and were harvested in all experiments from exponentially
growing cultures.
Necroptosis induction and cell viability assays
Necroptosis was induced by pre-treatment with z-VAD-fmk (20 μM) and Smac mimetic (10 μM) or cy-
cloheximide (5 μg/mL) for 30 min and followed bu TNF-α (20 ng/mL) for 12 or 16 h. Apoptosis was
induced by TNF-α (20 ng/mL) and Smac mimetic (10 nM) for 24 h. The compounds were incubated
with the cells exposed to one of the above combinations at the indicated concentrations for 16 h or 24 h.
Cell viability was then examined by using the CellTiter-Glo Luminescent Cell Viability Assay kit.
SIRS mouse model
All animal experiments were performed in accordance with the National Institutes of Health guidelines
and approved by the animal care and use committee of the Second Military Medical University (EC11-
055). For TNF-induced SIRS, female C57BL/6 J mice (6–8 weeks old) were purchased from Chang-
zhou Caven Laboratory Animal (Jiangsu, China) and raised in a pathogen-free environment (21-26 °C
and 40-70 % humidity). Compounds were suspended in 0.5% carboxymethyl cellulose sodium. Over-

night-fasted mice were randomized divided into vehicle and treatment groups (n=11 for each group). In
drug treatment groups, mice were given the test compound with different concentrations by oral gavage
2 h before TNF injection. mTNF-α was diluted in endotoxin-free PBS and intravenously injected (250
μg/kg) in a volume of 200 μL. Z-VAD-fmk was intraperitoneally given 200 μg 15 min before, and 75
μg 1 h after mTNF-α treatment. Body temperature was monitored with an electric thermometer.
MCAO rat model and treatment with CDDO analogues
Healthy adult male Sprague-Dawley rats weighing 180−220 g were purchased form Changzhou Caven
Laboratory Animal (Jiangsu, China) and raised in a pathogen-free environment (21-26 °C and 40-70 %
humidity). Rats were anesthetized with 10% chloral hydrate (300 mg / kg, i.p.), monofilament nylon su-
ture was inserted into the internal carotid artery (ICA), and then it was advanced to block the left middle
cerebral artery (MCA). After 2 h of occlusion, the thread was dismantled carefully to achieve the reper-
fusion for 24 h. Sham-operated mice were exposed to the same surgical procedure, except for arteries
occlusion. All rats were divided randomly into four groups as follows using a random number table:
sham operated (sham), I/R (vehicle), I/R + compound 20 (100 mg/kg), and I/R + nimodipine (1 mg/kg)
groups. Nimodipine, as the first-line treatment for stroke, an L-type voltage-gated calcium channel,
caused vasodilatation of vascular smooth muscle cells.[50, 51] Thus, nimodipine was used as a positive
control.
Neurological Deficit Score
Neurological deficits were evaluated blindly after 24 h of reperfusion with Zea-Longa score scoring sys-
tem.[52] The scoring system is as follows: 0 = No signs of impaired nerve function, normal activity; 1 =
Cannot fully extend contralateral forepaws; 2 = Circle to the opposite side while walking; 3 = Dump to
the opposite side; 4 = Cannot go away spontaneously and lose consciousness.
Infarct Volume Evaluation
After 24 h of reperfusion, six animals of each group were euthanized by cervical dislocation and decapi-
tated, then brains were used to measure infarct volume immediately. The animal brains were cut into 2
mm thick coronal sections, 5 - 6 pieces and exposed to TTC staining after immersion in 4% paraformal-

dehyde for 4 h at 4 °C. Unstained areas were defined as infarcts and measured using microscope image-
analysis software (Image-Pro Plus, U.S.A.). The proportion of cerebral infarction was calculated as fol-
low: Proportion of cerebral infarction (%) = Infarct area / whole brain area ×100%).
Hematoxylin-Eosin (HE) Staining
Rats were anesthetized with 10% chloral hydrate after 24 h of reperfusion, perfused with physiological
saline and 4% paraformaldehyde and then decapitated. Brains were dehydrated and hembedded in paraf-
fin and then cut into 5 μm coronal sections; subsequently, brain sections were deparaffinized and hy-
drated for HE staining.
Western Blotting
After CDDO analogues treatment, the protein samples were extracted from cells using RIPA lysis buffer
containing a protease inhibitor and phosphatase inhibitor cocktail (Beyotime Biotechnology, China),
The quantification of proteins was analyzed using BCA protein assay kit (Beyotime Biotechnology,
China). The proteins (30 μg) were resolved over sodium dodecyl sulfate-polyacrylamide gel electropho-
resis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Bedford, MA, U.S.A.). Then, the
membranes were blocked in 5% BSA for 2 h. Primary antibodies were prepared in 1% BSA at a dilution
of 1:1000. The blocked membranes were incubated with corresponding antibody overnight at 4℃, fol-
lowed by the incubation with marked secondary antibody (1:8000) for 1 h at room temperature. The
signals were captured and analyzed using the LI‐COR Odyssey system. Antibodies were from commer-
cial sources: anti-RIPK1 (Abcam Cat# ab178420); anti-human phospho-RIPK1 (Cell Signaling Tech-
nology, Cat# 65746); anti-human-RIPK3 (Abcam Cat# ab56164), anti-human phospho-RIPK3 (Abcam
Cat# ab209384), anti-human MLKL (Abcam Cat# ab184718), anti-human phospho-MLKL (Abcam
Cat# ab187091), anti-GAPDH (Abcam Cat# ab181602). Anti-HSP70 (CST Cat# 4872), anti-EGRF
(CST Cat# 4267).
Immunoprecipitation
Cells were lysed with Nonidet P-40 buffer (Beyotime Biotechnology, China) supplemented with 1 mM
PMSF, 1× protease inhibitor mixture (Roche), 10 mM β-glycerophosphate, 5 mM NaF, and 1 mM

Na3VO4. The lysates were centrifuged and the supernatants were incubated with anti-RIPK1 (CST Cat#
3493S) antibody overnight at 4 °C. The immunocomplex was captured by Protein A/G Agarose (Life
Technologies) overnight at 4 °C. Beads were washed three times with PBS, and the bound proteins were
removed by boiling in SDS buffer, and the samples were resolved in 10% SDS-polyacrylamide gels by
western blotting analysis.
Statistical Analysis.
The one-way analysis of variance was used to compare differences among groups represented by the
mean values ± SD. A log-rank (Mantel−Cox) test was performed for survival curve analysis. P < 0.05
was considered statistically significant.
ASSOCIATED CONTENT
Supporting Information
1H, 13C NMR spectra, MS, and HPLC of target compounds (DOCX).
ACKNOWLEDGMENTS
This work was funded by grants from the National Natural Science Foundation of China (82022065,
81872791, 82073696, 81872880 and 81973551); the Key Research and Development Program of Ning-
xia (2019BFG02017).
ABBREVIATIONS USED
ACD, accidental cell death; CDDO, bardoxolone; I/R, ischemia/reperfusion; HPLC, high-performance
liquid chromatography; MCAO, middle cerebral artery occlusion; MLKL, mixed lineage kinase do-
main-like protein; PCD, programmed cell death; PDAC, pancreatic ductal adenocarcinoma cancer;
RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; SIRS, sys-
temic inflammatory response syndrome; TC, TNF-α and cycloheximide; TCZ, TNF-α, cycloheximide,
and z-VAD-fmk; TNF-α, tumor necrosis factor-α; TS, TNF-α and Smac mimetic; TSZ, TNF-α, Smac
mimetic, and z-VAD-fmk; TTC, 2,3,5-triphenyltetrazolium chloride; TZ, TNF-α and z-VAD-fmk; UC,
ulcerative colitis

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